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Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change

Parvaiz Ahmad

M.N.V. Prasad

Editors

Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change

Editors Parvaiz Ahmad Department of Botany Amar Singh College University of Kashmir Srinagar, Jammu and Kashmir India [emailprotected]

M.N.V. Prasad Department of Plant Sciences University of Hyderabad Andhra Pradesh, Hyderabad 500 046 India [emailprotected], [emailprotected]

ISBN 978-1-4614-0814-7 e-ISBN 978-1-4614-0815-4 DOI 10.1007/978-1-4614-0815-4 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011938457 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Any external factor that imposes negative impact on growth and development of the plant is known as stress. Plants often experience abiotic stress like drought, salinity, alkalinity, temperature, UV-radiations, oxygen deficiency, etc. Abiotic stress is responsible for the huge crop loss and reduced yield more than 50% of some major crops. Ion imbalance and osmotic stress is the primary effect of abiotic stress. Prolonged exposure to primary stress causes secondary stress through the generation of reactive oxygen species (ROS). These are deleterious for the plants as it causes oxidative damage by reacting with biomolecules. Plants are able to perceive the external and internal signals and are then used by the plant to regulate various responses to stress. Plants respond the abiotic stress by up- and downregulation of genes responsible for the synthesis of osmolytes, osmoprotectants, and antioxidants. Stress-responsive genes and gene products including proteins are expressed and provide tolerance to the plant. To understand the physiological, biochemical, and molecular mechanisms for abiotic stress, perception, transduction, and tolerance is still a challenge before plant biologists. The chapters in this book deal with the effect of different abiotic stresses on plant metabolism and responses of the plants to withstand the stress. Chapter 1 describes involvement of different osmolytes, osmoprotectants, and antioxidants during abiotic stress. Chapter 2 deals with the role of halophytes in understanding and managing abiotic stress. Chapter 3 addresses the effect and defense mechanisms in plants under UV stress. Chapter 4 throws light on the potassium uptake and its role under abiotic stress. Chapters 5–7 deal with the effect of temperature (heat, chilling) on plants and their responses. Chapter 8 deals with the formation and function of roots under stress. Chapter 9 is concerned with role of ROS and NO under abiotic stress. Chapter 10 throws light on nitrogen inflow and nitrogen use efficiency (NUE) under stress. Chapter 11 addresses Am symbiosis and soil interaction under abiotic stress. Chapter 12 deals with the role of small RNA in abiotic stress. Chapter 13 describes the involvement of transcription factors (TFs) under abiotic stress. Chapters 14–17 deal with the involvement of different signaling molecules (Ca2+, H2O2, and phytohormones) under abiotic stress. Chapter 18 covers the role of ethylene and plant growth-promoting bacteria under environmental stress. Chapter 19 throws light on new approaches about metal-induced stress. Chapters 20 and 21 address the role of sulfur and salicylic acid in

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alleviating heavy metal-induced stress. Chapters 22 and 23 cover the bioremediation of organic contaminants and utilization of different weeds in removal of heavy metals. We hope that this volume will provide the background for understanding abiotic stress tolerance in plants. Srinagar, Jammu & Kashmir, India Hyderabad, Andhra Pradesh, India

Parvaiz Ahmad M.N.V. Prasad

Contents

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Abiotic Stress Responses in Plants: An Overview..................... Hans-Werner Koyro, Parvaiz Ahmad, and Nicole Geissler

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Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance .............................................................. Vinayak H. Lokhande and Penna Suprasanna

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UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants ..................................................................... Fernando E. Prado, Mariana Rosa, Carolina Prado, Griselda Podazza, Roque Interdonato, Juan A. González, and Mirna Hilal K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants ............................................................................. Manuel Nieves-Cordones, Fernando Alemán, Mario Fon, Vicente Martínez, and Francisco Rubio

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Temperature Stress and Responses of Plants ............................ Anna Źróbek-Sokolnik

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Responses and Management of Heat Stress in Plants .............. Abdul Wahid, Muhammad Farooq, Iqbal Hussain, Rizwan Rasheed, and Saddia Galani

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Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants ..................................... Dana Zoldan, Reza Shekaste Band, Charles L. Guy, and Ron Porat

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Root Form and Function in Plant as an Adaptation to Changing Climate .................................................................... Maria Rosa Abenavoli, Maria Rosaria Panuccio, and Agostino Sorgonà

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Contents

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9

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Reactive Oxygen Species and Nitric Oxide in Plants Under Cadmium Stress: From Toxicity to Signaling................ Luisa M. Sandalio, Maria Rodríguez-Serrano, Dharmendra K. Gupta, Angustias Archilla, Maria C. Romero-Puertas, and Luis A. del Río Reactive Nitrogen Inflows and Nitrogen Use Efficiency in Agriculture: An Environment Perspective ............................ Khalid Rehman Hakeem, Ruby Chandna, Altaf Ahmad, and Muhammad Iqbal Arbuscular Mycorrhizal Symbiosis and Other Plant–Soil Interactions in Relation to Environmental Stress ..................... Patrick Audet MicroRNAs and Their Role in Plants During Abiotic Stresses............................................................................. Praveen Guleria, Deepmala Goswami, Monika Mahajan, Vinay Kumar, Jyoti Bhardwaj, and Sudesh Kumar Yadav Transcription Factors Involved in Environmental Stress Responses in Plants ........................................................... Haibo Xin, Feng Qin, and Lam-Son Phan Tran

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Plant Signaling Under Abiotic Stress Environment.................. Parvaiz Ahmad, Renu Bhardwaj, and Narendra Tuteja

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Calcium Signalling in Plant Cells Under Environmental Stress ................................................................... Sylvia Lindberg, Md. Abdul Kader, and Vladislav Yemelyanov

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279 297

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Role of H2O2 as Signaling Molecule in Plants ............................ M.A. Matilla-Vázquez and A.J. Matilla

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Role of Phytohormone Signaling During Stress ........................ Mohammad Miransari

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Ethylene and Abiotic Stress Tolerance in Plants ....................... Elisa Gamalero and Bernard R. Glick

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New Approaches to Study Metal-Induced Stress in Plants......................................................................................... M.C. Cia, F.R. Capaldi, R.F. Carvalho, P.L. Gratão, and R.A. Azevedo

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Sulfur in the Alleviation of Cadmium-Induced Oxidative Stress in Plants ............................................................ Noushina Iqbal, Nafees A. Khan, Md. Iqbal R. Khan, Rahat Nazar, Asim Masood, and Shabina Syeed

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Contents

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21

Role of Salicylic Acid in Alleviating Heavy Metal Stress ......... Losanka P. Popova, Liliana T. Maslenkova, Albena Ivanova, and Zhivka Stoinova

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Bioremediation and Mitigation of Organic Contaminants in the Era of Climate Changes........................... Laura Coppola, Edoardo Puglisi, Costantino Vischetti, and Marco Trevisan

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Exploitation of Weeds and Ornamentals for Bioremediation of Metalliferous Substrates in the Era of Climate Change ..................................................... M.N.V. Prasad

Index ......................................................................................................

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Contributors

Maria Rosa Abenavoli Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale, Università Mediterranea di Reggio Calabria, Contrada Melissari – Lotto D, 89124 Reggio Calabria, Italy Altaf Ahmad Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 110062, India Parvaiz Ahmad Department of Botany, A.S. College, Srinagar 190008, Jammu & Kashmir, India Fernando Alemán Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain Angustias Archilla Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain Patrick Audet Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, QLD 4072, Australia R. A. Azevedo Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba 13418-900, SP, Brazil Reza Shekaste Band Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611, USA Jyoti Bhardwaj Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India Renu Bhardwaj Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India F. R. Capaldi Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba 13418-900, SP, Brazil

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R. F. Carvalho Departamento de Biologia Aplicada à Agropecuária, Universidade Estadual Paulista Júlio de Mesquita Filho, Jaboticabal 14884-900, SP, Brazil Ruby Chandna Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 110062, India M. C. Cia Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba 13418-900, SP, Brazil Laura Coppola Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italy Muhammad Farooq Department of Agronomy, University of Agriculture, Faisalabad 38040, Pakistan Mario Fon Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain Saddia Galani Khan Institute of Biotechnology and Genetic Engineering, University of Karachi, Karachi, Pakistan Elisa Gamalero Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale, Viale Teresa Michel 11, Alessandria 15121, Italy Nicole Geissler Institute of Plant Ecology, Justus Liebig University Giessen, Heinrich Buff-Ring 2632, 35392 Giessen, Germany Bernard R. Glick Department of Biology University of Waterloo N2L 3G1, Waterloo, ON, Canada Juan A. González Instituto de Ecología, Fundación Miguel Lillo, Miguel Lillo 251, CP 4000 Tucumán, Argentina Deepmala Goswami Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India P. L. Gratão Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba 13418-900, SP, Brazil Praveen Guleria Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India Dharmendra K. Gupta Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080, Granada, Spain

Contributors

Contributors

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Charles L. Guy Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611, USA Khalid Rehman Hakeem Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 110062, India Mirna Hilal Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000 Tucumán, Argentina Iqbal Hussain Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan Roque Interdonato Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000, Tucumán, Argentina Muhammad Iqbal Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 110062, India Noushina Iqbal Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Albena Ivanova Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev str, BL. 21 1113, Sofia, Bulgaria Md. Abdul Kader Department of Agronomy, Bangladesh Agricultural University Mymensingh, Mymensingh 2202, Bangladesh Md. Iqbal R. Khan Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Nafees A. Khan Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Hans-Werner Koyro Institute of Plant Ecology, Justus Liebig University Giessen, Heinrich Buff-Ring 2632, 35392 Giessen, Germany Vinay Kumar Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India Sylvia Lindberg Department of Botany, SU, SE-106 91 Stockholm, Sweden Vinayak H. Lokhande Functional Plant Biology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, Maharashtra, India Monika Mahajan Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India Vicente Martínez Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain

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Liliana T. Maslenkova Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev str, BL. 21 1113, Sofia, Bulgaria Asim Masood Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India A. J. Matilla Departamento de Fisiología Vegetal, Facultad de Farmacia, Universidad de Santiago de Compostela (USC), 15782, Santiago de Compostela, Spain M.A. Matilla-Vázquez Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK Mohammad Miransari Department of Soil Science, Shahed University, College of Agricultural Sciences, 18151/159 Tehran, Iran Rahat Nazar Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Manuel Nieves-Cordones Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain Maria Rosaria Panuccio Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale, Università Mediterranea di Reggio Calabria, Contrada Melissari – Lotto D, 89124 Reggio Calabria, Italy Griselda Podazza Instituto de Ecología, Fundación Miguel Lillo, Miguel Lillo 251, CP 4000 Tucumán, Argentina Losanka P. Popova Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev str, BL. 21, 1113 Sofia, Bulgaria Ron Porat Department of Postharvest Sciences of Fresh Produce, ARO, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel Carolina Prado Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000 Tucumán, Argentina Fernando E. Prado Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000 Tucumán, Argentina M.N.V. Prasad Department of Plant Sciences, University of Hyderabad, Prof. C.R. Rao Road, Gachibowli, Central University P.O., Hyderabad, AP 500 046, India Edoardo Puglisi Istituto di Chimica Agraria ed Ambientale, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza 29122, Italy

Contributors

Contributors

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Feng Qin Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100049, China Rizwan Rasheed Biology Department, Foreman Christian College, Lahore, Pakistan Luis A. del Río Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain Maria Rodríguez-Serrano Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain Maria C. Romero-Puertas Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain Mariana Rosa Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000 Tucumán, Argentina Francisco Rubio Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain Luisa M. Sandalio Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain Agostino Sorgonà Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale, Università Mediterranea di Reggio Calabria, Contrada Melissari – Lotto D, 89124 Reggio Calabria, Italy Zhivka Stoinova Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev str, BL. 21, 1113 Sofia, Bulgaria Penna Suprasanna Functional Plant Biology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400 085, Maharashtra, India Shabina Syeed Department of Botany, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India Lam-Son Phan Tran Signaling Pathway Research Unit, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan Marco Trevisan Istituto di Chimica Agraria ed Ambientale, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza 29122, Italy

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Narendra Tuteja Plant molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Costantino Vischetti Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italy Abdul Wahid Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan Haibo Xin Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100049, China Sudesh Kumar Yadav Plant Metabolic Engineering Laboratory, Biotechnology Division, Institute of Himalayan Bioresource Technology, Council of Scientific and Industrial Research, Palampur 176061, Himachal Pradesh, India Vladislav Yemelyanov Department of Genetics and Breeding, St. Petersburg State University, St. Peterburg 199034, Russia Dana Zoldan Department of Postharvest Sciences of Fresh Produce, ARO, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel Anna Źróbek-Sokolnik Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Plac Łódzki 1, 10-727 Olsztyn, Poland

Contributors

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Abiotic Stress Responses in Plants: An Overview Hans-Werner Koyro, Parvaiz Ahmad, and Nicole Geissler

Abstract

Plants are more and more affected by environmental stresses, especially by the devastating consequences of desertification and water scarcity which can be seen and felt all over the world. About 3.6 billion of the world’s 5.2 billion hectares of dryland used for agriculture have already suffered erosion, soil degradation, and salinization. Desertification can hinder efforts for sustainable development and introduces new threats to human health, ecosystems, and national economies. This problem is catalyzed by global climate change which exacerbates desertification and salinization. Therefore, solutions are desperately needed, such as the improvement of drought and salinity tolerance of crops, which in turn requires a detailed knowledge about tolerance mechanisms in plants. These mechanisms comprise a wide range of responses on molecular, cellular, and whole plant levels, which include amongst others the synthesis of compatible solutes/osmolytes and radical scavenging mechanisms. Regarding global change, elevated atmospheric CO2 concentrations can enhance salt and drought tolerance because oxidative stress is alleviated and more energy can be provided for energy-dependent tolerance mechanisms such as the synthesis of compatible solutes and antioxidants, thus increasing the suitability of plants as crops in future. A detailed knowledge of the physiological and biochemical basis of drought and salt tolerance and its interaction with elevated CO2 concentration can provide a basis for the cultivation of suitable plants in regions threatened by desertification and water scarcity under sustainable culture conditions. Even the drylands could offer tangible economic and ecological opportunities. H.-W. Koyro () • N. Geissler Institute of Plant Ecology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany e-mail: [emailprotected] P. Ahmad Department of Botany, Amar Singh College, Srinagar 190008, Jammu & Kashmir, India P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_1, © Springer Science+Business Media, LLC 2012

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H.-W. Koyro et al.

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The aim of this chapter is to uncover how compatible solutes and antioxidants alleviate environmental stress, especially drought and salt stress, and the role elevated CO2 concentrations can play in this context, so that early indicators allowing successful breeding can be identified and the potential of plants as crops in a CO2 rich world can be assessed. Keywords

Abiotic stress • Antioxidants • Osmolytes • Oxidative stress

1

Introduction

Plants are continuously affected by a variety of environmental factors. Whereas biotic environmental factors are other organisms such as symbionts, parasites, pathogens, herbivores, and competitors, abiotic factors include parameters and resources which determine plant growth like temperature, relative humidity, light, availability of water, mineral nutrients, and CO2, as well as wind, ionizing radiation, or pollutants (Schulze et al. 2002). The effect each abiotic factor has on the plant depends on its quantity or intensity. For optimal growth, the plant requires a certain quantity of each abiotic environmental factor. Any deviation from such optimal external conditions, that is, an excess or deficit in the chemical or physical environment, is regarded as abiotic stress and adversely affects plant growth, development, and/or productivity (Bray et al. 2000). Abiotic stress factors include, for example, extreme temperatures (heat, cold, and freezing), too high or too low irradiation, water logging, drought, inadequate mineral nutrients in the soil, and excessive soil salinity. As especially drought and salt stress are becoming more and more serious threats to agriculture and the natural status of the environment, this chapter will focus on these stress factors. They are recurring features of nearly all the world’s climatic regions since various critical environmental threats with global implications have linkages to water crises (Gleick 1994, 1998, 2000). These threats are collaterally catalyzed by global climate change and population growth.

The latest scientific data confirm that the earth’s climate is rapidly changing. Due to rising concentrations of CO2 and other atmospheric trace gases, global temperatures have increased by about 1°C over the course of the last century, and will likely rise even more rapidly in coming decades (IPCC 2007). Scientists predict that temperatures could rise by another 3–9°C by the end of the century with far-reaching effects. Increased drought and salinization of arable land are expected to have devastating global effects (Wang et al. 2003b). Abiotic stress is already the primary reason of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Bray et al. 2000; Wang et al. 2003b). It will soon become even more severe as desertification will further increase and the current amount of annual loss of arable area may double by the end of the century because of global warming (Evans 2005; Vinocur and Altman 2005). Simultaneously, rapid population growth increasingly generates pressure on existing cultivated land and other resources (Ericson et al. 1999). Population migration to those arid and semiarid areas increases the problems of water shortage and worsens the situation of land degradation in the destination, and in turn causes severe problems of poverty, social instability, and population health threats (Moench 2002). Water scarcity and desertification could critically undermine efforts for sustainable development, introducing new threats to human health, ecosystems, and national economies of various countries. Therefore, solutions to these problems are desperately needed, such as the improvement of salt and drought tolerance of crops, which in turn

1 Abiotic Stress Responses in Plants: An Overview

requires a detailed knowledge about salt and drought tolerance mechanisms in plants. The viability of plants in both dry and saline habitats depends on their ability to cope with (I) water deficit due to a low water potential of the soil and (II) restriction of CO2 uptake. Plants growing on saline soils are additionally confronted with (III) ion toxicity and nutrient imbalance. Water deficit (I) causes detrimental changes in cellular components because the biologically active conformation and thus the correct functioning of proteins and biomembranes depends on an intact hydration shell. As a consequence, severe osmotic stress can lead to an impairment of amino acid synthesis, protein metabolism, the dark reaction of photosynthesis or respiration and can cause the breakdown of the osmotic system of the cell (Larcher 2001; Schulze et al. 2002). Water deficit can be counteracted by compatible solutes, organic compounds which are highly soluble and do not interfere with cellular metabolism. They serve as a means for osmotic adjustment and also function as chaperons by attaching to proteins and membranes, thus preventing their denaturation. This protective function of compatible solutes can also alleviate ion specific effects of salt stress caused by ion toxicity and ion imbalance such as the precipitation of proteins due to changes in charge or the destruction of membranes caused by alterations of the membrane potential. Regarding the restriction of CO2 uptake (II), the negative effects of osmotic stress described earlier force plants to minimize water loss; growth depends on the ability to find the best tradeoff between a low transpiration and a high net photosynthetic rate (Koyro 2006). However, various plant species show a clearly reduced assimilation rate under osmotic stress conditions due to stomatal closure (Huchzermeyer and Koyro 2005). A consequence can be an excessive production of reactive oxygen species (ROS) which are highly destructive to lipids, nucleic acids, and proteins (Kant et al. 2006; Türkan and Demiral 2009; Geissler et al. 2010). However, generated ROS can be scavenged by the antioxidative system which includes nonenzymatic antioxidants and antioxidative enzymes (Blokhina et al. 2003).

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Ion toxicity (III) on saline habitats is caused by ion specific effects on membranes and proteins: On the one hand, changes of the ionic milieu lead to alterations of the membrane potential and thus to a destruction of biomembranes (Schulze et al. 2002). On the other hand, the hydration and charge of proteins are negatively influenced, so that their precipitation is promoted, but their activity is reduced (Kreeb 1996). These effects of salt stress can be alleviated by the protective chaperone function of compatible solutes, similarly as explained above for osmotic stress. When looking at drought and salt tolerance of plants in the face of global climate change, another important aspect should be considered: Compared to salinity and drought, elevated atmospheric CO2 concentrations have contrary effects on plants: They often improve photosynthesis while reducing stomatal resistance in C3 plants, thus increasing water use efficiency, but decreasing photorespiration and oxidative stress (Urban 2003; Kirschbaum 2004; Rogers et al. 2004). Furthermore, more energy can be provided for energy-dependent tolerance mechanisms such as the synthesis of compatible solutes and antioxidants. Therefore, the salt and drought tolerance and the productivity of these plants can be enhanced under elevated CO2 (Ball and Munns 1992; Wullschleger et al. 2002; Urban 2003), increasing their future suitability as crops. Against the background described earlier, this review uncovers how compatible solutes and antioxidants alleviate environmental stress, especially drought and salt stress, and the role elevated CO2 concentrations can play in this context.

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Compatible Solutes Which Can Prevent Detrimental Changes Under Environmental Stress

Severe osmotic stress can cause detrimental changes in cellular components. The best characterized biochemical response of plant cells to osmotic stress is the accumulation of high concentrations of either organic ions or other low

H.-W. Koyro et al.

4 Fig. 1.1 Chemical structure of some important compatible solutes in plants

molecular weight organic solutes termed compatible solutes. These compounds are highly soluble in water, electrically neutral in the physiological pH range, and noninhibitory to enzymes even at high concentrations, so that they do not interfere with essential metabolic (enzymatic) reactions (Rhodes et al. 2002). The structure of some important compatible solutes is shown in Fig. 1.1. Organic solutes play a crucial role in higher plants grown under dry or saline conditions. However, their relative contribution varies among species, cultivars, and even between different compartments within the same plant (Ashraf and Harris 2004). A wide range of metabolites which can prevent these detrimental changes in cellular components have been identified, including mono-, di-, oligo-, and polysaccharides (glucose, fructose, sucrose, trehalose, raffinose, and fructans), sugar alcohols (mannitol, glycerol, and methylated inositols), quaternary amino acid derivatives (Pro, GB, b-alaninebetaine and prolinebetaine), tertiary amines (1,4,5,6-tetrahydro2-mehyl-4-carboxyl pyrimidine), and sulfonium compounds (choline-O-sulphate, dimethylsulphoniopropionate) (Flowers and Colmer 2008; Vinocur and Altman 2005). The primary function of compatible solutes is to reduce water potential, to maintain turgescent cells, and to ensure balanced water relations (Wang et al. 2003a).

In addition, high concentration of compatible solutes exists primarily in the cytosol to balance the low water potentials achieved by high apoplasmic and vacuolar Na+ and Cl− concentration (Türkan and Demiral 2009). Recent studies indicate that compatible osmolytes also protect subcellular structures and mitigate oxidative damage caused by free radicals produced in response to salt stress (Slama et al. 2008; Smirnoff and Cumbes 1989). In many halophytes, organic osmolytes such as Pro or GB accumulate at suitably high concentrations to create osmotic potentials even below 0.1 MPa. In contrast to halophytes, in many glycophytes the concentrations of compatible solutes do not seem to be high enough to generate sufficiently low osmotic potentials (Türkan and Demiral 2009). This difference between halophytes and glycophytes can be used as an early indicator for salt resistance. Therefore, in the next chapters, the most important compatible solutes are described in detail.

2.1

Betaines

The quaternary ammonium compounds that function as effective compatible osmolytes in plants subject to salt stress are GB, b-alaninebetaine, prolinebetaine, choline-O-sulphate, hydroxyprolinebetaine, and pipecolatebetaine (Ashraf and

1 Abiotic Stress Responses in Plants: An Overview

5

Fig. 1.2 Biosynthetic pathway of glycinebetaine (adopted from Ahmad and Sharma 2008)

Harris 2004). GB occurs most abundantly in response to a variety of abiotic stress conditions by numerous organisms including bacteria, cyanobacteria, algae, fungi, animals, and many plant families such as Chenopodiaceae and Gramineae (Türkan and Demiral 2009). This metabolite is mainly located in chloroplasts and plays a vital role in the stroma adjustment and protection of thylakoid membranes, thereby maintaining the photosynthetic activity (Jagendorf and Takabe 2001). GB protects the photosystem II (PS-II) complex at high salinity (Murata et al. 1992) and at extreme temperatures or pH (Mohanty et al. 1993). GB also protects membranes against heat-induced destabilization and enzymes, such as RUBISCO, against osmotic stress (Mäkelä et al. 2000). In higher plants, GB is synthesized from serine via ethanolamine, choline by two-step oxidation reactions that were catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase, respectively (Russell et al. 1998; Ahmad and Sharma 2008; see Fig. 1.2). The insertion of serine and glycine can be taken as an indicator for the close relationship of the photorespiration (peroxisomes) to the synthesis of GB. Besides this, recently a biosynthetic pathway of GB from glycine with the involvement of two N-methyl transferase enzymes has been reported (Waditee et al. 2005). Highly tolerant genera such as Spartina and Distichlis accumulated the highest levels of GB, moderately tolerant species intermediate levels, and sensitive species hardly any GB (Rhodes and Hanson 1993). Genetic evidence that GB improves salinity tolerance has been obtained for many important

agronomical crops such as tobacco, tomato, potato, barley, maize, and rice. These plants have long been a potential target for engineering GB biosynthesis pathway and thus for resistance against different environmental stress conditions (Sairam and Tyagi 2004; Türkan and Demiral 2009). The importance of N-methyltransferase for stress tolerance could also be shown for Arabidopsis. Genetically modified plants of this genus accumulated betaine to significant levels at different environmental stress conditions and hence improved seed yield (Waditee et al. 2005). A moderate stress tolerance was noted in some transgenic lines based on relative shoot growth in response to salinity, drought, and freezing. Huang et al. (2000) reported metabolic limitation in betaine production in transgenic plants. In fact, Arabidopsis thaliana, Brassica napus, and Nicotiana tabacum were transformed with bacterial choline oxidase cDNA, and their levels of GB were only between 5 and 10% of the levels found in natural betaine producers. Beyond this, choline-fed transgenic plants synthesized substantially more GB. This result was taken as a hint that these plants require a distinct endogenous amount of choline to synthesize an adequate amount of GB (Sairam and Tyagi 2004). The protective effect of GB at salinity or drought could also be demonstrated by exogenous application at rice seedlings, soybean, and common beans (Ashraf and Foolad 2007; Demiral and Türkan 2006). GB pretreatment also alleviated salinity-induced peroxidation (oxidative damage) of lipid membranes of rice cultivars. Besides rice,

H.-W. Koyro et al.

6 Fig. 1.3 Biosynthetic pathway of proline (adopted from Ahmad and Sharma 2008)

the correlation between the protective effect of GB and the antioxidative defense system has been observed in chilling-stressed tomato (Park et al. 2006), drought- or salt-stressed wheat (Raza et al. 2007), and salt-stressed suspension cultured tobacco BY2 cells (Hoque et al. 2007).

2.2

Amino Acids, Proline, and Amides

It has been reported that amino acids (such as alanine, arginine, glycine, serine, leucine, and valine, the nonprotein amino acids citrulline and ornithine (Orn)), together with the imino acid Pro, and the amides such as glutamine and asparagine are accumulated in higher plants under salinity and drought stress (Dubey 1997; Mansour 2000). Pro is known to occur widely in higher plants and can be accumulated in considerable amounts in

response to salt stress, water deficit, and other abiotic stresses (Ali et al. 1999; Kavi Kishore et al. 2005; Koca et al. 2007; Ahmad and Sharma 2008). The Pro concentration is metabolically controlled. This imino acid is synthesized in plastids and cytoplasm while degraded to l -glutamate (Glu) in mitochondria. There are two different precursors of Pro in plants: Glu and Orn (Fig. 1.3). Pro is synthesized from Glu via glutamic-g-semialdehyde (GSA) and D1-pyrroline-5-carboxylate (P5C). P5C synthase (P5CS) catalyses the conversion of Glu to P5C, followed by P5C reductase (P5CR), which reduces P5C to Pro (Ashraf and Foolad 2007). The other precursor for Pro biosynthesis is Orn, which is transaminated to P5C by a mitochondrial Orn-g-aminotransferase (OAT) enzyme (Verbruggen and Hermans 2008). In the reverse reaction, Pro is metabolized to Glu in a feedback manner, via P5C and GSA with the aid of Pro

1 Abiotic Stress Responses in Plants: An Overview

dehydrogenase followed by P5C dehydrogenase (P5CDH) (Wang et al. 2003a). The contribution of Glu and Orn pathways to stress-induced Pro synthesis differs between species, and it has been shown that stress-tolerant plants are able to accumulate Pro in higher concentrations than stress-sensitive plants. Slama et al. (2008) showed a positive correlation between Pro accumulation and tolerance to salt, drought, and the combined effects of these stresses. Osmotic stress (particularly mannitol stress) led to a considerable increase of the Pro concentration in the obligatory halophyte Sesuvium portulacastrum, while the contents in soluble sugars and in Na+ remained unchanged. In drought-stressed plants, the concentration of K+, Na+, Cl−, and Pro, as well as ornithine-daminotransferase (d-OAT) activity increased significantly. Inversely, Pro dehydrogenase activity was impaired. Re-watering leads to a recovery of these parameters at values close to those of plants permanently irrigated with 100% of field capacity. The presence of NaCl and mannitol in the culture medium (ionic and osmotic stress) led to a significant increase of the Na+ and Pro concentration in the leaves, but it had no effect on leaf soluble sugar content. Slama et al. (2007a, b) assumed that the ability of NaCl to improve plant performance under mannitol-induced water stress is caused by an improved osmotic adjustment through Na+ and Pro accumulation, which is coupled with the maintenance of the photosynthetic activity. Similarly, the Pro concentration in the roots of salt tolerant alfalfa plants rapidly doubled under salt stress and was significantly higher than in salt sensitive genotypes (Petrusa and Winicov 1997). In addition to its role as an osmolyte for osmotic adjustment, Pro contributes to stabilizing subcellular structures (membranes and proteins) by forming clusters with water molecules which attach to proteins and membranes and prevent their denaturation (Koca et al. 2007; Ashraf and Foolad 2007; Lee et al. 2008). Due to its protective function on membranes it can also improve cell water status and ion homeostasis (Gadallah 1999; Gleeson et al. 2005), and it can scavenge free radicals and buffer cellular redox potential under stress conditions (Koca et al. 2007;

7

Ashraf and Foolad 2007; Lee et al. 2008). Pro is also involved in alleviation of cytoplasmic acidosis and sustaining NADP+/NADPH ratios at required levels for metabolism (Hare and Cress 1997), thus supporting redox cycling (Babiychuk et al. 1995). Transgenic approaches proved an enhancement of plant stress tolerance via overproduction of Pro. For instance, transgenic tobacco (N. tabacum), overexpressing the p5cs gene that encodes P5CS, produced 10- to 18-fold more Pro and exhibited better tolerance under salt stress (Kavi Kishor et al. 2005). In Arabidopsis, the overexpression of an antisense Pro dehydrogenase cDNA resulted in an increased accumulation of Pro and a constitutive tolerance to freezing and a higher salt tolerance (Nanjo et al. 2003). Similarly, Borsani et al. (2005) reported that the Arabidopsis P5CDH (D1-pyrroline-5-carboxylate dehydrogenase) and SRO5, an overlapping gene of unknown function in the antisense orientation, produced two types of siRNAs, 24-nt siRNA and 21-nt siRNA. In fact, they compared the levels of salt stress-induced Pro accumulation in various mutant plants (dcl2, sgs3, rdr6, and nrpd1a) which lacked SRO5P5CDH nat-siRNAs and cleavage of the P5CDH transcript, Pro accumulation was not significantly induced by salt stress or was induced to a lesser extent than in the corresponding wild type. This result is consistent with their inability to downregulate P5CDH under stress, thereby causing a continued Pro catabolism and reduced Pro accumulation. In contrast, the dcl1 and rdr2 mutants, which were able to degrade P5CDH mRNA, had the same Pro level as the wild type under salt stress. The wild-type level of Pro accumulation in dcl1 indicates that although the 21-nt P5CDH natsiRNAs were not produced, the 24-nt SRO5P5CDH nat-siRNA alone was sufficient to cause the downregulation of P5CDH (Fig. 1.4). An alternative approach to improve plant stress tolerance is the exogenous application of Pro, which can lead to either osmoprotection or cryoprotection. For example, in various plant species growing under salt stress, among them the halophyte Allenrolfea occidentalis, exogenous application of Pro led to a higher osmoprotection and an increased growth (Yancey 1994).

H.-W. Koyro et al.

8 Fig. 1.4 Diagram of phased processing of SRO5-P5CDH nat-siRNAs and its role in a salt-stress regulatory loop (Borsani et al. 2005)

2.3

Sugars and Sugar Alcohols

Several studies have been attempted to relate the magnitude of changes in soluble carbohydrates to salinity tolerance. Parida and Das (2005) found out that carbohydrates such as sugars (glucose, fructose, sucrose, and fructans) and starch are accumulated under salt stress. Furthermore, Megdiche et al. (2007) and Geissler et al. (2009a) proved that Cakile maritima and Aster tripolium plants accumulate high amounts of total soluble carbohydrates and Pro at high salinity (400 and 500 mM NaCl, respectively). The major functions of sugars and sugar alcohols are osmoprotection, osmotic adjustment, carbon storage, and radical scavenging (Adams et al. 2005; Ashraf et al. 2006; Messedi et al. 2006; Lee et al. 2008; Ahmad and Sharma 2008). Furthermore, there is a discussion about that they serve as molecular chaperones (Hasegawa et al. 2000; Liu et al. 2006).

There is a difference between starch and sugar accumulation in short- and long-term reaction (da Silva and Arrabaca 2004). In short-term water stress experiments, a decrease in sucrose and starch content was observed for Setaria sphacelata, a naturally adapted C4 grass while in longterm experiments, a higher amount of soluble sugars and a lower amount of starch were found. da Silva and Arrabaca (2004) assumed that the shift of metabolism towards sucrose might occur because starch synthesis and degradation are more affected than sucrose synthesis. Trehalose, a rare, nonreducing sugar, is present in several bacteria and fungi and in some desiccation-tolerant higher plants (Vinocur and Altman 2005). Under various abiotic stresses, the disaccharide trehalose accumulates in many organisms as an osmolyte and osmoprotectant, protects membranes and proteins in cells, and reduces the aggregation of denatured proteins

1 Abiotic Stress Responses in Plants: An Overview

(Ashraf and Harris 2004). In the transgenic plants, a comparatively moderate increase in trehalose levels lead to a higher photosynthetic rate and to a decrease in photooxidative damage during stress. Trehalose is thought to protect biological molecules from environmental stress (such as desiccation-induced damage), as suggested by its reversible water-absorption capacity (Penna 2003). It was shown that the contents of reducing and nonreducing sugars and the activity of sucrose phosphate synthase increase under salt stress, whereas starch phosphorylase activity decreases (Dubey and Singh 1999). In general, the sugar alcohols are divided in acyclic (e.g., mannitol) and cyclic (e.g., pinitol) polyols. Polyols can make up a considerable percentage of all assimilated CO2 and can have several functions such as compatible solutes, low molecular weight chaperones, and scavengers of stress-induced oxygen radicals (Bohnert et al. 1995). Polyols act in two indistinguishable ways, namely, osmotic adjustment and osmoprotection (Parida and Das 2005). In osmotic adjustment they act as osmolytes, facilitating the retention of water in the cytoplasm and enabling the sequestration of sodium into the vacuole or apoplast (cell wall). These osmolytes protect cellular structures by interacting with membranes, protein complexes, or enzymes. For instance, mannitol, a sugar alcohol that accumulates upon salt and water stress can alleviate abiotic stress. Transgenic wheat expressing the mannitol-1-phosphatase dehydrogenase gene (mtlD) of Escherichia coli was significantly more tolerant to water and salt stress (Abebe et al. 2003). Consequently, the transgenic wheat plants showed an increase in biomass, plant height, and number of secondary stems (tillers). The cyclic sugar alcohols pinitol and ononitol were accumulated in tolerant species such as the facultative halophyte Mesembryanthemum crystallinum when exposed to salinity or water deficit (Bohnert and Jensen 1996). Pinitol can be synthesized from myoinositol by the sequential catalysis of inositol methyl transferase and ononitol epimerase. An inositol methyl transferase (Imt) cDNA was isolated from transcripts in M. crystallinum growing under saline conditions (Vernon and Bohnert 1992), and transgenic tobacco for Imt has been obtained (Vernon et al. 1993).

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2.4

Polyamines

Under stressful conditions, different plant species respond differently towards levels of polyamines. Some might accumulate polyamines in response to stress, while others do not increase or even decrease their endogenous polyamine contents when exposed to harsh environments. It is proposed that PA play a defensive role during biotic stress responses (Walters 2003a, b). One of the examples is the hypersensitive response (HR) which consists of rapid cell death at the sight of pathogen entry, typically develops in the interaction between tobacco mosaic virus (TMV) and N resistance gene carrying N. tabacum and leads to enhanced polyamine synthesis and accumulation (Kusano et al. 2008). It is also believed that stress-induced polyamines tend to modulate the activity of a certain set of ion channels to adapt ionic fluxes in response to environmental changes. Many more examples of responses to biotic stress have been quoted by Kusano et al. (2008). Various abiotic stress conditions have been reported to alter the concentration of polyamines (Bouchereau et al. 1999; Walters 2003a). Exogenous polyamine application and/or inhibitors of enzymes involved in polyamine biosynthesis pointed out a possible role of these compounds in plant adaptation/defense to several environmental stresses (Bouchereau et al. 1999; Alcázar et al. 2006; Groppa and Benavides 2008; Alcázar et al. 2010). More recent studies using either transgenic overexpression or loss-offunction mutants support this protective/adaptive/defensive role of PAs in plant response to abiotic stress (Alcázar et al. 2006; Kusano et al. 2008; Gill and Tuteja 2010). For example, Arabidopsis plants overexpressing Cucurbita ficifolia Spd synthase gene were tolerant of multistresses (chilling, freezing, salinity, drought, and paraquat toxicity) (Kusano et al. 2007; Tassoni et al. 2010). According to Rhee et al. (2007), the basic principle underlying polyamine adaptive responses appears to be shared by the prokaryotic stringent response and the eukaryotic unfolded protein response (UPR). UPR is triggered when unfolded proteins and uncharged tRNAs accumulate in the endoplasmic reticulum (ER) due to ER stress or nutrient starvation.

H.-W. Koyro et al.

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As a result of this, cap-dependent translation of many mRNAs is suppressed and the expression of a certain set of genes including the luminal binding protein gene BiP is induced. The underlying mechanisms of UPR in yeasts and mammals have been well researched (Rutkowski and Kaufman 2004), although those in plants have not (Kamauchi et al. 2005; Urade 2007; Kusano et al. 2008). Recently, nitric oxide (NO), an endogenous signaling molecule in plants and animals, has gained considerable importance in the PA studies. It is known to mediate responses to biotic and abiotic stresses. It has been reported by Tun et al. (2006) that spermine and spermidine are potent inducers of NO in Arabidopsis, but putrescine and its biosynthetic precursor arginine are not. There are many more examples of NO affecting the concentrations of PAs and over the past few years studies on polyamines and NO are gaining attention (Kusano et al. 2008).

Fig. 1.5 Sites of reactive oxygen species (ROS) and the biological consequences leading to a variety of physiological dysfunctions that can lead to cell death (adopted from Ahmad et al. 2008)

3

Oxidative Stress and Antioxidative Responses to Environmental Stress

3.1

Production of ROS

Environmental stresses are responsible for the production of ROS. The production and removal of ROS is thought to be at equilibrium under normal conditions, whereas environmental stress disturbs this equilibrium by enhancing the production of ROS. ROS are very toxic for the organism as they affect the structure and function of the biomolecules. The main source of ROS production in plants is chloroplasts, mitochondria, and peroxisomes (Fig. 1.5). Mitochondria are responsible for the generation of oxygen radicals and hydrogen peroxide due to the overreduction of the electron transport chain.

1 Abiotic Stress Responses in Plants: An Overview

Chloroplasts are found to be the major producer of O2 and H2O2 (Davletova et al. 2005). This is because the oxygen pressure in the chloroplast is higher than in other organelles. Photoreduction of O2 to O2•− during the photosynthetic electron transport takes place and is called Mehler reaction. The production of superoxides is due to the reduction of molecular oxygen in the plastoquinone pool. This reduction may be due to the plastosemiquinone, by ferredoxin (Fd) or by sulfur redox centers in the electron transport chain within PSI (Dat et al. 2000). These superoxides are converted to hydrogen peroxide either spontaneously or by the action of the enzyme SOD. Hydrogen peroxide is also responsible for the production of hydroxyl radicals (OH•). The major producer of H2O2 in plant cells are peroxisomes. It has been reported that peroxisomes are also responsible for the production of superoxides (O2−). In peroxisomes, the production of O2− occurs in the peroxisomal matrix and the peroxisomal membrane. In the peroxisomal matrix, the oxidation of xanthine and hypoxanthine to uric acid in the presence of the enzyme xanthine oxidase generates O2− radicals (Halliwell and Gutteridge 2000). Peroxisomes have got two pathways for the production of H2O2. One is the disproportionation of O2− generated in this organelle and the other is a direct pathway. During photorespiration glycolate is catalyzed by glycolate oxidase, yielding H2O2. Fatty acid b-oxidation, the enzymatic reaction of flavin oxidases, can also produce H2O2 (Baker and Graham 2002; del Rio et al. 2002). ROS include 1O2, O2•−, H2O•, H2O2, OH•, RO• organic hydroperoxide (ROOH), excited carbonyl (RO•), etc. They cause damage to biomolecules like proteins, lipids, carbohydrates, and DNA, which ultimately results in cell death (Foyer and Noctor 2005). Fortunately, plants are equipped with an antioxidant machinery that scavenges the ROS and helps the plant to tolerate the stress conditions. The antioxidants include enzymatic antioxidants, viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), etc., and nonenzymatic antioxidants like ascorbic acid (AsA), vitamin E (a-tocopherol), reduced glutathione (GSH), etc.

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3.2

Enzymatic Antioxidants

3.2.1 Superoxide Dismutase SOD is one of the ubiquitous enzymes in aerobic organisms and plays a key role in cellular defense mechanisms against ROS. Within a cell, the SODs constitute the first line of defense against ROS. Its activity modulates the reactive amounts of O2•− and H2O2, the two Haber–Weiss reaction substrates, and decreases the risk of OH radical formation, which is highly reactive and may cause severe damage to membranes, proteins, and DNA (reviewed by Ahmad et al. 2010b). SOD was for the first time reported by Cannon et al. (1987) in maize and it catalyzes the dismutation of superoxide into hydrogen peroxide and molecular oxygen. 2H + + 2• O2 - ® 2H 2 O2 + O2 Different types of SOD isoforms have been observed in plants on the basis of metal cofactors attached to the active site. The isozyme containing Mn II at its active site is known as Mn-SOD. Similarly, the isozyme the active site of which contains Cu II and Zn II is known as Cu/Zn-SOD. The third isozyme contains Fe III and is referred to as Fe-SOD. The fourth SOD isoform contains Ni at the active site, is called Ni-SOD and is found in several Streptomyces species (Youn et al. 1996) and cyanobacteria (Palenik et al. 2003). Ni-SOD has not been reported in plants yet. Whereas only one type of SOD is found in most organisms, plants have multiple form of each type, which are encoded by more than one gene, indicating that plants have more complex antioxidant defense systems than other organisms. Several studies have reported enhanced stress tolerance related to overproduction of chloroplastic SOD (Pastori and Foyer 2002). In maize leaves, GR and DHAR were exclusively localized in mesophyll cells whereas most of the SOD and APX were localized in mesophyll and bundle sheath cells. Increased SOD activity was reported in Radix astragali under water deficit stress, which varied in three different genotypes (Tan et al. 2006). Chilling stress has a significant effect in the enhancement of SOD activity in cucumber

12

seedlings (Feng et al. 2003). The increase in SOD activity under drought stress was about 25% in soybean plants (Zhang et al. 2007). SOD activity was doubled in water stressed citrus plants when compared to well-watered control plants during seedling stage (Wu et al. 2006). SOD activity increased under drought stress in Euphorbia esula (Davis and Swanson 2001), maize (Pastori et al. 2000), Cassia angustifolia (Agarwal and Pandey 2003), wheat (Singh and Usha 2003), rice (Wang et al. 2005), P. acutifolius (Turkan et al. 2005), and Camellia sinensis (Chen et al. 2011), and the SOD activity was higher under salinity stress in C. roseus (Jaleel et al. 2008) and Morus alba (Ahmad et al. 2010a). While subjecting higher plants to water deficit stress SOD activity increases (Reddy et al. 2004). Koca et al. (2007) have shown that elevated SOD activity is accompanied with an increase in the activity of major H2O2 scavenging enzymes like APX, CAT, and POX in salt tolerant sesame cultivar Cumhuriyat as compared to cultivar Orhangazi. SOD activity increased by 1.6-fold in a salt tolerant mutant of Chrysanthemum compared to a nontolerant one under NaCl stress (Hossain et al. 2006). An increased activity of SOD enzyme has also been reported under different abiotic stresses in Catharanthus roseus (Jaleel et al. 2007), Pisum sativum (Ahmad et al. 2008), M. alba (Ahmad et al. 2010a), and Brassica juncea (Ahmad 2010; Ahmad et al. 2011). SOD activity has also been observed to increase by the application of heavy metals such as cadmium (Shah et al. 2001; John et al. 2009; Ahmad et al. 2011), lead (Verma and Dubey 2003; John et al. 2009), and copper (Lombardi and Sebastiani 2005). Canola overexpressing Mn-SOD confers tolerance to aluminum stress (Basu et al. 2001). Overexpression of Mn-SOD in transgenic Arabidopsis showed a twofold increase in Mn-SOD activity and higher tolerance to salt as compared to nontransgenic plants (Wang et al. 2004). Tanaka et al. (1999) demonstrated that expression of yeast mitochondrial Mn-SOD in rice chloroplasts led to a 1.7fold increase in Mn-SOD as compared to nontransgenic plants. Transgenic Arabidopsis with Mn-SOD confers tolerance to heat (Im et al. 2009). Wang et al. (2005) demonstrated that trans-

H.-W. Koyro et al.

genic rice plants expressing Mn-SOD have shown reduced injury and sustained photosynthesis under PEG stress. Overexpression of Cu/Zn-SOD and APX in transgenic tobacco enhanced seed longevity and germination rates after various environmental stresses (Lee et al. 2010). Transgenic tobacco expressing Cu/Zn-SOD have been shown to tolerate chilling and heat stress (Gupta et al. 1993) and enhanced tolerance to salt, water, and PEG stress (Badawi et al. 2004). Prashanth et al. (2008) have also demonstrated that Cu/Zn-SOD confers tolerance to salinity in rice plants.

3.2.2 Catalase Plant catalases are tetrameric iron porphyrins and play a role in stress tolerance against oxidative stress. Catalases are produced in peroxisomes and glyoxysomes. Catalases are involved in eliminating hydrogen peroxide generated by different environmental stresses (Kim et al. 2008; Ahmad et al. 2010b). Catalases decompose hydrogen peroxide to water and molecular oxygen without consuming reductants and may thus provide plant cells with an energy efficient mechanism to remove hydrogen peroxide (reviewed by Ahmad et al. 2010b). The enzyme is abundant in the glyoxysomes of lipid-storing tissues in germinating barley, where it decomposes H2O2 formed during the b-oxidation of fatty acids (Jiang and Zhang 2002) and in the peroxisomes of the leaves of C3 plants, where it removes H2O2 produced during photorespiration by the conversion of glycolate into glyoxylate (Kiani et al. 2008). This is also due to the fact that there is a proliferation of peroxisomes during stress, which might help in scavenging H2O2, which can diffuse from the cytosol (Lopez-Huertas et al. 2000; Kusaka et al. 2005). High temperatures affect the structure of most proteins and thus the activity of many enzymes. Hertwig et al. (1992) have demonstrated that the translation of catalase was hampered at 40°C. Anderson (2002) showed that high temperature is responsible for the decrease in catalase activity in pepper plants. In comparison, the desert plant Retama raetam exposed to heat shock temperature showed only a minor inactivation of catalase activity (Streb et al. 1997). Scandalios et al.

1 Abiotic Stress Responses in Plants: An Overview

(2000) have also observed a reduced catalase activity in maize on exposure to temperatures of 35–40°C. Sublethal doses of NaCl induce catalase activity in Nicotiana plumbaginifolia through activation of cat2 and cat3 genes (Savoure et al. 1999). However, catalase activity was found to decrease due to the salt stress because of accumulation of salicylic acid (Shim et al. 2003). Vaidyanathan et al. (2003) have demonstrated that salt tolerant rice cultivars contain higher levels of catalase activity compared to susceptible cultivars. Increase in catalase activity during salt stress has also been shown by other workers in maize (Azevedo-Neto et al. 2006; Arora et al. 2008), in sesame (Koca et al. 2007), and in mulberry (Ahmad et al. 2010a). Catalase activity has also been found to decrease in presence of heavy metal stress (Mishra et al. 2006; Khan et al. 2007; Mobin and Khan 2007; Ahmad et al. 2011). Verma and Dubey (2003) also demonstrated that the activity of catalase declines in rice plants with increasing concentration of Pb. John et al. (2009) also reported that an increase in Cd and Pb concentrations decreases the catalase activity in mustard. Decrease in catalase may be due to the inhibition of enzyme synthesis or change in assembly of enzyme subunits (Shah et al. 2001).

3.2.3 Ascorbate Peroxidase APX is an important antioxidant enzyme mainly found in higher plants and algae (Raven 2003). APX helps to detoxify H2O2 in the ascorbateglutathione (= Halliwell-Asada) pathway. APX utilizes ascorbic acid and reduces H2O2 to water and monodehydroascorbate (MDA). 2AsA + H 2 O2 ® 2MDA + 2H 2 O APX was first isolated from chloroplasts and algae (Shigeoka et al. 1980; Nakano and Asada 1981). Different isoforms of APX which include thylakoid (tAPX), glyoxisomal (gmAPX), stromal (sAPX), and cytosolic (cAPX) have been reported (Shigeoka et al. 2002; Mittler et al. 2004). In comparison to other antioxidants, APX

13

and guaiacol peroxidase (GPX) have a high affinity towards H2O2 (Mittler and Poulos 2005). APX isozymes have been found to be most stress responsive among the APX gene family during environmental stress (Mittler and Poulos 2005). APX1 has been found to enhance in response to environmental stress (Mittler 2002; Shigeoka et al. 2002). APX2 is expressed under stressful conditions and its expression is elevated in response to light stress or heat shock (Mullineaux and Karpinski 2002; Panchuk et al. 2002). Cytosolic APX1 has been found to protect Arabidopsis plants from a combination of stresses (Koussevitzky et al. 2008). Lu et al. (2007) demonstrated that cAPX improves salt tolerance in transgenic Arabidopsis. Mittler et al. (1999) have demonstrated that suppression of APX1 in tobacco leads to a higher sensitivity of the plant to pathogen attacks. Overexpression of APX1 resulted in enhanced tolerance to oxidative stress in tobacco (Yabuta et al. 2002). Biologists have demonstrated the importance of APX1 by using APX1 knockout mutants. The plants lacking APX1 have showed delayed growth, no response of guard cells towards light, and light stress resulted in an induction of catalase and heat shock proteins (Pnueli et al. 2003). The accumulation of H2O2 is responsible for the abnormal closure of stomata in knockout APX1 plants (Pnueli et al. 2003). The induction of heat shock proteins in knockout APX1 plants may be due to an enhanced level of H2O2 which is considered as an essential signaling molecule during abiotic stress (Mittler 2002; Neill et al. 2002).

3.2.4 Glutathione Reductase GR is a flavo-protein oxidoreductase and is found in both prokaryotes and eukaryotes (Romero-Puertas et al. 2006). GR is an important enzyme of the ascorbate–glutathione system and maintains the balance between reduced glutathione (GSH) and the ascorbate pool (reviewed by Ahmad et al. 2010b). Meldrum and Tarr (1935) for the first time reported GR in eukaryotes and yeast, and in 1951 it was also observed in plants (Conn and Vennesland 1951; Mapson and Goddard 1951). Later on GR has been isolated

H.-W. Koyro et al.

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from different plants and bacteria (Creissen et al. 1991; Creissen and Mullineaux 1995). GR is mainly found in chloroplasts (70–80%), and small amounts have been found in mitochondria, cytosol, and peroxisomes (Edwards et al. 1990; Romero-Puertas et al. 2006). GR catalyzes the reduction of glutathione in the cell. GSH is oxidized to GSSG which should be converted back to GSH in normal cells. Rapid recycling of GSH is more important than the synthesis of GSH. Hence GR and GSH have been found to play a very crucial role in stress tolerance in plants. GR plays an important role in alleviating oxidative stress in plants as evidenced by increased activities of GR during oxidative stress (Contour-Ansel et al. 2006; Khan et al. 2007; Mobin and Khan 2007; Hsu and Kao 2007). Increased activities of GR during drought stress were observed in different plants, e.g. in wheat (Selote and KhannaChopra 2006) and in rice (Selote and Khanna-Chopra 2004; Srivalli et al. 2003; Sharma and Dubey 2005). Salt stress also increased the GR activity in rice (Demiral and Turkan 2005; Tsai et al. 2005) and wheat (Sairam et al. 2005). A positive correlation between the increased activity of GR and chilling tolerance has been reported in rice (Guo et al. 2006), in maize (Hodges et al. 1997a, b) and tomato (Walker and McKersie 1993). Heavy metal stress is also responsible for the increase in the activity of GR in plants. Mulberry plants exposed to copper stress exhibit an increased GR activity (Tewari et al. 2006). A positive correlation of increased GR activity in presence of Cd has been reported in potato, radish, soybean, sugarcane, and mustard (Stroinski et al. 1999; Vitoria et al. 2001; Ferreira et al. 2002; Fornazier et al. 2002; Mobin and Khan 2007). Transgenic tobacco plants expressing the gor2 gene from E. coli showed an increase in GR activity (Stevens et al. 2000). Pilon-Smith et al. (2000) observed that cytosol GR increases by 2-fold and chloroplast GR increases by 50-fold in transgenic plants of B. juncea expressing the gor2 gene from E. coli. These transgenic plants showed an enhanced tolerance to Cd stress up to 100 mM. Expression of the gor2 gene from E. coli in tobacco (cv. Belw3) showed an increased activity of GR and increases

tolerance to paraquat and H2O2 stress (Lederer and Böger 2003).

3.3

Nonenzymatic Antioxidants

3.3.1 Ascorbic Acid Among the small molecular antioxidants in plants, ascorbate is most abundant and is most concentrated in leaves and meristems (reviewed by Ahmad et al. 2010b). It is about five to ten times more concentrated than GSH in leaves (Ishikawa et al. 2006). AsA is present in high concentration in fruits, especially citrus fruits, but the concentration in fruits is not always higher than in leaves (Davey et al. 2000). Some fruits such as blackcurrants and rose hips are famous for their exceptionally high ascorbate content (Ishikawa et al. 2006). AsA occurs in all subcellular compartments, and the concentration varies from 20 mM in the cytosol to 300 mM in chloroplasts (Noctor and Foyer 1998). The synthesis of AsA takes place in mitochondria and is transported to other cell compartments through a proton electrochemical gradient or through facilitated diffusion (Horemans et al. 2000). Franceschi and Tarlyn (2002) reported the presence of ascorbate in the phloem sap of A. thaliana. Other species of plants have also been reported to contain ascorbate in the phloem sap, e.g. cucurbita (Hanco*ck et al. 2008). This led to the conclusion that ascorbate is transported from source (leaves) to sink (meristem) (Ishikawa et al. 2006). Ascorbate plays an important role in plants as an antioxidant and as a cofactor of many enzymes (Ishikawa et al. 2006). As an antioxidant, ascorbate protects plants from oxidative stress. Ascorbate peroxidase utilizes ascorbic acid and reduces H2O2 to water, thereby generating monodehydroascorbate (MDA) in the ascorbate– glutathione cycle (Pan et al. 2003). MDA can also be reduced directly to AsA in the presence of the catalytic enzyme MDAR and the electron donor NADPH (Asada 1999). Maddison et al. (2002) have reported that ascorbate plays a role in the defense against ozone. AsA has the capability of donating electrons in various enzymatic

1 Abiotic Stress Responses in Plants: An Overview

and nonenzymatic reactions and is thus a powerful radical scavenger. It can directly scavenge 1O2, O2•−, and −OH radicals produced in the cell and can protect membranes against oxidative stress. In plant cells, the most important reducing substrate for H2O2 detoxification is ascorbic acid (Turkan et al. 2005). An increase in oxidized ascorbate during Cd stress has been reported by Demirevska-Kepova et al. (2006) in Hordeum vulgare. Yang et al. (2008) also reported that drought stress increases the ascorbate content in Picea asperata. Water stress results in significant increases in antioxidant AsA concentration in turfgrass (Zhang and Schmidt 2000; Vranova et al. 2002; Jaleel et al. 2007). Ascorbic acid shows a reduction under drought stress in maize and wheat, suggesting its vital involvement in oxidative response (Vertovec et al. 2001; Nayyar and Gupta 2006).

3.3.2 a-Tocopherol Plants have the capacity to synthesize a lipophylic antioxidant known as a-tocopherol or vitamin E. a-tocopherol scavenges free radicals in combination with other antioxidants (Munne-Bosch and Algere 2003; Massacci et al. 2008). It has also been reported that a-tocopherol protects the structure and function of PSII as it chemically reacts with O2 in chloroplasts (Lopez-Huertas et al. 2000; Nordberg and Arner 2001). Munne-Bosch and Algere (2003) reported that a-tocopherol helps in membrane stabilization and alleviates the tolerance of plants during oxidative stress. Environmental stresses are responsible for the generation of low molecular mass antioxidants such as a-tocopherol (Lowlor and Cornic 2002; Munne-Bosch and Algere 2003; Mahajan and Tuteja 2005; Martinez et al. 2007). Falk et al. (2003) reported the upregulation of genes of a-tocopherol synthesis during oxidative stress. Water stress resulted in elevated levels of a-tocopherol in Vigna plants (Manivannan et al. 2007) and turfgrass (Zhang and Schmidt 2000). 3.3.3 Reduced Glutathione Glutathione (l-glutamyl-l-cysteinylglycine, GSH) is a thiol compound composed of l-glutamic acid,

15

l-cysteine, and glycine. GSH is distributed universally in animals, plants, and microorganisms and has an established role as an essential compound of a free radical scavenger (Monneveux et al. 2006). GSH participates in numerous cellular processes and protects cells from the toxic effects of many ROS (Petropoulos et al. 2008). Additionally, GSH is involved in other biological functions, such as regulation of protein and DNA synthesis, protein activities, and maintaining membrane integrity (Cabuslay et al. 2002). Meyer et al. (2005) reported that levels of H2O2 are controlled by the action of glutathione. Reduction of glutathione (GSH) and oxidation of glutathione (GSSG) are necessary for controlling H2O2 levels in cells and have an important role in redox signaling (Pastori and Foyer 2002. Reduced glutathione is directly involved in the reduction of ROS in plants. Transgenic tobacco expressing glutathione gene withstands oxidative stress (Singh and Verma 2001). Glutathione is a tripeptide (a-glutamyl cysteinylglycine) and is found in the cytosol, chloroplasts, ER, vacuoles, and mitochondria (Sankar et al. 2007a, b). The nonprotein thiols are nucleophilic in nature and thus are important for the formation of mercaptide bonds with metals and for reacting with selective electrophiles (Rodriguez et al. 2005). In most plants, the major source of these nonprotein thiols is glutathione. Glutathione is considered the most important nonenzymatic antioxidant due to its relative stability and high water solubility (Samarah 2005). It can protect plant cells from environmental stress-induced oxidative stress (Samarah 2005).

4

The Effect of Elevated Atmospheric CO2 Concentration on Antioxidants and Osmolytes Under Environmental Stress

Elevated atmospheric CO2 concentration leads to a higher CO2 concentration gradient between the outside air and the intercellular spaces of the leaves, so that the diffusion of CO2 into the leaves

16

and the pCO2/pO2 ratio at the sites of photoreduction is increased (Robredo et al. 2007). Therefore, usually photorespiration and the rates of oxygen activation and ROS formation are reduced due to an increased NADPH utilization, whereas the net photosynthetic rate and thus the carbon supply is enhanced, especially in C3 plants (Polle 1996; Urban 2003; Kirschbaum 2004; Long et al. 2004; Hikosaka et al. 2005; Ignatova et al. 2005). Furthermore, we often find a lower stomatal resistance (Hsiao and Jackson 1999; Li et al. 2003; Marchi et al. 2004; Rogers et al. 2004), which together with the higher net assimilation also leads to a better water use efficiency of photosynthesis (Amthor 1999; Morgan et al. 2001; Urban 2003). As a consequence of these effects, on the one hand there might be less need for antioxidants as elevated CO2 ameliorates oxidative stress (Schwanz et al. 1996). On the other hand more energy can be provided for energy-dependent stress tolerance mechanisms such as the synthesis of osmolytes and antioxidants. Due to both effects mentioned earlier, elevated CO2 can increase plant survival under abiotic stress conditions (Ball and Munns 1992; Rozema 1993; Drake et al. 1997; Fangmeier and Jäger 2001; Wullschleger et al. 2002; Urban 2003; Geissler et al. 2010). Regarding oxidative stress, the antioxidant system can respond differently to elevated CO2 depending on species or even genotype as well as on treatment duration and growth conditions such as mineral nutrition (Schwanz et al. 1996; Polle et al. 1997; Sanità di Toppi et al. 2002; PérezLópez et al. 2009). Varying responses can be related to a species-specific differential regulation in order to maintain an adequate balance between ROS formation and antioxidant ability under the actual conditions (Pérez-López et al. 2009). However, many studies have reported an increased tolerance to various abiotic stresses under elevated CO2 due to an alleviation of oxidative stress: In chestnut trees, photoinhibition due to high irradiance stress was ameliorated, and higher GSH levels were found in juvenile leaves (Carvalho and Amâncio 2002). Sgherri et al. (2000) reported that CO2 enrichment led to an

H.-W. Koyro et al.

improved water use efficiency and a decreased photorespiration in Medicago sativa under drought stress. As a consequence, the cells showed a higher reducing status, increased ascorbate/dehydroascorbate and GSH/GSSG ratios. There was no demand for a higher GR activity (no CO2 effect) and less requirement for Ca2+ ATPase activity to maintain Ca2+ homeostasis under stress conditions. Similarly, in cold stressed maize elevated CO2 had no effect on SOD, CAT, and APX activities, but the formation of superoxide radicals and membrane injury was reduced (Baczek-Kwinta and Ko cielniak 2003). The alleviation of oxidative stress was probably due to a higher CO2 assimilation, overcoming the Rubisco limitation under low temperature. In some cases elevated CO2 even leads to reduced activities of antioxidative enzymes because there is less need for antioxidants: Pérez-López et al. (2009) reported that barley plants exposed to NaCl stress under ambient CO2 exhibited enhanced activities of SOD, APX, CAT, GR, and dehydroascorbate reductase (DHAR), which was accompanied by ion leakage and lipid peroxidation. Furthermore, the expression ratio of enzyme isoforms changed, e.g. a relatively higher contribution of GR1 relative to GR2 and of Cu/Zn-SOD (which seems to be especially important for salt tolerance in Hordeum vulgare) was observed. Elevated CO2 ameliorated ion leakage and lipid peroxidation, while the plants showed a lower upregulation of the antioxidant enzymes and an even higher relative contribution of GR1 and of Cu/Zn-SOD. The authors explain these results with less ROS generation and a better maintenance of redox homeostasis due to an enhanced photosynthesis and a reduced photorespiration. Similar results were found for Solanum lycopersicum by Takagi et al. (2009). NaCl salinity decreased plant biomass, net assimilation, and the transport of assimilates to the sink (stem), while CAT and APX activities increased. Under elevated CO2 the negative effects of salinity were alleviated, especially when the sink activity was relatively high, and CAT and APX activities decreased compared to ambient CO2. The improvement of oxidative stress (and of water relations) seemed to cause an activation of sink activity under elevated CO2.

1 Abiotic Stress Responses in Plants: An Overview

17

Fig. 1.6 Antioxidant enzyme expressions (relative volume percentages of the spots) in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Superoxide dismutase, (b) ascorbate peroxidase, (c) glutathione-S-transferase. Values represent mean ± SD values of eight gels per

treatment. Significant differences (P £ 0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment

In contrast to the studies mentioned earlier, in some cases antioxidant activities are enhanced by elevated CO2. In ozone stressed Betula pendula, elevated CO2 eliminated the chloroplastic accumulation of H2O2, which could be explained by a higher photosynthetic rate, leading to a higher NADPH formation and a more efficient enzymatic detoxification (e.g., via the ascorbate–glutathione cycle; Oksanen et al. 2005). Marabottini et al. (2001) found a higher activity of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) in drought-stressed Quercus under elevated CO2. Rao et al. (1995) observed a more persistent high activity of glutathione reductase (GR), APX, und SOD in ozone-stressed wheat. Schwanz and Polle (2001) examined the drought tolerant species Quercus robur and the sensitive Pinus pinaster. They found out that Q. robur generally exhibits a higher activity of several antioxidative enzymes; furthermore, elevated CO2 concentration ameliorated damage caused by drought stress in both species due to a higher stability of antioxidative enzymes and an enhanced SOD activity. Similar results were reported for the facultative halophyte A. tripolium. Under ambient CO2 concentration

salt stress led to an overexpression and thus to higher relative activities of the antioxidative enzymes APX, SOD, and glutathione-Stransferase (GST), while under elevated CO2 the expression and activities of these enzymes were further increased (Fig. 1.6; Geissler et al. 2010). Similarly, elevated CO2 concentration led to a significantly higher content of carotenoids – nonenzymatic antioxidants – in the salt treatments (Geissler et al. 2009b). These results implicate that the enhancement of enzyme expression and activity and the carotenoid content were not high enough to sufficiently eliminate ROS under ambient CO2 concentration. Under elevated CO2, however, a higher supply of energy-rich organic substances due to a significantly enhanced net assimilation rate (Geissler et al. 2009a, b) enabled the plants to invest more energy in the energydependent synthesis of enzymatic and nonenzymatic antioxidants. Therefore, ROS could be detoxified more effectively, so that salinity tolerance could be improved, manifesting itself in a higher survival rate of the salt-treated plants (Geissler et al. 2009a). Furthermore, investigations about A. tripolium showed that elevated CO2 concentration does not

18

H.-W. Koyro et al.

Fig. 1.7 Content of osmolytes in controls and salt treatments (75% seawater salinity) of Aster tripolium under ambient and elevated CO2. (a) Proline, (b) total soluble carbohydrates. Values represent mean ± SD values of six measurements per treatment. Significant differences

(P £ 0.05) between the salinity treatments (within one CO2 treatment) are indicated by different letters, significant differences between the CO2 treatments (within one salt treatment) are indicated by an asterisk. ctr control, sal salt treatment

only have an effect on antioxidants, but on osmolytes as well. This halophyte employed its additional carbon gain under elevated CO2 concentration also for a higher synthesis of compatible solutes (Geissler et al. 2009a): Salinity (under ambient CO2) led to an accumulation of proline in all plant organs and of soluble carbohydrates in the main roots (Fig. 1.7). Under elevated CO2 concentration, the plants accumulated a higher amount of proline, especially in the leaves which are the primary areas of influence of CO2. In the main root, there was no necessity of an additional accumulation of proline because this organ is well protected against salt damage

due to a high content of compatible solutes even at ambient CO2 concentration. Furthermore, a higher amount of soluble carbohydrates under elevated CO2 was found in all plant organs due to the increased photosynthesis and a lower conversion of saccharides to starch. These results are in accordance with the study of Abdel-Nasser and Abdel-Aal (2002) investigating Carthamus mareoticus under drought stress: Elevated CO2 concentration increased the accumulation of total soluble carbohydrates in well watered as well as in stressed plants due to a higher amount of assimilates. The drought-induced inhibition of the sucrose phosphate synthase activity was

1 Abiotic Stress Responses in Plants: An Overview

annihilated under elevated CO2, and the droughtinduced increase in sucrose content was further enhanced. The content of total amino acids and especially of proline behaved similarly to sucrose, as well as the activities of the proline synthesizing enzymes 1-pyrroline-5-carboxylate reductase (P5CR) and the ornithine aminotransferase (OAT). In contrast, the activity of the proline degrading enzyme proline dehydrogenase (PDH) was reduced by drought stress and further decreased under elevated CO2. In contrast to C. mareoticus, proline (and other amino acids) do not seem to contribute to salt tolerance in barley, but to reflect a reaction to stress damage, as shown by Pérez-López et al. (2010): Although a better osmotic adjustment (more negative osmotic potential) of salt-stressed plants was recorded under elevated CO2, the proline content decreased, showing less stress damage. Instead, the accumulation of soluble sugars and other unidentified osmolytes (possibly polyols and/or quaternary nitrogen compounds) was actively enhanced under elevated CO2, and these substances played an important role in osmotic adjustment and as compatible solutes under saline conditions. Elevated CO2 provided a higher carbon and ATP supply for salt tolerance mechanisms, enabling the plants to actively increase their compatible solute concentration, which in turn leads to a better water uptake and turgor maintenance for plant growth. As a summary, it can be concluded that elevated CO2 concentration can enhance salt and drought tolerance of plants by alleviating oxidative stress, increasing the activity of the antioxidative system, and/or increasing the accumulation of compatible substances, having a positive effect on their suitability as crops on dry and saline soils in future.

5

Conclusion and Future Perspective

Abiotic stresses, especially osmotic and ionic stresses, are responsible for the decrease in yield especially in arid and semiarid regions. It is estimated that 45% of the world’s agricultural land

19

experience drought and 19.5% of the irrigated land are affected by salinity. These problems will be further catalyzed by global climate change. Prolonged environmental stresses are responsible for the production of ROS in different cell compartments like chloroplasts, mitochondria, peroxisomes, etc. ROS attack biomolecules, viz., DNA, lipids, proteins, carbohydrates, and disturb the normal functioning of the cell. Under severe stress conditions, ROS ultimately lead to cell death. In order to withstand oxidative stress, plants are equipped with enzymatic and nonenzymatic antioxidants. Many workers have reported the positive effects of SOD, CAT, APX, GR, MDHAR, AsA, glutathione, etc., in combating oxidative damage to the cell. To overcome the deleterious effects of abiotic stresses, plants also accumulate osmolytes and osmoprotectants such as proline and glycine betaine. These compounds are thought to play a role in osmotic adjustment and protect subcellular structures. Elevated atmospheric CO2 concentration can alleviate oxidative stress, increase the activity of the antioxidative system, and/or increase the accumulation of compatible substances, so it can enhance salt and drought tolerance of plants and their suitability as crops in a future world of climate change. The biggest challenge to the modern plant scientists is to develop stress-tolerant plants without compromising yield. There can be no doubt that transgenic plants will be invaluable in assessing precisely the role that main antioxidants, ROS, and osmolytes play in the functional network that controls stress tolerance. Researchers should look for defined sets of markers to predict tolerance towards a particular type of stress. While manipulating genes for stress tolerance in important crops, the genes incorporated should contribute to tolerance not only at a certain plant growth stage of interest but also at the whole plant level, because achieving maximum crop yield under saline conditions is the principal objective of all agriculturists. Modern techniques like genomics, proteomics, ionomics, and metabolomics will be helpful to study plant responses to abiotic stresses. Regarding global climate change, it would be desirable to develop model plants not only for understanding stress tolerance mechanisms, but

20

also their interaction with elevated atmospheric CO2 concentration in order to assess the suitability of plants as crops in future. Acknowledgments The authors would like to thank Mr. Jürgen Franz, Mr. Wolfgang Stein, Mr. Gerhard Mayer, Mrs. Angelika Bölke, Prof. Dr. Edwin Pahlich, PD Dr. Christian Zörb, Mrs. Anneliese Weber (Giessen University), and Mr. Steffen Pahlich (Zürich University) for technical assistance and scientific advice regarding the experiments with Aster tripolium.

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1 Abiotic Stress Responses in Plants: An Overview Sgherri CLM, Salvateci P, Menconi M, Raschi A, NavariIzzo F (2000) Interaction between drought and elevated CO2 in the response of alfalfa plants to oxidative stress. J Plant Physiol 156:360–366 Shah K, Ritambhara GK, Verma S, Dubey RS (2001) Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Sci 161:1135–1144 Sharma P, Dubey RS (2005) Drought induced oxidative stress and enhances the activities of antioxidant enzymes in growing rice seedlings. Plant Growth Regul 46:209–221 Shigeoka S, Nakano Y, Kitaoka S (1980) Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase. Biochem J 186:377–380 Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, Yoshimura K (2002) Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot 53:1305–1319 Shim IS, Momose Y, Yamamoto A, Kim DW, Usui K (2003) Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants. Plant Growth Regul 39:285–292 Singh B, Usha K (2003) Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress. Plant Growth Regul 39:137–141 Singh BG, Verma DPS (2001) Glutathione: An antioxidant to withstand oxidative stress in transgenic lines of tobacco. Ind J Plant Physiol 6:229–232 Slama I, Ghnaya T, Hssini K, Messedi D, Savouré A, Abdelly C (2007a) Comparative study of mannitol and PE osmotic stress effects on growth, and solute accumulation in Sesuvium portulacastrum. Environ Exp Bot 61:10–17 Slama I, Ghnaya T, Messedi D, Hssini K, Labidi N, Savoure A, Abdelly C (2007b) Effect of sodium chloride on the response of the halophyte species Sesuvium portulacastrum grown in mannitol-induced water stress. J Plant Res 120:291–299 Slama I, Ghnaya T, Savouré A, Abdelly C (2008) Combined effects of long-term salinity and soil drying on growth water relations, nutrient status and proline accumulation of Sesuvium portulacastrum. C R Biol 331:442–451 Smirnoff N, Cumbes QJ (1989) Hydroxyl radical scavenging activity of compatible solutes. Phyotochemistry 28:1057–1060 Srivalli B, Chinnusamy V, Khanna-Chopra R (2003) Antioxidant defense in response to abiotic stresses in plants. J Plant Biol 30:121–139 Stevens RG, Creissen GP, Mullineaux PM (2000) Characterization of pea cytosolic glutathione reductase expressed in transgenic tobacco. Planta 211: 537–545 Streb P, Tel-Or E, Feierabend J (1997) Light stress effects and antioxidative protection in two desert plants. Funct Ecol 11:416–424 Stroinski A, Kubis J, Zielezinska M (1999) Effect of cadmium on glutathione reductase in potato tubers. Acta Physiol Plant 21:201–207

27 Takagi M, El-Shemy H, Sasaki S, Toyama S, Kanai S, Saneoka H, Fujita K (2009) Elevated CO2 concentration alleviates salinity stress in tomato plant. Acta Agric Scand B Soil Plant 59:87–96 Tan Y, Liang Z, Shao H, Du F (2006) Effect of water deficits on the activity of anti-oxidative enzymes and osmoregulation among three different genotypes of Radix Astragali at seeding stage. Colloids Surf B Biointerfaces 49:60–65 Tanaka Y, Hibino T, Hayashi Y, Tanaka A, Kish*tani S, Takabe T, Yokota S, Takabe T (1999) Salt tolerance of transgenic rice overexpressing yeast mitochondrial Mn-SOD in chloroplasts. Plant Sci 148:131–138 Tassoni A, Franceschetti M, Bagni N (2010) Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers. Plant Physiol Biochem 46:607–613 Tewari RK, Kumar P, Sharma PN (2006) Antioxidant responses to enhanced generation of superoxide anion radical and hydrogen peroxide in the copper-stressed mulberry plants. Planta 223:1145–1153 Tsai YC, Hong CY, Liu LF, Kao CH (2005) Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2. J Plant Physiol 162:291–299 Tun NN, Santa-Catarina C, Begum T, Silveira V, Handro W, Floh EIS, Scherer GFE (2006) Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant Cell 47:346–354 Türkan I, Demiral T (2009) Recent developments in understanding salinity tolerance. Environ Exp Bot 67:2–9 Turkan I, Bor M, Ozdemir F, Koca H (2005) Differential responses of lipid peroxidation and antioxidants in the leaves of drought tolerant P. acutifolius Gray and drought sensitive P. vulgaris L. subjected to polyethylene glycol mediated water stress. Plant Sci 168:223–231 Urade R (2007) Cellular response to unfolded proteins in the endoplasmic reticulum of plants. FEBS J 274: 1152–1171 Urban O (2003) Physiological impacts of elevated CO2 concentration ranging from molecular to whole plant responses. Photosynthetica 41:9–20 Vaidyanathan H, Sivakumar P, Chakrabarty R, Thomas G (2003) Scavenging of reactive oxygen species in NaCl stressed rice (Oryza sativa) differential response in salt tolerant and sensitive varieties. Plant Sci 165: 1411–1418 Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759 Verma S, Dubey RS (2003) Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant Sci 164: 645–655 Vernon DM, Bohnert HJ (1992) A novel methyltransferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO J 11:2077–2085 Vernon DM, Taraczynski MC, Jensen RG, Bohnert HJ (1993) Cycl*tol production in transgenic tobacco. Plant J 4:199–205

28 Vertovec M, Sakcali S, Ozturk M, Salleo S, Giacomich P, Feoli E, Nardini A (2001) Diagnosing plant water status as a tool for quantifying water stress on a regional basis in Mediterranean drylands. Ann For Sci 58: 113–125 Vinocur B, Altman A (2005) Cellular basis of salinity tolerance in plants. Environ Exp Bot 52:113–122 Vitoria AP, Lea PJ, Azevedo RA (2001) Antioxidant enzymes responses to cadmium in radish tissues. Photochem 57:701–710 Vranova E, Inze D, Van Brensegem F (2002) Signal transduction during oxidative stress. J Exp Bot 53: 1227–1236 Waditee R, Bhuiyan NH, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf AT, Takabe T, Takabe T (2005) Genes for direct methylation of glycine provide high levels of glycine betaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci USA 102:1318–1323 Walker MA, McKersie BD (1993) Role of ascorbateglutathione antioxidant system in chilling resistance of tomato. J Plant Physiol 141:234–239 Walters DR (2003a) Resistance to plant pathogens: possible roles for free polyamines and polyamine catabolism. New Phytol 159:109–115 Walters DR (2003b) Polyamines and plant disease. Phytochem 64:97–107 Wang HY, Huang YC, Chen SF, Yeh KW (2003a) Molecular cloning, characterization and gene expression of a water deficiency and chilling induced proteinase inhibitor I gene family from sweet potato (Ipomoea batatas Lam.) leaves. Plant Sci 165:191–203 Wang W, Vinocur B, Altman A (2003b) Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta 218:1–14 Wang Y, Ying Y, Chen J, Wang XC (2004) Transgenic Arabidopsis overexpressing Mn-SOD enhanced salttolerance. Plant Sci 167:671–677

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2

Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance Vinayak H. Lokhande and Penna Suprasanna

Abstract

Halophytes are a diverse group of plants with tolerance to high salinity. While most of our crops are glycophytes lacking the genetic makeup for salt tolerance, halophytes are endowed with ability to seize NaCl into their cell vacuoles as an osmoticum. The sensitivity of crops to environmental extremities has become a major limitation to worldwide food production. Study of halophytes can be rewarding as the mechanisms by which halophytes survive and maintain productivity on saline water can be understood to define and manage adaptations in glycophytes. The adaptation mechanisms include ion compartmentalization, osmotic adjustment, succulence, ion transport and uptake, antioxidant systems, maintenance of redox and energetic status, and salt inclusion/excretion. Real benefits can be accrued if sustained efforts are in place to investigate the speciesspecific regulation during abiotic stresses and extend genetic resource and manipulate stress tolerance mechanisms. Halophytes are also an important plant species with potential for the purposes of desalination and restoration of saline soils, withstand high soil salinity and saline water irrigation, phytoremediation and wetland restoration. It will be invaluable to develop these strategies to ensure sustainability, and future efforts to improve crop performance on marginal and irrigated land. Keywords

Halophytes • Abiotic stress • Compatible solutes • Antioxidants • Phytoremediation

P. Suprasanna () • V.H. Lokhande Functional Plant Biology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, Maharashtra, India e-mail: [emailprotected] P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_2, © Springer Science+Business Media, LLC 2012

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1

Introduction

Environment basically consists of balanced interaction between biotic and abiotic factors, and often abrupt perturbations in abiotic factors surrounding the biotic organisms lead to change the homeostasis, consequently creating a stressful condition for the survival of living organisms. Environmental stresses represent the most limiting factors for agricultural productivity. The abiotic stresses such as shade or high light levels, subzero, low or high temperatures, drought, flooding, high salinity, inorganic nutrient imbalance, infection, predation, and natural or man-made toxic compounds and oxidative stress (Bohnert and Sheveleva 1998) cause losses worth hundreds of million dollars each year due to reduction in crop productivity and crop failure (Zhu 2001; Flowers 2004). In view of their sessile nature, plants should have developed some adaptation strategies to manage the changing environmental conditions particularly with the available resources. Therefore, foremost adaptation carried out by terrestrial plants to its surrounding is adjustment in their water potential as low as that of soil in which they are able to grow. In the course of evolution, some plants have evolved and adapted to freshwater habitat for acquiring nutrients from the low concentrations of minerals present in fresh water such as glycophytes, whereas the plants which retained their habitat in nutrientrich marine environment were found more successful to combat the adverse abiotic stresses and are referred as “halophytes” (Flowers et al. 2010). These plants can be grown using land and water unsuitable for conventional crops and can provide food, fuel, fodder, fiber, resins, essential oils, and pharmaceutical feedstocks (Table 2.1).

2

Halophytes

Soil salinity and irrigated agriculture have coexisted since ancient times, and ever since the problem of salinity in agriculture has become a challenge. Soils are generally classified as saline when the electrical conductivity of the saturated

paste extract (ECe) is 4 dS m−1 or more (which is equivalent to 40 mM NaCl) and generate an osmotic pressure of approximately −0.2 MPa. Based on this, plants differ greatly in their growth response to saline conditions and therefore classified as “glycophytes” or “halophytes” referring to their capacity to grow on highly saline environments (Munns and Tester 2008). Halophytes are remarkable plants which have the ability to complete their life cycle in a substrate rich in NaCl that normally found toxic to other species and destroy almost 99% of their population (Flowers and Colmer 2008). These are highly evolved and specialized organisms with welladapted morphological, anatomical, and physiological characteristics allowing them to proliferate in the soils possessing high salt concentrations (Flowers et al. 1977; Flowers and Colmer 2008). Moreover, some halophytes consistently require a particular concentration of NaCl in the growth medium are referred as “obligate halophytes” or “true mangroves” and, apart from their growth in highly saline environment, some halophytes have capacity to grow on the soil devoid of salt are called as “facultative halophytes” or “mangrove associates.” This presence or absence of substrate in the form of salt offers advantages for the halophytes in the competition with salt–sensitive plants (glycophytes) for the management of abiotic stress tolerance and utilization of these species for the improvement of crop yield. In this regard, it is essential to understand the adverse effects of abiotic stresses and tolerance mechanisms developed by the halophytes and exploit such knowledge for the improvement of crop plants which can meet the demand of food, feed, fodder, and industrial raw material. The standard approach to this problem would be to increase the tolerance capacity of conventional crop plants, which otherwise are high yielders. An alternative strategy is to make use of halophytes that already have the required level of stress tolerance and are still productive at high external adverse conditions. Salinity is one of the major abiotic constraints, affecting almost every aspect of plant’s physiology at both whole plant and cellular level through osmotic stress in an earlier phase and ionic stress at a later stage of

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

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Table 2.1 List of halophytes used for saline agriculture in Pakistan and other countries (modified from Khan and Qaiser 2006) Uses Food

Fodder

Forage

Ornamental

Chemicals

Plant species A. hortensis, Aizoon canariense, Apium graveolens, Arundo donax, Atriplex halimus, Avicennia marina, Cocos nucifera, Cynamorium coccinium, Echinochloa crusgalli, Glinus lotoides, Glossonema varians, H. stocksii, Haloxylon griffithii ssp griffithii, N. schoberi, Neurada procumbens, Nitraria retusa, Ochradenus baccatus, Oxystelma esculentum, P. sylvestris, Pedalium murex, Pentatropis nivalis, Pheonix dactylifera, Pisonia grandis, Polypogon monspeliensis, Portulaca oleracea, Rumex vesicarius, S. brachiata, S. persica, Salicornia bigellovi, Salvadora oleoides, Sesuvium portulacastrum, Solanum incanum, Suaeda fruticosa, Triglochin maritime, Zizyphus nummularia, Zygophyllum simplex A. griffithii, A. halimus, A. leucoclada, A. tatarica, Aegiceras corniculatus, Alhaji maurorum, Anagallis arvensis, Artemisia scoparia, Arthrocnemum indicum, Atriplex canescens, Avicennia marina, B. glaucus, Beta vulgaris ssp maritma, Bienertia cycloptera, Bolboschoenus affinis, Caesalpinea bonduc, Camphorosma monspelictum, Carex divisa, Chloris virgata, Cressa cretica, Dalbergia sissoo, Glinus lotoides, Halocnemum strobilaceum, Haloxylon stocksii, Lobularia maritime, Lolium multiflorum, Neurada procumbens, Orthochloa compressa, P. farcta, P. juliflora, Populus euphratica, Prosopis cineraria, Raphanus raphanistrum, Rhizophora mucronata, Salsola tragus, Seidlitzia florida, Seriphidium quettense, Suaeda fruticosa, T. repens, T. triquetra, Trianthema portulacastrum, Trifolium fragiferum, Vicia sativa, Zaleya pentandara, Zygophyllum simplex A. littorali, A. macrostachys, Aeluropus lagopoide, Agrostis stolonifera, Aristida adsceshoines, Aristida mutabilis, Atriplex dimorphostegia, C. ciliaris, C. pennesittiformis, Cenchrus biflorus, Chloris gayana, Cynodon dactylon, D. aristatum, D. scindicum, Dactyloctenium aegyptium, Desmostachya bipinnata, Dichantheum annulatum, Diplachne fusca, E. crusgalli, E. japonica, E. superba, Echinochloa colona, Eleusine indica, Eragrostis curvula, Festuca rubra, Halocharis hispida, Halopyrum mucronatum, Haloxylon persicum, Lasiurus scindicus, Nitraria retusa.Oligomeris linifolia, P. minor, P. pratensis, Paspalum pasploides, Phalaris arundinacea, Poa bulbosa, S. helvolus, S. ioclados, S. kentrophyllus, S. tourneuxii, S. tremulus, S. virginicus, Sacchraum bengalense, Salvadora persica, Sporobolus coromandelianus, Urochondra setulosa Achillea millefolium, Alhaji maurorum, Ammi visnaga, Artemisia scoparia, Avicennia marina, Caesalpinea bonduc, Calotropis procera, Camphorosma monspelictum, Cassia italic, Centella asiatica, Ceriops tagal, Chenopodium ambrosoides, Corchorus depressus, Cressa cretica, Cynamorium coccinium, Erythrina herbacea, Evolvulus alsinoides, Glinus lotoides, Halogeton glomeratus, Imperata cylindrical, Inula brittanica, Ipomoea alba, L. gilsei, L. sinuatum, L. stocksii, Leptadenia pyrotechnica, Limonium axillare, Melhania denhamii, Microcephala lamellate, Neurada procumbens, olanum surrattense, Oligomeris linifolia, Oxystelma esculentum, P. oleracea, Pedalium murex, Pentatropis nivalis, Populus euphratica, Portulaca quadrifida, Psylliostachys spicata, Rumex vesicarius, S. quettense, Seriphidium brevifolium, Solanum incanum, Sonneratia caseolaris, Thespesia populneoides, Trianthema portulacastrum, Tribulus terrestris, Urginea indica,Verbena officinalis, Withania sominifera, Z. simplex, Zaleya pentandara, Zygophyllum propinquum Aeluropus lagopoides, Ardisia solanacea, Calotropis procera, Cenchrus ciliaris, Clerodendrum inerme, Dalbergia sissoo, Euphorbia thymifolia, Ficus microcarpa, Halocnemum strobilaceum, Ipomoea pes-caprae, K.iranica, Knorringia sibirica subsp. Kochia indica, Mesembryanthemum crystallinum, N. schoberi, Nitraria retusa, Phyla nodiflora, Polypogon monspeliensis, Raphanus raphanistrum, S. taccada, Scaevola plumier, Sessuvium sessuvioides, T. passernioides, T. ramosissima, T. szovitsiana, T. tetragyna, Tamarix mascatensis, Thomsonii, Trianthema portulacastrum

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plant growth (Munns and Tester 2008) and leads to a series of morphological, physiological, biochemical, and molecular changes. In the past 2–3 decades, considerable progress has been made in the evaluation of halophytes to understand their survival mechanisms to be used as crop plants. In the present article, we document different aspects of halophytes, with an emphasis on mechanism of tolerance to salinity, drought and heavy metal tolerance, and their exploitation to manage the problems associated with the abiotic stresses as well as for environmental protection. Halophytes respond to salt stress at cellular, tissue, and the whole plant level (Epstein 1980). In response to salt stress, the general physiology of halophytes has been reviewed occasionally (Flowers et al. 1977; Epstein 1980; Flowers 1985, 2004) and since then other reviews have examined their eco-physiology (Ball 1988; Rozema 1991; Breckel 2002), photosynthesis (Lovelock and Ball 2002), response to oxidative stress (Jitesh et al. 2006), and flooding tolerance (Colmer and Flowers 2008). Therefore, studies on the halophytes can be instructive from three prospects: first, the mechanism by which halophytes survive and maintain productivity under abiotic constraints can be used to define a minimal set of adaptations required in tolerant germplasm. This knowledge can help to focus the efforts of plant breeders and molecular biologists working with conventional crop plants (Glenn and Brown 1999). Second, halophytes grown in an agronomic setting can be used to evaluate the overall feasibility of high-salinity agriculture, which depends on more than finding a source of tolerant germplasm (Glenn et al. 1997). Third, halophytes may become a potential source of new crops.

3

Halophytes Diversity

Halophytes show immense diversity in habitat and behavior to tolerate the abiotic stress conditions with uneven distribution across the taxa of flowering plants (Flowers et al. 2010). This group of plants has been classified based on their tolerance capacity to salinity stress. Aronson (1989) listed approximately 1,550 species as salt-tolerant

based on their capacity to tolerate the salt concentration more than 80 mM NaCl (equivalent to EC 7.8 dS m−1), whereas, plants limiting the growth beyond this concentration were categorized as glycophytes or salt-sensitive. Using similar features, Menzel and Lieth (2003) recorded total 2,600 species as salt-tolerant. However, considering the salt tolerance limit proposed by Aronson (1989) and Menzel and Lieth (2003) which is found to be significantly lower than the salt concentration of seawater (~480 mM Na+ and 580 mM Cl−), Flowers and Colmer (2008) defined the halophytes as plants that have evolved and tolerate to complete their life cycle in at least ~200 mM NaCl. Applying the new definition to Aronson’s database, Flowers et al. (2010) further classified a total of 350 species as halophytes with major species distributed in 20 orders including 256 families. It has also been suggested that salt tolerance was widely distributed among flowering plant families and had a polyphyletic origin. The authors also stated that distribution and development of evolutionary link of halophytes may account to not more than ~0.25% of the known species of angiosperms.

4

Adaptations to Abiotic Stresses

Halophytes have evolved a number of adaptive traits which allow them to germinate, grow, and achieve their complete life cycle of development under such harsh conditions (Flowers et al. 1977). A variety of studies performed on glycophytes and halophytes subjected to abiotic stresses has demonstrated that impairment in growth under stress condition results from various responses induced through both osmotic effects related to disturbance in plant–water relationships and ionic effects associated with mineral toxicity and deficiency (Lauchli and Epstein 1990). Associated with these primary stresses, higher plants also suffer from secondary stresses provoked by cellular damages especially those induced by oxidative stresses due to imbalance between production and destruction of reduced reactive oxygen species (Zhu 2001).

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

In order to achieve the tolerance status, three interconnected aspects of plant activity are significant: damage must be prevented, homeostatic condition must be re-established, and growth must resume. At present, there are different mechanisms of abiotic stress tolerance in halophytes that have been proposed which include ion compartmentalization, osmotic adjustment through osmolytes accumulation, succulence, selective transport and uptake of ions, enzymatic and nonenzymatic antioxidant response, maintenance of redox and energetic status, salt inclusion/excretion and genetic control (Flowers and Colmer 2008). Understanding the mechanism of tolerance in halophytes at morphological, anatomical, physiological, biochemical, and molecular levels is crucial to improve the tolerance of the crop plants and their adoption under abiotic stress conditions to exploit such problem soils. A generalized scheme for the plant’s response to abiotic stresses and mechanism of stress tolerance is presented (Fig. 2.1).

4.1

Ion Compartmentation

The sensitivity of cytosolic enzymes to salt is similar in both glycophytes and halophytes, indicating that the maintenance of high cytosolic K+/ Na+ ratio is a key requirement for plant growth under salt conditions (Glenn and Brown 1999). While dealing with Na+, the cell must also acquire nutrient K+. The Na+ ion is the foremost inorganic ion and a cheap source of osmoticum in the halophytes to maintain the osmotic balance under abiotic stresses. Under typical physiological conditions, plant cells require high K+ (100– 200 mM) and lower Na+ (less than 1 mM) and accordingly the high cytosolic K+/Na+ ratio to maintain the osmotic balance (Tester and Davenport 2003) for proper functioning of the cell. Na+ competes with K+ for intracellular influx since both these are transported by common channels present on the membranes and, thus, subsequently increase K+ efflux from intracellular stores as against the higher Na+ stress built

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up outside the cell. To maintain a high K+/Na+ ratio in the cytosol, plant cell employs primary active transport, mediated by channels and cotransporters for Na+ extrusion and/or the intracellular compartmentalization of Na+ into the vacuole (Blumwald 2000). When halophytes are exposed to saline condition, a large increase in extracellular Na+ level establishes the Na+ electrochemical potential gradient more than the actual negative electrical membrane potential difference at the plasma membrane (−140 mV) which favor the passive transport of sodium ions from the outer environment inside the cell. Recently, uniporter or ion channel type transporters have been identified for the entry of Na+ into the cell; these are high-affinity potassium transporter (HKT), low-affinity cation transporter (LCT1), nonselective cation channels (NSCC) like cyclic nucleotide-gated channels (CNGCs) and glutamate-activated channels (GLRs) (Apse and Blumwald 2007). HKTs have been shown to function as Na+/K+ symporter and as Na+ selective uniporters (Horie and Schroeder 2004). In the process of elevated levels of Na+ outside the cell, the electrochemical gradient makes the sodium uptake passive; however, the efflux of Na+ outside cell is an active process and requires energy in the form of ATP. In this process, the Na+/H+ antiporter (NHX) present on the plasma membrane facilitates the Na+ efflux. This electroneutral exchange of sodium for protons to facilitate efflux is the only mode of transport that has been measured for efflux under physiological conditions (Apse and Blumwald 2007). Besides the efflux of Na+, some halophytes have developed mechanism to sequester the Na+ into the vacuoles as an efficient mechanism to avoid the toxic effects of Na+ in the cytosol. The transport of Na+ into the vacuoles is mediated by cation/H+ antiporters that are driven by the electrochemical gradient of protons generated by the vacuolar H+ translocating enzymes such as H+-ATPase and H+-PPiase (Gaxiola et al. 2007). These transporters play an important role in the sequestration of Na+ ions into the vacuole or exclusion outside the cell of the halophytes.

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Fig. 2.1 Generalized scheme for plant responses to abiotic stresses and mechanism of stress tolerance. Plants exposed to various abiotic stresses (salinity, drought, extreme temperatures, toxic metals, etc.) initiate the cascade of changes in plants’ functioning such as imbalanced water and nutrient uptake, stomatal closure, altered gaseous exchange, improper functioning of photosynthetic systems due to over-reduction of electron transport chains in chloroplast and mitochondria causing generation of reactive oxygen species (ROS). The integrative effect of these factors leads to induce the oxidative damage to functional and structural molecules (DNA, proteins, lipids, and carbohydrates) making the changes in the redox, osmotic,

V.H. Lokhande and P. Suprasanna

ionic, and energetic homeostasis of the plant. These stress signals triggers the downstream signaling processes and gene activation through transcription factors. Activation mechanisms involve enzymatic and nonenzymatic antioxidants for detoxification of ROS, osmolytes (proline, glycine betaine, sugar polyols) synthesis for osmotic balance and protection to structural molecules, ion compartmentation for ionic homeostasis and maintenance of redox and energetics through the higher ratios of GSH/GSSG, ASC/DHA, NADP/NADPH, and ATP/ADP. The coordinated action leads to re-establish the cellular homeostasis, protection of functional and structural proteins and membranes, and ultimately the tolerance to abiotic stresses

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

4.2

Succulence

Succulence is commonly called as halosucculence and found to occur within a range of salt concentrations optimal for growth. The sequestration of saline ions into the vacuoles leads to the plant to increase the succulence, one of the common characteristics of the halophytes (Flowers et al. 1977). Succulence minimizes the toxic effects of excessive ion accumulation and has been reported to be associated with accretion of osmotically active solutes for the maintenance of cell turgor pressure. The succulent halophytes unlike glycophytes tend to accumulate sodium in the vacuole to higher levels than in the cytoplasm and as the volume of the vacuole is much greater than that of the cytoplasm in fully expanded cells, the total sodium content of the root will approximate to the sodium content of the vacuole (Yeo and Flowers 1986). Succulent halophytes generally have thick leaves and stems, mainly associated with an increase in the size of their mesophyll cells along with smaller intracellular spaces. It has also been shown that succulent leaves have more and large-sized mitochondria because the succulent halophytes require excess energy for salt compartmentalization and excretion. Whether succulence is a response to salinity or adaptation to salinity is debatable. But as halophytes tend to become succulent in response to salinity (due to physiologically less available water which affects the changes in the integral part of the plant development), the succulence might be the adaptation to salinity stress (Waisel 1972). This adaptive nature of succulence made the halophytes more successful in the course of evolution exposing to various environmental constraints. Most of the halophytes such as Sesuvium portulacastrum, Suaeda sps., Lobularia maritime, Mesembryanthemum crystallinum, Halosarcia pergranulata subsp. Pergranulata, etc. were found more amenable to accumulate the excess Na+ in their leaves and stems and increase the succulence under optimum NaCl concentrations in the range of 100–400 mM which leads to sequester these saline ions into the vacuole and become more successful for their growth in saline environment (Qi et al. 2009; Lokhande et al.

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2010a). Thus it appears that increased succulence could be due to a “diluting” effect on the ion content of cells which might otherwise rise to toxic levels, and sodium acts as a specific stimulant of growth which can be considered as tending to reduce the turgor pressure component of the water potential of the cell (Jennings 1968). The succulent halophytes are able to balance the growth and ion accumulation through its sequestration into the vacuole; however, some of the halophytes were adapted to saline environment through secretion of salts from salt glands, cuticles or in guttation fluid, re-transported back to the roots and soil via the phloem or become concentrated in salt hairs. Salt glands act as transient cells because they are devoid of vacuole and have a large number of mitochondria and other organelles. The halophytes which secretes the saline ions include Limonium latifolium, Spartina sps., Sporobolus spicatus, Atriplex sps., etc. (Ramadan 2000). However, not all halophytes have salt glands; neither do they all discard salt saturated tissue, demonstrating that individual halophytes utilize different salt tolerance traits under different stress periods.

4.3

Osmotic Adjustment

Osmotic adjustment in response to abiotic stresses is an adaptive mechanism in the halophytes in order to maintain their water balance (Flowers and Colmer 2008). Besides the accumulation of inorganic ions and its sequestration in the vacuole, the osmotic balance between vacuole and cytoplasm is also maintained through the synthesis of organic solutes to retain the stability of the proteins in cells in response to drop in the water potential of the environment (Glenn and Brown 1999). Plant cells synthesize a variety of organic solutes such as proline, sucrose, polyols, trehalose and quaternary ammonium compounds (QACs) such as glycine betaine, alaninebetaine, prolinebetaine, choline-O-sulfate, hydroxyprolinebetaine, and pipecolatebetaine (Rhodes and Hanson 1993). These are low molecular weight, highly soluble compounds and are nontoxic even at high cellular concentrations (Ashraf and Foolad 2007) without

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disturbing intracellular biochemistry and cellular functions (Cushman 2001), protects subcellular structures, mitigate oxidative damage caused by free radicals (Attipali et al. 2004), maintains the enzyme activities under salt stress and protection of cellular components from dehydration injury (Ashraf and Foolad 2007). The osmolytes accumulation is frequently reported in glycophytes and halophytes being continuously exposed to abiotic stresses; however, synthesis of these osmolytes is an energy-dependent process which consumes large number of ATP molecules (Raven 1985), thus affecting the growth. Osmolytes synthesis and their overproduction in transgenic plants has been achieved in transgenic crop plants, however little success has been achieved on the desired protective levels of these osmolytes in plants. In contrast, some plants showed increased tolerance to abiotic stresses after exogenous application of these organic solutes (Ashraf and Foolad 2007). Although increased accumulation of these osmolytes by the plants exposed to abiotic stresses has been reported, not all plant species synthesize the all kinds of osmolytes at a time; some plant species synthesize and accumulate very low quantity of these compounds while some plant species not do so (Ashraf and Foolad 2007).

4.3.1 Proline Similar to glycophytes, proline accumulation is a common adaptive response to various abiotic stresses. Several studies using transgenic plants or mutants demonstrated that proline metabolism has a complex effect on development and stress responses, and that proline accumulation is important for the tolerance to certain adverse environmental conditions (Hong et al. 2000; Miller et al. 2010). In plants, proline is mainly synthesized from glutamate using two important enzymes such as pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR). Proline is synthesized in cytoplasm; however in mitochondria, the catabolism occurs via sequential action of proline dehydrogenase (PDH) producing pyrroline-5-carboxylate (P5C) and its conversion to glutamate using P5C dehydrogenase (P5CDH) (Szabados and Savoure 2009). Halophytes have shown vast diversity for

V.H. Lokhande and P. Suprasanna

the accumulation of proline in response to abiotic stresses, wherein plants from the Aizoaceae family accumulate large quantities of proline showing its role in osmoprotection (Delauney and Verma 1993). Proline concentrated in the cytosol, chloroplast and vacuoles and compatible with enzyme activity in the cytoplasm showed its significant contribution to osmotic adjustment. Besides being an osmoprotectant, proline also has a role in detoxification of reactive oxygen species and act as an antioxidant, stabilization of proteins and protein complexes and as a signaling/regulatory molecule (Szabados and Savoure 2009). It also function as a protein-compatible hydrotrope (Srinivas and Balasubramanian 1995), alleviating cytoplasmic acidosis, and maintaining appropriate NADP+/NADPH ratios compatible with metabolism (Hare and Cress 1997). Also, rapid breakdown of proline upon relief of stress provides sufficient reducing agents that support mitochondrial oxidative phosphorylation and generation of ATP for recovery from stress and repairing of stress-induced damages (Hare and Cress 1997). In halophytic plant species in response to abiotic stresses, proline accumulation in the cytosol has been shown to contribute substantially to cytoplasmic osmotic adjustment. For example, in cells of Distichlis spicata treated with 200 mM NaCl, the cytosolic proline concentration was estimated to be more than 230 mM (Ketchum et al. 1991). In Sesuvium portulacastrum, Lokhande et al. (2010a, b, 2011a) found an extensive increase in proline content when the callus and axillary shoot cultures exposed to salt and drought stress alone or under iso-osmotic stress conditions of NaCl and PEG. Higher proline accumulation has also been shown in S. portulacastrum plants exposed to various abiotic constraints that include salinity, drought, and heavy metals (Messedi et al. 2004; Slama et al. 2008; Ghnaya et al. 2007; Moseki and Buru 2010; Lokhande et al. 2011b). Such an osmotic adjustment through proline accumulation is also evident in other species like Plantago crassiflora, Salicornia europaea, Atriplex halimus, A. halimus subsp. schweinfurthii, Avicennia marina, Hordeum maritimum, Ipomoea pes-caprae, Paspalum vagin*tum, Phragmites australis, and Suaeda sps.

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

(Vicente et al. 2004; Reda et al. 2004; Nedjimi and Daoud 2009; Pagter et al. 2009; Lefevre et al. 2009; Sucre and Suarez 2010). Among different halophytic plants, S. portulacastrum has been reported as a high proline accumulator, with levels reaching 300 mmol g−1 leaf dry matter (Slama et al. 2008). Such a pronounced accumulation of proline and its physiological role in osmotic adjustment may have made the halophytes more successful to grow under adverse environmental stresses.

4.3.2 Glycine Betaine Among the variety of quaternary ammonium compounds, glycine betaine (GB) is one of the most abundantly occurring and synthesized at higher concentrations in the plants exposing to dehydration stress due to adverse environmental calamities. GB is located in chloroplast where it plays an important role in osmotic adjustment and protection of thylakoid membrane, by maintaining the photosynthetic machinery in active state (Robinson and Jones 1986). GB is synthesized mainly from choline, which is converted to betaine aldehyde and then to GB through sequential enzymatic action of choline monooxyegenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Although other pathways such as direct N-methylation of glycine are also known, the pathway from choline to GB has been identified in all GB-accumulating plant species (Ashraf and Foolad 2007). It is widely believed that synthesis and accumulation of GB protects cytoplasm from ion toxicity, dehydration and temperature stress and helps normal functioning of the metabolic machineries in the cell during stressed conditions by stabilizing macromolecule structures, protecting chloroplast and photosynthesis system II (PSII) by stabilizing the association of the extrinsic PSII complex proteins and indirectly interacting with phosphatidylcholine moieties of membranes to alter their thermodynamic properties (Subbarao et al. 2001). It has been shown that tolerant species are more amenable to accumulate higher GB in comparison to sensitive species as a response to abiotic stress imposition. Based on the GB and proline accumulation potential,

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Tipirdamaz et al. (2006) categorized the halophytes from inland and salt marsh habitats of Turkey. The studies have shown that the species that behaved as GB accumulators appeared poor proline accumulators and vice versa. The GB accumulation reported in the halophytes is generally in the range of 1.5–400 mmol g−1 DW and some of the highest GB accumulating halophytes are members of the Chenopodiaceae (Halocnemum strobilaceum, Petrosimonia brachiata, Suaeda confuse), Compositae (Artemisia santonicum), and Frankeniaceae (Frankenia hirsuta). Increased accumulation of GB has also been demonstrated in other halophytes such as Beta vulgaris (Subbarao et al. 2001), Spartina anglica (Mulholland and Otte 2002), Atriplex halimus (Martinez et al. 2005), A. Nummularia (Silveira et al. 2009), and S. portulacastrum (Lokhande et al. 2010a, b). Increased GB accumulation has also been correlated with increased betaine aldehyde dehydrogenase gene expression (BADHmRNA) in Salicornia europaea and Suaeda maritima leaves exposed to salt stress (Moghaieb et al. 2004). Considering the significance of GB in the osmotic balance of the halophytes under stressful environment, different methods can be derived to enhance the concentration of this compound in crop plants to increase their stress tolerance. The approaches can include breeding of sensitive cultivars with their tolerant relatives from halophytes with natural abilities to produce high levels of GB or genetically engineer the sensitive species through transformation of the genes responsible for GB synthesis. Although some progress has been made in introducing the genes for the production of these compounds in naturally accumulating or low-accumulating plant species, levels of these compounds’ accumulation in transgenic plant have often been low or insufficient to the plant stress tolerance (Ashraf and Foolad 2007).

4.3.3 Soluble Sugars In general, modulations in the carbon metabolism and the levels of carbohydrates (sugars) are seen due to changes occurring in the process of photosynthesis and carbon partitioning of the plant at organ level and in whole plants exposing

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to abiotic stresses (Gonzalez et al. 2009). Soluble sugars function as metabolic resources and structural constituents of cells, besides acting as signals regulating various processes associated with plant growth and development. Such signaling can modulate stress pathways into a complex network to further orchestrate metabolic plant responses. A variety of sugar compounds such as sucrose, glucose, mannose, maltose, trehalose, and many other sugar alcohols have been studied in response to abiotic stresses (Briens and Larher 1982; Yuanyuan et al. 2009) and the accumulation of soluble sugars has been attributed as an important parameter of osmotic adjustment in the halophytes. Briens and Larher (1982) screened different organs of 16 halophyte species for soluble carbohydrates and other osmolytes and found that all the species accumulated sucrose, fructose and glucose whereas Plantago maritime, Juncus maritimus, Phrgamites communis and Scripus maritimus showed the highest accumulation of soluble sugars. The presence of higher amounts of soluble sugars has been reported as main contributors to osmotic adjustment in the Atriplex halimus plants exposed to PEG and NaCl stresses and it is correlated with the response of NaCl stress on soluble sugar synthesis (Martinez et al. 2005). The accumulation of total soluble sugars has also been correlated with the variations at genotypic level among two genotypes of Cakile maritime namely Jebra and Tabarka which showed differences in the total soluble carbohydrate concentrations. While the content of the sugars was unaffected in the leaves of Jerba plants at moderate salinity, the plants of the saltsensitive Tabarka showed a slight increase in soluble carbohydrate contents during leaf development. The contribution of this compatible solute group to the “osmotic pool” was found higher in the salt-tolerant Jerba than in the salt-sensitive Tabarka seedlings exposed to 400 mM NaCl stress (Megdichi et al. 2007). Further, Sesuvium portulacastrum axillary shoots exposed to salinity stress showed optimum growth at 200 mM NaCl in comparison to control and exhibited increased synthesis of total soluble sugars over proline and glycine betaine (Lokhande et al. 2010b). Salinity-induced soluble sugar accumulation has

also been observed in P. euphratica (Zhang et al. 2004). Accumulation of soluble sugars has been observed in plants undergoing drought, flooding, and water logging conditions (Chai et al. 2001; Munns 2002; Li and Li 2005). Chenopodium quinoa exposed to water deficit and water-logging stresses showed no changes in starch, sucrose, or fructose content but showed increased glucose and total soluble sugar content in stressed plants in comparison to control (Gonzalez et al. 2009). These studies in halophytes demonstrate that soluble sugars play a significant role besides other osmolytes in the osmotic adjustment.

4.4

Antioxidant Systems

The halophytic plants display a cascade of events upon exposure to environmental stresses leading to metabolic disturbance. The cascade of events include physiological water-deficit abscisic acidregulated stomatal closure in leaves, limited CO2 availability, over-reduction of electron transport chain in the chloroplast and mitochondria and finally generation of reactive oxygen species (ROS). These ROS are highly toxic and in the absence of protective mechanism in the plant can cause oxidative damage to proteins, DNA, and lipids (Mittler 2002; Miller et al. 2010). Additionally, this may also lead to alteration in the redox state resulting in further damage to the cell (Mittler et al. 2004). To regulate the ROS levels, plant cells are evolved with complex enzymatic and nonenzymatic antioxidant defense mechanisms, which together help to control the cellular redox state under changing environmental conditions. A correlation between enzymatic and nonenzymatic antioxidant capacitance and abiotic stress tolerance has been reported in several plant species such as Crithmum maritimum, C. maritime, Plantago genus, Sesuvium portulacastrum, Mesembryanthemum crystallinum (Ben Amor et al. 2005; Jitesh et al. 2006; Sekmen, Turkan and Takio 2007; Ashraf 2009; Lokhande et al. 2010a, b, 2011a-c). Superoxide dismutase (SOD) constitutes the first line of defense converting O2•− to H2O2, which is further reduced to

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

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Table 2.2 Examples of halophytic plant species studied for the antioxidant responses in response to abiotic stresses Plant species Avicennia marina Bruguiera parviflora, B. gymnorrhiza Beta vulgaris, B. maritime Crithmum maritimum Hordeum vulgare H. maritimum M. crystallinum

Enzyme/protein/gene studied SOD, CAT, POX, APX, MDHAR SOD, CAT, APX

References Cherian et al. (1999), Jitesh et al. (2006), Kavitha et al. (2008), and Kavitha et al. (2010) Takemura et al. (2000) and Parida et al. (2004)

SOD, CAT, APX, GR SOD, CAT, POX SOD, POX, CAT, GR SOD, CAT, GPX, APX, MDHAR, DHAR, GR SOD, ferritin, CAT, Mn, Fe, Cu/Zn SOD

Bor et al. (2003) Ben Amor et al. (2005) Patra and Panda (1998) Hafsi et al. (2010)

Phaseolus vulgaris Sesuvium portulacastrum Suaeda nudiflora, S. salsa

SOD, CAT, APX SOD, CAT, APX, POX, DHAR, GST SOD, APX, CAT, GPX SOD, CAT, APX, GR SOD, CAT, POX

T. halophila

SOD, APX, POX

Nitraria tangutorum Phragmites australis

water and oxygen by ascorbate peroxidase (APX) and catalase (CAT). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), and glutathione peroxidase (GPX) are an important enzymes involved in regeneration of ascorbate and GSH for the proper functioning of ASC-GSH cycle (Noctor and Foyer 1998; Mittler 2002; Miller et al. 2010). Halophytes have evolved various mechanisms of adaptations of which increased antioxidant enzyme activities was found one of an important mechanism of stress tolerance. Halophytes have capacity to maintain high metabolic activity even at inhibitory concentrations of intracellular Na+ due to enhanced antioxidant mechanism (Jitesh et al. 2006). Most of the early studies in halophytes were on photosynthesis and respiration and related to ion compartmentation, osmotic adjustment (Flowers et al. 1977; f*ckushima et al. 1997). However, recently, more emphasis has been given on abiotic stresses in relation to antioxidant enzymes in halophytes (Takemura et al. 2000; Cherian and Reddy 2003; Parida et al.

Slesak et al. (2002, 2008), Slesak and Miszalski (2003), Hurst et al. (2004), and Parmonova et al. (2004) Yang et al. (2010) Carias et al. (2008) Jebara et al. (2005) Lokhande et al. 2010a, b, 2011a-c) Cherian and Reddy (2003), Wang et al. (2004a), and Fang et al. (2005) Taji et al. (2004), Wang et al. (2004a, b), and M’rah et al. (2006)

2004; Slesak et al. 2002, 2008; Slesak and Miszalski 2003; Jitesh et al. 2006; Lokhande et al. 2010a–b, 2011a, c). The response of antioxidant enzyme systems in the halophytes exposed to abiotic stresses has been reviewed by Jitesh et al. (2006) and is summarized in Table 2.2. Most of the halophytes have shown increased efficiency of antioxidant enzyme machinery thus removing the ROS levels to a greater extent and maintain the plants survival under stressful conditions.

4.5

Redox and Energetics

The cellular redox state is made tangible in terms of the redox state of the individual redox-active molecules in a cell. For each redox-active molecule, its redox state can be defined as the proportion of reduced molecules relative to the total pool size, or alternatively as the ratio between reduced and oxidized molecules within a pool (Potters et al. 2010). A large number of redox-active compounds such as ascorbate (ASC), glutathione

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(GSH), pyridine nucleotides (NADH and NADPH), carotenoids, tocopherols, distinct redox-active phenolics, polyamines and proteins carrying redox-active S-groups are contained in plant cells (Smirnoff 2005; Queval and Noctor 2007). The enzymatic and nonenzymatic antioxidants involved in ROS scavenging significantly contribute to the redox state maintenance of the cellular environment through continuous channeling of these redox-active components which facilitate the proper functioning of the cell under stressful conditions. In general, the maintenance of redox state is correlated with the energy metabolism of the plant cell in terms of ATP/ ADP ratio. Under stressful conditions, the ROS produced due to oxidative stress requires more energy in the form of ATP to maintain the cellular homeostasis such as ion compartmentation, osmolytes synthesis, etc. The ATP requirement for these processes is different, such as 3.5, 41 and 50 ATP molecules are required for the synthesis or accumulation of one molecule of Na+, proline, and glycine betaine, respectively (Raven 1985). Thus, the energetic status of the plant is also dependent on the type of molecule synthesized or accumulated by the plant. It is possible that halophytes evolved to survive under abiotic stress conditions through proper maintenance of a higher redox and energetic status which could have conferred a plasticity to grow under stress. Not much information is available on the redox signaling and energetics in case of the halophytes. Recently, Lokhande et al. (2010c) for the first time demonstrated that maintenance of redox and energy state plays a major role in mediating salinity tolerance and in achieving a balance between tolerance and growth in Sesuvium portulacastrum. The plants under optimum levels of NaCl (250 mM) showed retention in the growth whereas significantly toxic levels of NaCl (1,000 mM) disturbed the homeostasis of the plant due to abrupt changes in the ratios of redox-active compounds (ASC/DHA, GSH/GSSG, NADP/ NADPH) and energy molecules (ATP/ADP) (Fig. 2.2a–f). Further to stick this work, more efforts could be initiated to unravel the redox and energetic of the halophytes to gain the knowledge of redox control. The concept of a cellular redox

state, as proposed by Foyer and Noctor (2005), is very useful in terms of elucidating the importance of redox reactions in gene expression, metabolism control, signal transduction, and cellular defense. The concept must now be developed to identify and quantify those redox-active compounds that are the specific regulators of cellular responses (Potters et al. 2010).

4.6

Genomic Approaches

Plant adaptation to environmental stresses is controlled by a cascade of molecular networks. In this regard, the application of genomic technologies has made more impact on understanding the plant responses to the abiotic stresses (Cushman 2003). The technology has made remarkable success in understanding the abiotic stress tolerance at genome level with potential to modify plants’ tolerance for increasing yield under stressful conditions (Bohnert et al. 2006). In contrast to traditional breeding and marker-assisted selection programs, the direct introduction of a small number of genes by genetic engineering has also become tangible and attractive as a rapid approach to improve the plants’ stress tolerance (Cushman and Bohnert 2000; Popova et al. 2008) to re-establish homeostasis and to protect and repair damaged proteins and membranes (Wang et al. 2003). In the course of studies on mechanism of abiotic stress tolerance, Arabidopsis thaliana has emerged as an excellent model system (Zhu 2001) because most of the crop plants are glycophytes. However, study of some novel mechanisms unique to halophytes or stress-tolerant plants may be difficult with Arabidopsis and this has been made possible by the available genome information on Mesembryanthemum crystallinum, which, when compared with the Arabidopsis genome, seems to contain a number of transcripts that have no counterparts (Wang et al. 2004b). Thereafter, several halophytes such as M. crystallinum, Suaeda species, Atriplex species have been employed to dissect out the molecular basis of stress tolerance mechanism of the halophytes. Recently, Thellungiella halophila (salt cress), a member of the Brassicaceae, has emerged as a

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

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Fig. 2.2 The salinity stress responses (growth, redox, and energy status) of Sesuvium portulacastrum exposed to optimum (250 mM) and supra-optimal (1,000 mM) concentrations of NaCl

model for understanding adaptation of the halophytes to abiotic stress tolerance due to its hom*ology with the glycophyte model, A. thaliana (Wang et al. 2004b; Amtmann 2009). This halophyte has the ability to grow in high salt concentrations which otherwise become inhibitory for the growth of its salt-sensitive relative A. thaliana

and other crop plants (Zhu 2001; Nah et al. 2009). The salient features of T. halophila such as small diploid genome (240 Mb and 2n = 14), short and self-fertile life cycle and ease of floral dipping method of transformation have enabled it as a successful candidate for molecular detailing of its response to abiotic stress tolerance and relative

42

comparison with A. thaliana. The comparative genomics of T. halophila and A. thaliana revealed extensive and novel information on presence of differential genes responsible for abiotic stress tolerance in T. halophila in comparison to A. thaliana (Nah et al. 2009). Taji et al. (2004) studied the differences in the regulation of salt tolerance between salt cress and Arabidopsis by analyzing the gene expression profiles using a full-length Arabidopsis cDNA microarray. Only a few genes were induced by 250 mM NaCl in salt cress stress compared to Arabidopsis. Even in the absence of stress, a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, b-glucosidase, and SOS1, were expressed at high levels. The study also found salt cress to be more tolerant to oxidative stress than Arabidopsis. The salt tolerance mechanisms between salt-sensitive glycophytes and salt-tolerant halophytes could result from alterations in the regulation of the same basic set of genes involved in salt tolerance among these plants. Kant et al. (2006) used gene-specific primers of Arabidopsis that showed similar realtime PCR amplification efficiencies with both A. thaliana and T. halophila cDNA and concluded that the expression of specific salt tolerance orthologues differs between unstressed and stressed plants of both species. The development of expressed sequence tags (ESTs) and cDNA libraries using various genomic approaches such as suppressive subtractive hybridization (SSH), differential display reverse transcription-polymerase chain reaction (DDRTPCR), representational difference analysis (RDA), serial analysis of gene expression (SAGE), and cDNA microarray (Breyne and Zabeau 2001) provided an enormous databases for understanding the genetic network involved in abiotic stress tolerance mechanism of halophytes (Wang et al. 2004b; Kore-eda et al. 2004; Popova et al. 2008). Using this approach, various genes responsible for stress tolerance have been isolated from halophytes and cloned or overexpressed in the bacterial systems as well as sensitive cultivars of glycophytes to enhance the stress tolerance capacity and improve crop yield (Table 2.3). Transcript-profiling experiments in Arabidopsis in response to drought, cold, or salinity

V.H. Lokhande and P. Suprasanna

stresses using the Arabidopsis GeneChip array or full-length cDNA microarrays have shown that extensive changes occur in the transcriptome of Arabidopsis (Fowler et al. 1999; Kreps et al. 2002; Seki et al. 2002). It is known that approximately 30% of the transcriptome on the Arabidopsis GeneChip 8 K oligoarray changed in stress treatments (Kreps et al. 2002). The expressed sequence tag analyses of Thellungiella clones revealed 90–95% identities between Thellungiella and Arabidopsis cDNA sequences (Wang et al. 2004a, b; Wong et al. 2006). In a comparison of three stresses (cold, low water availability, and saline conditions) as well as recovery from water deficits in Thellungiella, Wang et al. (2006) employed an expression profiling strategy to identify stress responses. There was not much degree of overlap among genes responsive to drought, cold, or salinity suggesting relatively few common end responses triggered by these stresses existed in this halophyte. While Thellungiella had shown activation of the expression of some well-known stress-responsive genes, it was found to downregulate a large number of biotic stress-related genes under drought and salinity treatments. The study has made a significant step in showing the emergence of Thellungiella as a model species for the molecular elucidation of abiotic stress tolerance, and that Thellungiella responds precisely to environmental stresses, thereby conserving energy and resources and maximizing its survival potential. SOS1 (Salt Overly Sensitive 1) is known to play key role in the ion homeostasis mechanism movement (Shi et al. 2000). Although SOS1 has been intensely studied in Arabidopsis, its involvement in the salt tolerance of halophytes is not much known. Oh et al. (2009) investigated the role(s) by which ThSOS1, the SOS1 hom*olog in Thellungiella, was involved in modulating the halophytic character using ectopic expression of the gene in yeast and in Arabidopsis and Thellungiella SOS1-RNA interference (RNAi) lines. The knockdown of SOS1 expression totally altered Thellungiella into a salt-sensitive plant like Arabidopsis. The authors found that the activity of ThSOS1 could limit Na+ accumulation and the distribution of Na+ ions.

Peroxiredoxin Q gene SsPrx Q

Suaeda salsa

Thellungiella halophila

IMT1, myo-Inositol O-methyl-transferase FLC gene

Controls vernalization response pathway Thioredoxin-dependent peroxidase activity

Ascorbate regeneration and ROS scavenging Strongly expressed in roots than in leaves and stems under abiotic stresses Inositol methylation

Monodehydroascorbate reductase (MDHAR) Fructose-1,6-bisphosphate aldolase SpFBA

Betaine aldehyde dehydrogenase AcBADH Choline monooxygenase CMO

Trait improved Eightfold higher activity of the vacuolar-type Na+/H+ antiporter Increased osmotic adjustment and MDA content Compartmentalize more Na+ in roots and keep a relative high K+/Na+ ratio in the leaves Improved synthesis of glycine betaine Three- to sixfold increased activity of CMO increases glycine betaine synthesis

Genes Vacuolar Na+/K+ antiporters AgNHX1 Vacuolar Na+/K+ antiporters MsNHX1 Vacuolar Na+/K+ antiporters AlNHX1

Mesembryanthemum crystallinum

Sesuvium portulacastrum

Avicennia marina

Suaeda liaotungensis, Beta vulgaris, Atriplex hortensis, A. nummularia

Atriplex centralasiatica

Aeluropus littoralis

Medicago sativa

Source organism Atriplex gmelini

Table 2.3 Source of genes from halophytes for the improvement of abiotic stress tolerance

E. coli

T. halophila

E. coli

Escherichia coli

N. tabacum

N. tabacum

Guo et al. (2004)

Fang et al. (2006)

Rammesmayer et al. (1995)

Fan et al. (2009)

Russell et al. (1998), Shen et al. (2001), Tabuchi et al. (2005), and Li et al. (2003, 2007) Kavitha et al. (2010)

Yin et al. (2002)

Zhang et al. (2008)

N. tabacum

N. tabacum

Bao-Yan et al. (2002)

References Ohta et al. (2002)

A. thaliana

Target organism Oryza sativa

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance 43

V.H. Lokhande and P. Suprasanna

44

5

Role of Halophytes in Abiotic Stress Management

5.1

Desalination and Stabilization of Saline Soils

The problem of salinity is widespread covering at least 75 countries (Goudie 1990). Various physical, chemical, and biological approaches have been developed for the reclamation of such saline soils (Shahid 2002). Biological methods include organic manure, crop rotation, salt-tolerant crops (Shahid 2002), as well as vegetative bioreclamation (Qadir and Oster 2004). The reclamation of saline soil using such biological means is also referred as desalinization (Zhao 1991), biodesalination, and desalination of salt-affected soils by halophytes (Rabhi et al. 2009). The potential of plants to accumulate enormous salt quantities depends often on the capacity of their aboveground biomass (hyper-accumulating plants) (Rabhi et al. 2010). This ability could be significant particularly in the arid and semi-arid regions where insufficient precipitations and inappropriate irrigation systems are unable to reduce the salt burden in the rhizosphere of plants and suitable physicochemical methods are too expensive (Shahid 2002). The plant-based method of saline soil stabilization is of importance especially in several developing countries where chemical amendments are getting more and more expensive. In this regard, the use of Na+ and Cl− hyperaccumulating plants for soil desalination is often suggested as a strategy (Ravindran et al. 2007). A large number of species has been utilized for soil desalination based on their suitability and capacity to accumulate the salt. Halophytes are one of the important categories of plant species extensively used for this purpose with rice as the only one glycophytic exception (Iwasaki 1987). In order to be useful for desalination purpose, the plant species to be used should have high salt resistance, high biomass production, considerable shoot sodium content, and high degree of economic utilization (such as fodder, fuel, fiber, essential oil, and oil seeds) (Rabhi et al. 2010). Shoot-succulent halophytes such as Sesuvium

portulacastrum and Suaeda sps. meet these criteria since they are able to accumulate enormous Na+ quantities within their above-ground organs. They can become useful candidates for the desalination of salt-affected soils under nonleaching conditions. Zhao (1991) calculated that Suaeda salsa produced about 20 ton DW ha−1 and withdraw 3–4 ton NaCl. Ravindran et al. (2007) estimated that Suaeda, Sesuvium, Excoecaria, Clerodendron, Ipomoea, and Heliotropium species could remove 504, 474, 396, 360, 325, and 301 kg NaCl, respectively, from 1 ha land in 4 months. Selection of suited species is the first step for affordable soil desalination at a wider scale in the arid and semi-arid regions (Rabhi et al. 2009). In a case study, a significant decrease in electrical conductivity of the soil having a 50% saturation percentage was recorded from 33 to 20 dS m−1 in the presence of single growth cycle of J. rigidus in Egypt (Zahran and Wahid 1982). Suaeda salsa showed its potential to reduce the soil Na+ content at depth 0–10 cm by 2.4 with a density of 15 plants m−2 and by 3.8 with a density of 30 plants m−2 (Zhao 1991). It has also been demonstrated that the growth of annual glycophytes (Medicago ssp.) was much better on the soil previously desalinated with perennial halophytes in saline ecosystems (Abdelly et al. 1995). It was concluded that perennial halophytes desalinize and fertilize the rhizosphere, offering a favorable microhabitat for a better growth of annual glycophytes. Ravindran et al. (2007) evaluated the capacity of six halophytic species (Suaeda maritima, Sesuvium portulacastrum, Clerodendron inerme, Ipomoea pes-caprae, Heliotropium curassavicum and one tree species Excoecaria agallocha) to desalinize the upper 40 cm of soil under field conditions in India. This study demonstrated that after 120 days of cultivation of the halophytes, Suaeda maritima and Sesuvium portulacastrum showed a decrease in electrical conductivity of saline soil from 4.9 to 1.4 and 2.5 dS m−1, respectively. The potential of native halophytes Arthrocnemum indicum and Suaeda fruticosa to desalinize saline soils was compared with that of an introduced halophyte, S. portulacastrum. In this study, Rabhi et al. (2009) confirmed S. portulacastrum as the most suitable

2 Prospects of Halophytes in Understanding and Managing Abiotic Stress Tolerance

plant with higher accumulation of Na+ in its shoot parts for desalination purpose in arid and semiarid regions where precipitation is too low to leach salts from rhizosphere. Similarly, successful germination and growth of Hordeum vulgare (barley) was observed on the soil desalinated with salt accumulator halophyte S. portulacastrum (Rabhi et al. 2010). Taken together, the reports suggest that salt accumulator halophytes can be exploited as a potential source for desalination of agricultural land in the arid and semi-arid regions as well as for the stabilization of saline lands along the coastal regions of the world.

5.2

Phytoremediation

Contamination of agricultural soil by heavy metals (such as Cu, Cd, Zn, Mn, Fe, Pb, Hg, As, Cr, Se, Ur, etc.) has become a serious environmental concern due to their potential impact on the ecosystems. Such toxic elements are considered as soil and water pollutants due to their widespread occurrence, and their acute and chronic toxic effect on plants grown in such soils as well as on humans living in their surrounding (Yadav 2010). Plants, as sessile organisms have developed diverse detoxification mechanisms against absorbing a diversity of natural and man-made toxic compounds. Pollutant-degrading enzymes in plants are a natural defense system against a variety of allelochemicals released by competing organisms, including microbes, insects and other plants. Therefore, plants act as natural, solarpowered pump-and-treat systems for cleaning up contaminated environments, leading to the concept of phytoremediation (Aken 2008). A variety of plant systems have been studied for phytoremediation practices of contaminated soil; however, each species has limitations to accumulate the toxic metals and detoxify to nontoxic compounds through the enzymatic actions. In the course of evolution from marine to freshwater habitat, halophytes are found most successful group of plants which have shown adaptations to a variety of abiotic stresses, tolerance to heavy metal stress is one of these. In recent years, more emphasis has been placed to remove the toxic

45

metals from contaminated soil and water bodies and reclamation of such lands for sustainable agriculture. In this regard, extensive research is undertaken to exploit the use of metal hyperaccumulating plants and search for a suitable plants that can significantly accumulate heavy metals and metalloids (Zabłudowska et al. 2009). However, phytoremediation constitutes a group of strategies meant not only to reduce the metal load at the contaminated site but also to stabilize the site. These strategies are referred as “phytoextraction” or “phytostabilization” and the selection of a plant may depend on the level of contamination at the site of concern. Both strategies can be integrated into operation at highly contaminated mine sites with a plant that may not be a hyper-accumulator but can tolerate even very high concentrations of toxic metals (Lokhande et al. 2011b). Various halophytes have evolved distinct morphological specializations for dealing with abiotic stressed environments such as presence of “aerial stilts” in the members of families Rhizophoraceae and “pneumatophores” in the members of Avicennaceae and Sonneratiaceae which enable gaseous exchange and oxygenation for respiration in an anoxic environment (Hutchings and Saenger 1987); however, the members of Myrsinaceae, possess no aerial roots. Table 2.4 presents phytoremediation potential of some halophytes. Numerous laboratory-based trials suggested that the concentrations of metals required to show significant negative effects on halophytes may be significantly higher when compared to their aquatic and terrestrial floral counterparts (MacFarlane et al. 2007). For example, there were no adverse effects on the growth of Rhizophora mucronata and Avicennia alba seedlings treated with Zn (10–500 mg ml−1) and Pb (50–250 mg ml−1). In Kandelia candel seedlings, only at the highest applied metal concentrations (400 mg kg−1 Cu and Zn) inhibition of leaf and root development was observed (Chiu et al. 1995). Similarly, Pb (0–800 mg g−1) had little negative effect on Avicennia marina seedlings (MacFarlane and Burchett 2002). Studies have demonstrated the accumulation of metals (Cu, Zn, Pb, Fe, Mn, and Cd) predominantly in root

V.H. Lokhande and P. Suprasanna

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Table 2.4 Examples of halophytic plant species used for the purpose of phytoremediation Plant species Sesuvium portulacastrum Mesembryanthemum crystallinum Halimione portulacoides, Spartina maritima Arthrocnemum macrostachyum, Spartina argentinensis Triglochin maritima, Juncus maritimus, Sarcocornia perennis, Halimione portulacoides Atriplex halimus subsp. Schweinfurthii A. halimus Spartina densiflora, S. maritima Aster tripolium Sarcocornia perennis Halimione portulacoides Tamarix smyrnensis Juncus maritimus Sporobolus virginicus, Spartina patens, and Atriplex nammularia Salicornia europaea

Phytostabilization/phytoextraction/ phytoexcretion of heavy metals Cd, Pb and As Cd

References Ghnaya et al. (2007), Nouairi et al. (2006), Zaier et al. (2010a, b), and Lokhande et al. (2011b) Ghnaya et al. (2007) and Nouairi et al. (2006)

Cd, Cu, Pb, and Zn

Reboreda and Caçador (2007, 2008)

Cd and Cr

Redondo-Gómez et al. (2010a, b)

Hg

Castro et al. (2009)

Cd

Nedjimi and Daoud (2009) and Lefevre et al. (2009) Manousaki and Kalogerakis (2009) Cambrolle et al. (2008)

Pb and Cd As, Cu, Fe, Mn, Pb, and Zn Cu and Pb Fe, Mn, and Hg Zn, Pb, Co, Cd, Ni, and Cu Pb and Cd Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn Zn, Cu, and Ni

Cd

tissue, rather than in foliage, in numerous mangrove species grown in the field conditions, such as Avicennia sps., Rhizophora sps. and Kandelia sps. (Peters et al. 1997). It has also been observed that for some mangroves, concentrations of translocated metals are low, with bio-concentration factors (BCF; ratio of leaf metal to corresponding sediment metal concentration) ranging from 25 dS m−1 and produce 0.5–5 ton of edible dry matter year−1. The potentials of five halophytic plant species namely Diplachne fusca, Spartina patens, Sporobolus virginicus (Smyrna-smooth), Sporobolus virginicus (Dixe-coarse), and Medicago sativa have been studied as a source of forage plants in Egypt on the soils irrigated with different concentrations of seawater. The studies showed that S. virginicus (Dixe) produced the highest biomass upon irrigated with either 25 or 37.5% sea water, followed by S. patens and D. fusca whereas S. virginicus showed the lowest yield. The studies conducted on cultivation of Salvadora persica in semi-arid saline and alkali soils showed its efficiency for growth and as a source of industrial oil on both saline and alkali soils for economic and ecological benefits which is otherwise not suitable for conventional arable farming (Reddy et al. 2008). Therefore, considering the approach of biosaline agriculture more efforts should have been undertaken on the cultivation of nonarable lands with nonconventional plant resources such as halophytes to bring the uncultivable land for the use of economic purposes of the human being. This strategy will help to improve the gross economy of the developing countries.

6

Conclusions and Future Perspectives

Worldwide food production is affected to a large extent by environmental extremities and the sensitivity of crops is a major limitation for achieving higher plant productivity. Plants have evolved adoptive mechanisms which can be understood and exploited as an important resource for development of crops tolerant to extremities. Halophytes show a diversity of growth responses to increasing salinity. Halophytes have evolved to the changing environmental conditions through developing

49

variety of tolerance mechanisms such as halosucculence, ion compartmentation (exclusion/inclusion), osmoregulation, enzymatic and nonenzymatic antioxidants and maintenance of redox and energy status. Research on understanding the abiotic stress tolerance mechanism of halophytes has been on the upfront using wide array of physiological, biochemical, and molecular tools. Some of the halophytes (e.g., Thellungiella halophila) that tolerate adverse conditions have become the choice model systems for unraveling the different pathways associated with halophytic behavior. However, research in the context of progress in metabolomics, genomics, and proteomics has to be initiated in diverse halophytic species with the use of advanced techniques to gain detailed knowledge of abiotic stress tolerance. Halophytes have also been utilized practically for managing the stressful environment and shown to be involved in increasing the economy of developing countries in many parts of the world. Halophytes have shown their role in desalination of saline lands from arid and semi-arid regions as well as the stabilization of saline lands along the coastal sides, phytoremediation of heavy metal contaminated sites and wetland restoration and revegetation through introduction of variety of halophyte species. This has led to developing agriculture on saline lands for supporting the sources of food, forage, fodder, medicine, ornamental and important plant-based chemicals to ever-growing human population. In addition, this may also help in reducing the burden on the crop plants which are facing the productivity problems due to exposure to various abiotic stresses. Considering these applications, constructive strategies have to be developed and implemented for the protection of world’s nonrenewable resources, wherein available halophyte diversity can be utilized as an important source. Investigation of the gene regulation and the balance of individual stress tolerance mechanisms will aid in translating the information to other salt-sensitive crops. Such studies will be helpful for ensuring sustainability of future research efforts to improve and manage crop performance on marginal and irrigated land through genetic manipulation.

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UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants Fernando E. Prado, Mariana Rosa, Carolina Prado, Griselda Podazza, Roque Interdonato, Juan A. González, and Mirna Hilal

Abstract

The UV-B is an important component of solar radiation to which all terrestrial and aquatic plants were exposed during the early evolutionary phase of the Earth. Hence the plants, principally terrestrial, have evolved different mechanisms to avoid and repair the UV-B damage; therefore, it is not surprising that photomorphogenic responses to the solar UV-B are erroneously assumed to be adaptations to the harmful UV radiation. The responses to UV-B enhancement include changes in the leaf area, leaf thickness, stomatal density, wax deposition, stem elongation, and branching pattern, as well as in the synthesis of secondary metabolites, alterations in plant–pathogen and plant–predator interactions, and in gene expression. However, under field conditions the ambient solar UV-B provides an important signal for the normal plant development and may be perceived by the plants through nondestructive processes involving both UV-B specific and UV-B nonspecific signaling pathways. The specific signaling pathways include the components UVR8 and COP1 which regulate the expression of a set of genes that are essential for the plants’ protection. The nonspecific signaling pathways involve DNA damage, reactive oxygen species (ROS), hormones, and wound/defense signaling molecules. Indeed under the field conditions, the ambient UV-B might more properly be viewed as a photomorphogenic signal than as a stressor. Therefore, it might not be appropriate to evaluate the adaptive roles of plant responses to UV-B cues upon stress tolerance by the simultaneous application of both solar radiation and supplemental UV-B. In this chapter, we analyzed the information regarding F.E. Prado () • M. Rosa • C. Prado • R. Interdonato • M. Hilal Cátedra de Fisiología Vegetal, Facultad de Ciencias Naturales e IML, Miguel Lillo 205, CP 4000 Tucumán, Argentina e-mail: [emailprotected] G. Podazza • J.A. González Instituto de Ecología, Fundación Miguel Lillo, Miguel Lillo 251, CP 4000 Tucumán, Argentina P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_3, © Springer Science+Business Media, LLC 2012

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physiological and morphogenic responses of the terrestrial plants to the UV-B radiation, as well as the events related to UV-B perception, signal transduction, gene expression, and ROS formation from different studies carried out in greenhouses, growth chambers, and field conditions. Keywords

UV-B radiation • DNA damage • DNA repair • Metabolites • Signaling • Secondary metabolites • Morphogenic responses

1

Introduction: Knowing the Solar UV-B Radiation, a Historical Background

Within the electromagnetic radiation spectrum, the UV radiation describes a spectral range between 200 and 400 nm, which borders on the visible range. The UV radiation is divided into three effective types: UV-A (320–400 nm), UV-B (280–320 nm), and UV-C (200–280 nm). Less than 7% of the sun’s radiation reaching the Earth’s surface falls approximately in the range between 295 and 400 nm (UV-A and UV-B); the shorter UV wavelengths get filtered out by the stratospheric ozone. Therefore, the lower limit of shorter wavelengths of the solar UV radiation reaching the surface is determined by the stratospheric ozone layer. The stratospheric ozone absorbs virtually all the UV radiation ranging approximately 295 nm and lesser. Although the stratospheric ozone determines the amount of UV-B radiation that reaches the surface of the Earth, its level is significantly affected by variations in latitude and altitude. The level of UV-B radiation over tropical latitudes is higher than in temperate regions due to lesser atmospheric UV-B absorption determined by the solar angle and the ozone layer itself which is thinner in equatorial regions. Thus, the UV-B radiation is relatively high in tropical areas and relatively low in the polar regions. Increases of the UV-B irradiance with increasing elevation above sea level is also known, i.e., measurements of the UV-B irradiance show an average increase between 10 and 19% for every 1,000 m increase in elevation. Besides geographical factors, the atmospheric

pollutants (e.g., smoke, aerosols) and especially the weather factors (e.g., clouds, haze) greatly decrease the level of UV-B reaching the Earth’s surface. Depending upon the type and height of clouds, liquid water content, and particle distribution, the cover of clouds can attenuate over 70% of the incident UV-B radiation (McKenzie et al. 2007). Over 35 years ago, it was warned that manmade nitrous oxide and chlorine-containing compounds (e.g., chlorofluorocarbons, CFCs) produce the breakdown of large amounts of ozone in the stratosphere (Crutzen 1972; Molina and Rowland 1974; in Velders et al. 2007). This fact causes the depletion of the stratospheric ozone layer increasing the UV-B radiation at the ground level, especially in Antarctic and Arctic regions as well as in high altitude areas (Ryan and Hunt 2005). By their contributions on chlorine-containing compounds and the depletion of the ozone layer, Crutzen, Molina, and Rowland were awarded with the Nobel Prize for chemistry in 1995. After the Molina and Rowland’s work, Farman, Gardiner, and Shanklin, scientists of the British Antarctic Survey, shocked the scientific community during the early middle 1980s by publishing the results of a study showing a springtime ozone hole in the Antarctic ozone layer (Farman et al. 1985, in Ryan and Hunt 2005). This fact rang alarm bells worldwide and within the same year, 20 nations including most of the major CFCs producers signed the Vienna Convention, which established a framework for negotiating an international regulation on the ozone-depleting substances. After that, on September 16, 1987 the Montreal Protocol on “Substances that Deplete the Ozone Layer” was opened for nations signature

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

and entered into force on January 1, 1989 (Velders et al. 2007). At present, after several amendments, all the countries in the United Nations have ratified the Montreal Protocol (http://ozone.unep.org/ Meeting_Documents/). However, today the ozone depletion is a global phenomenon and according to the European Ozone Research Coordinating Unit (EORCU) its amount approximately reaches 0.6% per year. The level of UV-B radiation in the biosphere varies, spatially and temporally, quite considerably but the depletion of the stratospheric ozone strongly affects its penetration (Ryan and Hunt 2005). Thus, despite reductions in the production and use of ozone-depleting chemicals, the potential of ozone depletion by anthropogenic emissions or natural causes (e.g., volcanoes) still remains. In this scenario, the level of stratospheric ozone will continue decaying with a severe decline occurring between 2010 and 2019 in the northern hemisphere that may result up to 50–60% increase in the springtime UV-B radiation according to the Global Climate Model (GCM) that is based on the simplified ozone-depletion chemistry (Taalas et al. 2000). Furthermore, the recovery of the stratospheric ozone to early 1980s levels is not predicted until roughly 2050.

2

Solar UV-B Radiation and the Life of Terrestrial Plants

Since the discovery of the ozone layer depletion (~30 years ago) the responses of microorganisms, animals, and terrestrial plants to solar UV-B radiation have been active subjects of many studies (Rozema 2000; Björn et al. 2002; Ryan and Hunt 2005). Prior to building of the atmospheric oxygen and then the stratospheric ozone layer, the UV-C radiation and high levels of both UV-B and UV-A would probably have reached the Earth’s surface relatively unattenuated affecting all the living organisms. In this context, the UV radiation seems to be a ubiquitous factor in the course of terrestrial biota? Plant evolution from the early Archean era began as solitary photosynthetic cells (co*ckell and Horneck 2001). The UV effects on terrestrial plants, which are principally detrimental, have been demonstrated

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with some of the most essential components of the biochemical machinery, i.e., DNA molecule and photosystem II (PSII) (Singh et al. 2008). However, when the stratospheric ozone layer developed, the UV-A radiation and a minor portion of the UV-B wavelengths only could reach the Earth’s surface due to the atmospheric absorption and scattering of the UV-C radiation. Therefore, how the UV radiation has altered the Earth’s environment over geological time periods is essential for understanding the evolutionary history of the earth and also to understand how the UV-B radiation has contributed as selection pressure on the development of terrestrial plants (Björn and McKenzie 2007). In fact, Sagan (Sagan 1973 in Singh et al. 2008) first considered the UV radiation as a selection pressure on the early photosynthetic organisms, when our knowledge on biological effects of the UV radiation on plants was in its infancy. During the evolutionary history of the Earth, the terrestrial plants coevolved under different solar UV-B levels and may have experienced significantly higher UV-B irradiances than the current surface UV-B level (co*ckell and Horneck 2001; Rozema et al. 2002). Thereby, the UV-B tolerance acquired earlier probably helps to explain why plants are distributed at lower latitudes or higher elevations, where UV-B irradiances are greater, are less sensitive to high levels of the UV-B radiation than those at higher latitudes and/or lower elevations (Turunen and Latola 2005; Ren et al. 2010). The present rate of atmospheric changes is so rapid that evolution may not keep up with it, particularly in long-living plants like trees. The UV-B environment of terrestrial plants is presently quite variable in both time and space, and thus organisms experience different UV-B doses and adapt to UV-B radiation at different levels (Rozema 2000). In this context it is expected that terrestrial plants respond differentially to increasing solar UV-B. Nevertheless, although studies assessing possible consequences of the ozone depletion have greatly increased our understanding on how living organisms are affected by the UV-B radiation, the focus of these researches may also have distracted the attention from the UV-B radiation as a component of the ambient light environment involved in the evolution of life on the Earth’s surface (co*ckell and Horneck 2001).

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3

UV-B Radiation as a Modulator of the Plant Function

Due to their absolute sunlight requirement for survival, the plants are inevitably exposed to solar UV-B radiation. However, from equatorial to the polar regions and from the sea level to high mountains, the terrestrial plants are exposed to greatly different UV-B irradiances, given the geographical differences in UV-B irradiances is much greater than corresponding differences in the total solar radiation (Rozema 2000). The plant chemical photoprocesses respond differently to different UV wavelengths as the biological damage exacerbated as wavelength becomes shorter. Thus, the relative effectiveness of UV-B-ranging wavelengths (effective UV-B irradiance) must be known in order to assess the responses to ozone changes. The effective UV irradiance (E) or dose rate exposure is given by E = ∫ F(λ)W(λ)dλ, where W(l) is the weighting function (action spectrum) for a specific biological or chemical effect and F(l) is the spectral irradiance, either computed or measured, for a given time (e.g., hour, day, year) and location. As a result, the biological effectiveness of the weighted UV-B irradiances (UV-BBE, biologically effective UV-B radiation) related to different action spectra has different responses to atmospheric ozone changes (Flint and Caldwell 2003). It has been estimated that 1% decrease in the stratospheric ozone concentration would result in nearly 2% increase in the UV-BBE at mid-latitudes. Therefore, the recently projected 15% stratospheric ozone reduction would result in up to 30% increase in the value of UV-BBE in the next three decades (McKenzie et al. 2007).

4

Solar UV-B Radiation: Stress Factor or Beneficial Signal Factor?

From the ozone depletion perspective, the UV-B radiation is considered as an environmental stressor of photosynthetic organisms (Jordan 2002;

Rozema et al. 2002; Ballaré 2003; Caldwell et al. 2007). However, from an evolutionary perspective this assumption is questionable. The terrestrial plants have always developed under the solar UV-B and then their genetic machinery coevolved together with the ambient UV-B level. Therefore, it can be hypothesized that the metabolic machinery of plants contains all the necessary elements for a normal coexistence with the current UV-B level and so the solar UV-B radiation should not be considered as an environmental stress factor. In fact, the current level of the ambient UV-B radiation should be considered as a signal factor that induces the expression of genes related to the normal plant development (Jenkins 2009). While the UV-B exclusion must be considered as an anomalous signal factor that induces the expression and/or repression of another set of genes (Brosché et al. 2002; Stratmann 2003; Hectors et al. 2007). In this context, the solar UV-B radiation appears as a reliable plant effector, but it is not always possible to identify a unique particular reason as explanation of the underlying UV-B effects. In nature, the terrestrial plants are seldom affected by only a single environmental factor; they typically respond to several environmental factors acting in concert (Bruno et al. 2003). Therefore, the influence of changing UV-B in natural ecosystems must be evaluated considering two opposite processes: (a) facilitation; (b) competition. These processes have been recognized as key drivers in a wide range of natural communities and hence, effectiveness of the UV-B radiation will be greatly modified by other environmental factors, in some cases aggravating and in others, ameliorating the overall UV-B effect (Bruno et al. 2003).

5

Responses of Terrestrial Plants to Ambient Solar UV-B

The most extended researches relating to the effects of increasing solar UV-B radiation on terrestrial plants have been performed in both austral and boreal polar regions (Day et al. 2001; Phoenix et al. 2003; Robson et al. 2003; Rozema et al. 2006; Newsham and Robinson 2009). The depletion of the stratospheric ozone is greater in both

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Antarctic and Arctic regions than in other nonpolar latitudes where it is less pronounced and subject to other atmospheric factors such as horizontal and vertical ozone transport. In the Antarctic zone, the complete breakdown of the stratospheric ozone occurs only during few springtime days, but the springtime ozone depletion reaches 50–60% on average (Rozema et al. 2005). This event has occurred uninterruptedly for at least 30 years, leading to a marked increase of the solar UV-B irradiance. Since the 1990s frequent occurrence of the springtime ozone hole over the Arctic also occurs resulting in significant ozone depletion and increasing the UV-B irradiance at the ground level (Rex et al. 2004). Similar to the Antarctic area, the ozone loss over the Arctic area is higher in the early springtime than in the growing plant season (late springtime and summer). The Arctic springtime ozone depletion is lower than the Antarctic one and rarely reaches 40–50% on average (Rex et al. 2004). Also, the Arctic ozone loss is extremely sensitive to frequency of sudden stratospheric warming due to the greenhouse effect. Because of the influence of increasing greenhouse gases, the ozone holes may worsen leading to greater ozone depletion over the Arctic and increasing the severity and duration of the Antarctic ozone depletion (Rozema et al. 2005). At present both Antarctic and Arctic polar regions represent one the most extreme UV-B environment and constitute an excellent site to study the responses of terrestrial plants to increased solar UV-B. Although almost all the investigations were carried out in the Antarctic area this ecosystem only has two species of higher plants: Deschampsia antarctica and Colobanthus quitensis (Convey and Smith 2006). While the terrestrial Arctic ecosystem has more than 160 higher plant species, allowing more species interactions and feedbacks and perhaps providing a more general representative ecosystem response to enhanced UV-B than the more simple two-species Antarctic ecosystem (Rozema et al. 2006). Short- and longterm studies have shown different and controversial effects of both enhanced and excluded solar UV-B radiation on the Antarctic and Arctic flora species (Searles et al. 2002; Phoenix et al. 2003; Robson et al. 2003). However, in three extensive overviews, Dormann and Woodin (2002) and

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Rozema et al. (2005, 2006) claimed the finding that neither flowering plants nor mosses and lichen species of the polar ecosystems are markedly affected by the enhanced solar UV-B. Almost all the plant parameters related to the growth and photosynthesis were not significantly affected by elevated UV-B simulating 15, 30, or may be higher (e.g., 50%) of the ozone depletion (Rozema et al. 2005). In fact, these overviews contradict many authors who hypothesized that stressful harsh climatic and environmental polar conditions would make the polar plants vulnerable to the enhanced UV-B, and that the repair of UV-Binduced damage could be hampered by the low polar temperatures (Newsham and Robinson 2009; Snell et al. 2009). The absence of significant UV-B effects on polar plants could imply that they are better adapted to high UV-B regimes and capable of preventing and/or effectively repairing the UV-B damage (Rozema et al. 2005). Although this fact may be interpreted that terrestrial plants from polar ecosystems are particularly tolerant to the ozone depletion, in a more generalized way it has been applied to all the plants and ecosystems, especially those located in high UV-B environments (e.g., tropical and subtropical mountain areas). This assumption implies that terrestrial plants occurring naturally in the high UV-B habitats would undoubtedly have evolved specific adaptations that protect them against the deleterious effects of the UV-B radiation. Hence such plants could show a reduced responsiveness mainly due to their reduced sensitivity to UV-B radiation. Similarly, the plants growing in habitats with low UV-B irradiances (e.g., forest underground) could suffer changes even under small variations in the stratospheric ozone layer (Turunen and Latola 2005). The solar UV-B radiation cannot be regarded as merely an environmental factor causing plant damages because it can also act as an informational signal leading to morphogenic effects on the structure of plants and the overall function of forest ecosystems (Julkunen-Tiitto et al. 2005). For many years both field and laboratory experiments have focused on the UV-B increased scenario, being scarce those on the responses of plants to the current level of solar UV-B (Searles et al. 2001). This lack of information constitutes

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an important gap that impedes us to understand the responses of terrestrial plants to solar UV-B changes completely. Many reports consider the solar UV-B as an environmental stressor that affects the development of plants (Láposi et al. 2002; Kadur et al. 2007), while others have communicated no detrimental effects of the solar UV-B on the plant growth (Amudha et al. 2010). Some have even reported protective and/ or beneficial effects of the solar UV-B radiation (Winter and Rostás 2008). Although there is no conclusive explanation for these contradictory effects, they could obey to variations in the UV-B sensitivity among different species and even among cultivars of the same species (Gilbert et al. 2009; González et al. 2009). In this context, the terrestrial plants have developed different strategies to avoid UV-B radiation reaching the most sensitive cellular targets. A major strategy against penetration of the solar UV-B is based on epidermal screening of the incident radiation (Tattini et al. 2005). The mechanisms that inhibit the penetration of UV-B radiation inside the leaf tissues comprise different leaf structural features such as leaf surface reflectance due to the leaf surface wax and hairs (trichomes) (Liakopoulos et al. 2006; González et al. 2007), epidermal thickness (Hilal et al. 2004), epidermal terpenoids (resin) accumulation (Zavala and Ravetta 2002), and epidermal accumulation of UV-absorbing compounds (Burchard et al. 2000; Agati and Tattini 2010). However, despite largely evolved UV-protection mechanisms, complete UV-B protection is not achieved and a small percentage of the solar UV-B radiation penetrates inside the leaf (Krauss et al. 1997). It is generally accepted that a gradient exists in the ability to screening of UV; the herbaceous plants (being least efficient) towards woody and perennial plants, with the conifers being the most efficient (Krauss et al. 1997). Moreover, the proportion of UV-B radiation reaching the leaf photosynthetic mesophyll is significantly higher in the deciduous broadleaf trees than in the evergreen conifer trees (Julkunen-Tiitto et al. 2005; Turunen and Latola 2005). This indicates a greater susceptibility of the deciduous trees to the enhanced UV-B radia-

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tion as well as a greater cost of maintenance (Snell et al. 2009). The reason for the low UV-B transmittance in the conifer needles is that UV-absorbing compounds are located in both vacuoles and epidermal cell walls, whereas in herbaceous plants these are located primarily inside the vacuoles of epidermal cells (JulkunenTiitto et al. 2005). Moreover, the soluble flavonoids can be actively and rapidly mediated by the exposure to UV-B radiation whereas the cell-wall bound insoluble phenyl-propanoids represent a more passive UV-screening mechanism (Krauss et al. 1997; Clarke and Robinson 2008). These compounds absorb the UV-B wavelengths effectively, but they also transmit the visible PAR inside the mesophyll cells (Krauss et al. 1997). Interestingly, excess of penetrating UV radiation (UV-A and UV-B) could be converted into visible PAR radiation through both yellow and green fluorescence emission from the epidermal cell-wall bound UV-absorbing compounds (Hoque and Remus 1999). Although the epidermal thickness and concentration of UV-absorbing compounds seems to be the strongest predictors of epidermal transmittance and depth of the UV-B penetration, clear relationships between effectiveness of the accumulation of UV-absorbing compounds and epidermal morphological changes have still not been established, suggesting that other intrinsic plant factors are also important in determining the UV-B screening efficiency. Moreover, the endogenous constitution of plants can affect the chemical composition at both whole and organ level. Even within an individual plant the quality and quantity of secondary metabolites may differ between young and old leaves, as well as between the leaves exposed to the sun and those that remain in the shade (Brenes-Arguedas et al. 2006). Alteration in the accumulation of species-specific UV-absorbing compounds may result in changes in the tissue attractiveness or palatability to insects and herbivores (Izaguirre et al. 2007), pathogen attacks (Stratmann 2003), plant–plant interactions (Sullivan 2005), and changes in litter decomposition processes (Pancotto et al. 2003). Because most of these studies have been conducted on

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crop monocultures or isolated pot grown plants, the extrapolation of their responses to natural ecosystems is difficult (Phoenix et al. 2003). One previous ecosystem study found little effect of the ambient solar UV-B on Sphagnum bog and Carex fen in Tierra del Fuego-Argentina (Searles et al. 2002). Moreover, in related studies the solar UV-B reduced the herbivory, but increased the damage of DNA in the perennial herb Gunnera magellanica and reduced both the leaf number and length of the Antarctic species D. antarctica and C. quitensis (Ballaré et al. 2001). Although overall these studies expand our limited knowledge on how the exposure to natural ambient UV-B can modify the biomass accumulation, population dynamics, and competitive interactions in nonagricultural species and thereby how ecosystems may respond to future UV-B fluctuations. Presently long-time studies are very scarce, only a few studies with more than 4 years under continuous monitoring have been communicated (Robson et al. 2003; Rozema et al. 2006; Trošt-Sedej and Gaberščik 2008). Although the visible radiation can often penetrate dense canopies deeper than the UV-B because of its higher transmittance through leaves, in less dense canopies the situation may be reversed. This implies that the UV-B/PAR ratio should change with the canopy leaf area and leaf architecture (Shulski et al. 2004). In order to understand how the natural ecosystems respond to the ambient solar UV-B radiation, many additional well-designed long-term studies with various plant species are needed in order to understand the different behavior of UV-B and PAR inside the canopy as well as to obtain a complete picture of the gene–environment interactions.

6

Effects of Artificially Enhanced UV-B Radiation

Different to polar studies, earlier researches on nonpolar terrestrial plants were mainly focused on the effects of artificially increased UV-B radiation on crop species rather than ecosystems (Flint et al. 2003). Although, such researches

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were important to understand the physiological responses and identify possible targets for the UV-B radiation, extensive recent studies have shown that effects of the artificially manipulated UV-B have been often overestimated (Rozema 2000). Moreover, the responses of terrestrial plants to simulated solar UV-B enhancement vary greatly due to artifacts derived from the experimental conditions (Musil et al. 2002a; Flint et al. 2003). A critical point besides the variability of experimental conditions in the evaluation of simulated solar UV-B enhancement is the use of lamps to provide the UV-B radiation. Both UV-fluorescent and broadspectrum xenon-arc lamps are the most commonly used sources of UV radiation in UV-B enhancement experiments (Flint et al. 2009). In terrestrial studies, the UV-fluorescent lamps are usually used, but in aquatic experiments the xenon-arc lamps are preferred. Although the UV-fluorescent lamps are widely used, they supply more short- than long-wave UV-B radiation compared with the solar spectrum (Musil et al. 2002a). In addition, all the UV lamps emit small but biologically effective UV-C radiation, which is not present in the solar radiation reaching the Earth’s surface (Flint et al. 2009). Other debatable question in the studies on UV-B effects is the use of UV filters. The major filters used to exclude either UV-A or UV-B in UV-exclusion studies are: (a) cellulose diacetate, CA, that is commonly used to exclude the UV-C radiation and transmit both UV-A and UV-B; (b) polyester, the generic name for Mylar (trade name of the DuPont Co.) that is used to exclude both UV-C and UV-B and transmit the UV-A only; (c) polychlorotrifluoroethylene, PCTFE (Aclar 22 C) that transmits all the UV radiation (UV-A, UV-B, and UV-C); (d) copolymers of tetrafluoroethylene and hexafluoropropylene, Teflon FEP (trade name of the DuPont Co.) that transmits the radiation at 245 nm and above; (e) polyvinyl fluoride, Tedlar TUT (trade name of the DuPont Co.) that blocks wavelengths in the UV-B region; (f) clear polyethylene, DuraFilm Super 4 (trade name of the AT Plastics Inc.) that blocks the UV radiation up to 380 nm; polymethylmethacrylate, Plexiglas (trade name

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of the Arkema): the standard Plexiglas excludes the UV-B wavelengths and a portion of the UV-A region, whereas the UV-T Plexiglas transmits all the wavelengths in both UV-B and UV-A regions (Krizek et al. 2005). Although these filters have been widely used in UV-B studies, their transmittance properties vary leading to erroneous interpretations of the UV effects in long-term experiments (Day et al. 2001). Moreover, CA, the most widely used UV filter produces detrimental effects on plants (Krizek and Mirecki 2004). On the other hand, in greenhouses or growth chambers unrealistic balances frequently occur among the different light spectral regions: UV-B/UV-A/PAR (photosynthetic active radiation (PAR), 400–700 nm) and often levels of PAR lower than in the field conditions are also observed. Low levels of PAR increase the sensitivity of plants to UV-B-induced damages (Pradhan et al. 2006). Additionally, to calculate and compare the doses of UV-B under different spectral regimes the UV-B radiation is weighted (UV-BBE) according to a suitable biological action spectrum or biological weighting function (BWF). To obtain the UV-BBE there are different BWFs available but there is a generalized consensus for the use of the Caldwell’s BWF (Flint and Caldwell 2003). Several results, however, have shown that this very steep action spectrum may lead to over- or underestimation of the UV-B effects (Micheletti et al. 2003). Moreover, differences in climatic conditions can also affect the interpretations and comparisons among different studies based on the BWF (Musil et al. 2002b; Flint et al. 2009). Therefore, all the quantitative predictions relating to UV-B enhancement effects could be greatly affected. On the other hand, more recent studies have proposed that increases of the ambient solar UV-B radiation at magnitudes anticipated under the current stratospheric ozone projections will not significantly have large-scale deleterious effects on terrestrial plants even though some species may suffer photosynthesis decreases and growth reductions (Rozema et al. 2006; Xu and Qiu 2007; Newsham and Robinson 2009).

7

Physiological and Morphological Responses to UV-B Enhancement

From indoor and outdoor studies, there is a general consensus that UV-B enhancement produces physiological, biochemical, morphological, and anatomical changes in the plants (Searles et al. 2001). According to the literature, the enhancement of UV-B radiation can affect the terrestrial plants at different functional levels involving conformational changes and damages to different molecules such as DNA, proteins, and lipids (Li et al. 2010). As a result, if damage to macromolecules, that is, DNA is not effectively repaired, the UV-B effect will be translated to the biochemical level with the consequent alteration and/or impairment of the plant functionality (e.g., photosynthetic process, growth, yield). Although it is clear that there is a wide range of both intra- and interspecific sensitivity to UV-B radiation (Gilbert et al. 2009) the terrestrial plants through the evolution have acquired different protective strategies to avoid the adverse effects of UV-B radiation. The two major protective mechanisms are: (a) shielding through the production of soluble phenolics (e.g., flavonoids, anthocyanins, hydroxycinnamic acid derivatives), insoluble polyphenols (e.g., lignin), and cell-wall bound UV-absorbing compounds (Hilal et al. 2004; Clarke and Robinson 2008), as well as by reflection of the UV-B radiation by epicuticular waxes and cuticular structures (Hada et al. 2003; Schmitz-ho*rner and Weissenböck 2003; Agati and Tattini 2010); (b) removal and direct reversion of the DNA lesions induced by UV-B radiation (Tuteja et al. 2001; Britt 2004; Kimura et al. 2004).

7.1

DNA: Damage and Repair

The more important UV-B-induced DNA alterations are the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidine dimers (6–4 photoproducts, 6-4PPs) (Dany et al. 2001). The DNA repair mechanisms operating in

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

plants include the following processes: (a) direct reversal (DR); (b) photoreactivation that induces photolyases; (c) dark repair (Tuteja et al. 2001; Britt 2004). The DR is a simple mechanism that involves a single-enzyme reaction for the removal of certain types of DNA damage. Alkyltransferases simply extract alkyl groups from the alkylated bases that are transferred to internal cysteine residues and thus inactivate themselves. The best example for DR is the correction of miscoding alkylation lesion O6-methylguanine, which is generated endogenously in small amounts by the reactive cellular catabolites. This reaction is catalyzed by a specific enzyme, called methylguanine methyltransferase (MGMT), which removes a methyl group from a guanine residue of the DNA molecule and transferring it to one of its own cysteine residues in a rapid and error-free repair process (Tuteja et al. 2001). The photoreactivating enzyme DNA photolyase (PRE) is a DR phenomenon performed by the combined action of one or more photolyases and the visible light (blue, violet, or long-wave UV) (Hidema et al. 2007). Photolyases specifically recognize and bind the pyrimidine dimers to form a complex molecular structure which is stable in absence of the light. After absorbing a blue light photon the pyrimidine dimers are reversed to pyrimidine monomers without excision of the damaged base (Tuteja et al. 2001). The repair reaction is fast and requires about 1 h for completion (Takeuchi et al. 2007). In plants, two specific types of photolyases have been characterized: (a) CPDphotolyase; (b) 6-4PP-photolyase (Tuteja et al. 2001). The dark repair processes include the nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and other DNA repair pathways. These mechanisms have been observed in several plant species and some of the genes required for the processes were identified (Kimura et al. 2004). The wide class of helix-distorting lesions such as CPDs and 6-4PPs are repaired by the NER process. It is one of the most versatile DNA repair pathway operating in plants. Unlike other DNA repair pathways that are specific repair processes, the NER pathway is capable of removing various DNA damage classes, including those induced by the UV-B

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radiation (pyrimidine dimers) and chemical agents (bulky DNA adducts) (Kimura et al. 2004). The NER pathway sequentially involves recognition of the DNA damage, incision on the damaged strand, excision of the damage-containing oligonucleotides, and the DNA synthesis and ligation (Liu et al. 2003). Also, the NER pathway is a slow process (about 24 h for completion) and includes several enzymes. There are two subpathways of the NER process that are designated as: (a) global genomic repair (GGR); (b) transcription-coupled repair (TCR). While the GGR pathway repairs the DNA damage over the entire genome, the TCR pathway is selective for the transcribed DNA strand in expressed genes (Kimura et al. 2004). Oxidized or hydrated bases and single-strand breaks are repaired by the BER pathway that is considered an essential process for maintenance of the DNA molecule. The BER mainly removes the DNA damages that are arising spontaneously in the cell from hydrolytic events such as deamination or base loss, fragmented bases resulting from ionizing radiation (e.g., UV-B radiation), and oxidative damage or methylation of the ring nitrogen by endogenous agents. The process that involves the BER mechanism is initiated by DNA glycosylases that release the damaged base by cleavage of the sugarphosphate chain followed by excision of the abasic residue or abasic residue containing oligonucleotides and then the synthesis and ligation of the DNA occurs. The BER pathway involves several enzymatic steps and depends strongly on the presence of nicotinamide adenine dinucleotide (NAD+). Also, the BER process comprises two subpathways that are designated as: (a) BERshort-path; (b) BERlong-path. The BERshort-patch is a DNA polymerase beta-dependent mechanism, while the BERlong-patch is a DNA polymerase delta/ epsilon-dependent mechanism (Kimura et al. 2004). The major difference between BER and NER pathways is the way by which the DNA damage is removed. The NER pathway cuts out the damage as a part of an oligonucleotide fragment, while the BER mechanism excises only one nucleotide (Tuteja et al. 2001). The excision repair processes (NER and BER) are very important for maintaining the genome stability

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and essential for the survival of plants. The mismatch repair pathway (MMR) is also important in the DNA repair processes when by errors of replication or hom*ologous recombination can be produced mismatched bases. The MMR pathway basically discriminates between correct and incorrect bases and after DNA synthesis the error is corrected (Tuteja et al. 2001). Although most of the present understanding of the eukaryotic MMR has come from studies of the E. coli MutS and MutL proteins (Kolodner and Marsischky 1999), recent studies carried out in Arabidopsis and rice have reported interesting findings on the MMR pathway operating in plant cells (Tuteja et al. 2001; Kimura et al. 2004). According to the E. coli model, the MutS dimer recognizes mispairs and then binds on it followed by the MutL binding, which activates the MutH (endonuclease) that makes a single-strand incision (nick). The MutH incision can be done on either side of the mismatch. Subsequent to incision the excision is initiated and proceeds toward mismatch. To fill the gap (100–1,000 nucleotide gap), the original template strand can then be replicated and finally sealed by ligation. The proteins involved in the last step of eukaryotic MMR are: (a) DNA polymerase d, RP-A (replication protein); (b) PCNA (proliferating cell nuclear antigen); (c) RFC (replication factor) (Kolodner and Marsischky 1999). Although the UV-A wavelengths can mediate the photoxidative damage (Turcsányi and Vass 2000) the UV-B radiation is the most important photooxidant agent for terrestrial plants. The DNA damage can also be caused by reactive oxygen species (ROS) and free radicals produced by the UV-B radiation. This damage includes several modifications such as cross-linking, aggregation, denaturation, and degradation (Hidema et al. 2007). The formation of 7,8-dihydro-8oxoguanine (GO) is a common oxidative DNA lesion generated by a direct modification mediated by ROS. The GO is mutagenic and can mispair with adenine (A) during the DNA replication (Yang et al. 2001). If the resulting A/GO is not repaired before the next round of the DNA replication, a C/G → A/T transversion occurs and the opportunity for repair is lost. The A/GO is

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repaired via the BER which is initiated by the DNA repair enzyme adenine-DNA glycosylase (Yang et al. 2001). The UV-absorbing compounds (e.g., flavonoids, anthocyanins, hydroxycinnamic acid derivatives, phenolics) accumulating in epidermal and subepidermal cell layers have traditionally been thought to function as UV-B filters, but also play an important role as quenchers of the ROS and free radicals in the amelioration of the UV-B-induced DNA oxidative damage (Agati and Tattini 2010). The UV-absorbing compounds are also effective in reducing the induction of cyclobutane pyrimidine dimers (CPDs) in plants exposed to high UV-B levels (Hidema et al. 2007). The inhibition of CDP formation seems to be high enough to compensate the DNA damage arising even from unusually strong solar irradiations (Tuteja et al. 2001). Other related UV-absorbing compounds, that is, anthocyanins through an anthocyanin-DNA complex could also provide protection against the oxidative damage. Since both anthocyanins and DNA mutually protect each other in vitro, it is likely that such protection mechanism may also operate in vivo (Sarma and Sharma 1999). In the plant cells, anthocyanins are predominantly localized inside the vacuoles and thus their putative role in the protection of DNA should be critically examined. Accepting this fact, it has been demonstrated that the excess accumulation of anthocyanins reduces the amount of blue/UV-A radiation reaching the cell and may sometimes lower the ability to photorepair the damaged DNA. For example, the purple rice is a highly UV-B sensitive species despite possessing an elevated level of anthocyanins in their leaves (Hada et al. 2003). Although significant amounts of flavonoids have been found in the chloroplasts or etioplasts isolated from a wide range of plants growing under both ambient and enhanced UV-B irradiances (Tattini et al. 2005; Agati et al. 2007), it is also likely that some amount of anthocyanins can be present in the nuclei and organelles and then may associate with the DNA molecule, offering to it a certain protection against the oxidative damage (Feucht et al. 2004). In this context, the UV-absorbing compounds seem to have an important protective function against the DNA

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

damage induced by shorter solar wavelengths (Schmitz-ho*rner and Weissenböck 2003) and so the speculations concerning a great biological risk with regard to increases in solar UV-B radiation after the depletion of the ozone layer are presumably premature. In the natural populations, both protection and DNA repair are complementary and necessary processes for the plant development. Thereby, it is expected that the plants growing under different UV-B irradiances can exhibit different levels of the DNA protective mechanisms (Turunen and Latola 2005). Under field conditions, the observed DNA damage can often be modified by climatic conditions and then a direct extrapolation of the DNA changes obtained in controlled-environment experiments under artificially enhanced UV-B radiation to plants growing under the ambient solar UV-B is complex and unrealistic. Differences between damage, repair, and defense can be subtle and identification of a particular mechanism does not always occur as the explanation underlying a given phenomenon. For example, the UV-induced degradation of the D1 protein of the PSII can be seen either as damage or as a part of the repair mechanism leading to substitution of the damaged components of the PSII (Turcsányi and Vass 2000). Consequently, understanding these differences and potentially using the DNA repair mechanisms could become very important for producing UV-B-tolerant plants.

7.2

Secondary Metabolites: Flavonoids and Anthocyanins

The increase of secondary metabolites synthesis has been recognized as one of the most frequently observed plant response to UV-B enhancement (Searles et al. 2001; Rozema et al. 2002; Bassman 2004). Considerable attention has been focused, over the past two decades, on the UV-B-induced biosynthesis of phenylpropanoid-derivative compounds, particularly flavonoids and hydroxycinnamic acid derivatives (Jordan 2002; Rozema et al. 2002; Bassman 2004). Although these compounds exhibit important interspecific differences induced by the UV-B radiation, they are often

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derivatives of the flavonols quercetin and kaempferol (Buer et al. 2010). Quercetin- and kaempferol-derivative flavonoids are usually glycosylated and frequently contain a hydroxycinnamic acid moiety esterified to one of the glycosyl groups (orthodihydroxy B-ringsubstituted flavonoids) (Tattini et al. 2004). The flavonoids are ubiquitous molecules occurring in the vacuoles and cell walls of epidermal cells and in nonsecretory and glandular trichomes, and it has been assumed that they primarily have the function of attenuating the shorter solar wavelengths due to their good quantum efficiency (Burchard et al. 2000). In this way, the location of flavonoids in trichomes (Tattini et al. 2004), cuticular wax layers (f*ckuda et al. 2008), and epidermal cells (Burchard et al. 2000) may largely prevent that the UV-B radiation reaches sensitive targets within the leaf. However, the flavonoids also have another protective function against the shorter solar wavelengths. Considering that the flavonoids with orthodihydroxylated B-ring may efficiently dissipate the excess of energy through tautomeric interconversions (Smith and Markham 1998), scavenge the ROS through the quenching mechanism (Yamasaki et al. 1997; Hilal et al. 2008), and inhibit the formation of free radicals (Neill and Gould 2003; Xu et al. 2008), they can also act as effective antioxidant molecules (Jordan 2002; Tattini et al. 2005; Buer et al. 2010). However, a major criticism regarding functions of the flavonoids is the use of mutants that lack or possess the ability to synthesize flavonoids, which may oversimplify the plant model system for quantifying the UV-B-tolerance/flavonoidbiosynthesis relationships (Bieza and Lois 2001). In this context, contrary to determination of the flavonoid concentration at the whole-leaf level, less attention has been devoted to analyzing the tissue-specific location of individual flavonoids, which may clarify their complex functional roles in both attenuation and antioxidant mechanisms against the high UV-B irradiances (Tattini et al. 2004). Furthermore, the short-term experiments and inappropriate microscopy techniques for visualizing the flavonoids also greatly contributed to this superficial conclusion. More recent studies, however, suggest that the biosynthesis of

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flavonoids, particularly internal flavonoid glycosides may be largely controlled by constitutive morphoanatomical and biochemical features, primarily intended both to prevent the light penetration (Burchard et al. 2000) and to remove the consequent oxidative damage (Apel and Hirt 2004). Agreeing with these findings, Semerdjieva et al. (2003) showed in the Vaccinium spp. an inverse relationship between cuticule thickness (primary barrier to UV-B penetration) and the mesophyll accumulation of UV-B-induced flavonoids. Regarding to ROS scavenging activity of the flavonoids, Yamasaki et al. (1997) proposed a model to address major criticisms on the antioxidant functions of the flavonoids compartmentalized in epidermal vacuoles, and at the same time to explain the preferential UV-B-induced synthesis of flavonoids with effective antioxidant properties in vitro. According to Yamasaki’s model, the orthodihydroxy B-ring-substituted flavonoids, not their monohydroxy B-ring-substituted counterparts, are effective substrates for the class III peroxidases, which quench the H2O2 freely diffusing from the mesophyll cellular organelles to vacuoles of the epidermal cells. The model was remarkable in calling out the question whether vacuolar flavonoids could be effective in protecting underlying tissues from the damaging shorter solar wavelengths, while not protecting the epidermal cells from the oxidative damage. Epidermal cells and glandular trichomes usually contain much higher concentrations of flavonoids than the mesophyll cells (Burchard et al. 2000), then the H2O2 leaked out from the mesophyll cells under high UV-B irradiances can be scavenged by the flavonoid-peroxidase system in the epidermal cells according to the proposal of Yamasaki et al. (1997). Consistent with this idea blackening of the epidermis after a severe light stress is frequently observed in many species under the field conditions. This phenomenon has been ascribed to the polymerization of vacuolar phenolics that result from the penetration of H2O2 inside the epidermal cells (Yamasaki et al. 1997). Nevertheless, it cannot be excluded the possibility that other apoplastic flavonoid-depending peroxidases such as guaiacol peroxidase and

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syringaldazine peroxidase associated with the process of lignification may be involved in the mechanism of ROS scavenging (Hilal et al. 2004). The concept of delocalized scavenging of H2O2 by the vacuoles can be applied not only to the organelle–organelle interactions but also to the cell–cell interactions (Yamasaki et al. 1997). It is also acknowledged as a controversy matter whether the ability to accumulate flavonoids, particularly flavonoids with orthodihydroxylated B-rings, and the tolerance to UV-B radiation is highly correlated (Dixon et al. 2001; Musil et al. 2002b; Hofmann et al. 2003). The orthodihydroxylated B-ring-derivative flavonoids such as quercetin and luteolin glycosides are accumulated in the vacuoles of the mesophyll cells in Ligustrum vulgare leaves exposed to the full sunlight, in presence or absence of the UV-B radiation (Agati and Tattini 2010). This finding, which is consistent with previous reports indicating that the UV-B radiation is not a prerequisite for the synthesis of flavonoids (Tattini et al. 2004, 2005; Jenkins 2009), leads to the conclusion that the light-induced oxidative damage may regulate the biosynthesis of flavonoids, irrespective of the presence of UV-B radiation. The flavonoids and other UV-absorbing phenolics (e.g., hydroxycinnamic acid derivatives) are also synthesized in other abiotic/biotic unfavorable conditions such as drought, salinity, low temperature, heavy metal pollution, pathogen attack, and as feeding deterrent. Besides their antioxidant abilities the flavonoids might exert modulatory effects in the cell through selective actions at different components by cell interactions (Buer et al. 2010). This fact has become increasingly important because attention focuses on the new concept of flavonoids as potential modulators of the intracellular signaling cascades that are vital for the cell functionality (Jenkins 2009). The anthocyanins, other members of the phenol family, have generally been included into photodamaging-protective compounds (Gould 2004). The anthocyanins show a weak absorption in the shorter UV region (270–290 nm), but their acylated counterparts (hydroxycinnamic acid derivatives) exhibit an increased absorption in the longer UV-B region (310–320 nm) (Neill and

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

Gould 2003). Because anthocyanins are photoinduced many researchers surmise that they must either have a photoprotective function against the light-induced photooxidation or against the UV-B damage (Hughes et al. 2005). Even without acylation the anthocyanins can significantly attenuate the visible radiation. In fact, the more clear evidences really support the theory of the photooxidative protection, while the role in UV-B protection seems to be much less apparent (Kytridis and Manetas 2006). This assumption, however, contradicts the theory that anthocyanins have a UV-B-filtering role (Neill and Gould 2003). Disagreeing with the last theory the UV-B vulnerability is poorly correlated with the content of anthocyanins. For example, an Arabidopsis mutant with enhanced sensitivity to UV-B radiation was found deficient in certain flavonoids, whereas the amount of anthocyanins displayed unchanged. Similarly the responses of a Brassica rapa mutant to the supplementary UV-B treatment were mostly independent of the anthocyanin level in leaves (Gould 2004). Agreeing with these findings the anthocyanins often occur in very low concentrations compared to other UV-absorbing compounds, and require a long exposure to the UV-B radiation to be synthesized (Neill and Gould 2003). On the other hand, the red-leafed plants of Impatiens capensis and rice displayed significantly worse performances under the UV-B enhancement than their green-leafed counterparts (Dixon et al. 2001). Moreover, it has been communicated that the accumulation of anthocyanins can cause deleterious effects on terrestrial plants after a long-term UV-B exposure (Gould 2004). It has been noted that the DNA damage after a prolonged UV-B treatment was substantially greater in the purple-leafed rice than in the nearisogenic green line. Anthocyanins in the purple rice prevented the photoactivation of photolyases by absorbing some of the incident blue/UV-A light on leaves. Thus, any short-term gain from the absorption of UV-B radiation by anthocyanins would be offset by their property to absorb the visible light and thereby limit the rate of DNA repair (Hada et al. 2003). Furthermore, it has been demonstrated that other abiotic and biotic stresses produce changes in the chemical pattern

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of anthocyanins (Close and Beadle 2003), then it is obvious that such changes will influence the absorption spectra of anthocyanins under the UV-B enhancement. Also absorptive artifacts due to the dissociation of covalent bonds can occur during the improper isolation of anthocyanins and misread absorption spectra will be generated (Gould 2004). The accumulation of anthocyanins is usually transient and generally occurs in the vacuoles of peripheral tissues such as palisade and/or spongy mesophyll exposed to high light irradiances, but there are some exceptions (e.g., accumulation in the abaxial leaf tissues and in obligatory shade plants) (Kytridis and Manetas 2006). Perhaps the improved solubility of anthocyanins that in contrast to other flavonoids are nearly always glycosylated allows them to be stored in the vacuole more efficiently than the nonglycosylated flavonoids (Winefield 2002). In fact, the importance of flavonoids should not be overlooked in the discussion of anthocyanin production and UV-B protection. In this context, the flavonoids induced by the UV-B radiation (Agati and Tattini 2010; Buer et al. 2010) are recognized as strong UV-B absorbers, and their UV-B absorption capacity is much stronger than that of anthocyanins (Bieza and Lois 2001). Since the production of anthocyanins represents a conversion of flavonoid precursors that themselves are strong UV-B absorbers, a conundrum appears: if one of the effects of UV-B radiation on plants is to induce the UV-B-protective pigments, why are anthocyanins produced instead of their flavonoid precursors? Characteristics of both flavonoids and anthocyanins absorption spectra must be analyzed to respond this conundrum (Solovchenko and Merzlyak 2008). The flavonoids exhibit two bands in the UV region: (a) short-wave peaking around 280 nm; (b) long-wave situated in the range of 300–360 nm. However, the exact positions of the maxima vary for different flavonoid derivatives. The anthocyanins also have two maxima: one in the UV-B region (270–320 nm) and another in the visible region with a maximum located in the blue-green part of the visible wavelengths (500–540 nm) (Gould 2004). In this way, the UV-B component of the solar spectrum can

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be screened by both flavonoids and anthocyanins. However the UV-A radiation, whose proportion in the solar spectrum could be tenfold higher as compared with the UV-B spectrum region also exerts significant effects on plants (Krizek 2004). For example, maximum inhibition of the photosynthesis under natural radiation fluxes is induced by radiation in the UV-A region (Ivanova et al. 2008). Although this fact supports the importance of the UV-protection provided by the flavonoids in the range of 300–360 nm, the high visible fluxes (400–700 nm) also induce a photodamage in plant tissues, especially in the chloroplast (Krizek 2004). The anthocyanins are able to intercept a great proportion of the solar radiation in the range of 500–600 nm, which correspond to the maximum solar energy reaching the Earth’s surface (Gould 2004). This finding therefore contributes to support the role that anthocyanins play in the photoprotection of plant tissues (Solovchenko and Merzlyak 2008). In this context, the accumulation of anthocyanins requires visible light and generally coincides with the period of high excitation pressure and the increased potential for the photooxidative damage. The photooxidative damage is produced by an imbalance between the light capture, CO2 assimilation, and carbohydrate utilization (e.g., greening of developing tissues, senescence, adverse environmental conditions) (Hughes et al. 2005). Thereby, the attenuation of light by anthocyanins may help to reestablish this balance and to reduce the excitation pressure (Kytridis and Manetas 2006). Then, the risk of cellular photooxidative damage is lowered. Also, it would seem that the anthocyanin biosynthesis can enhance under the high light, but it is not usually a prerequisite for the protection against the oxidative stress (Gould 2004). Like the colorless flavonoids the colored anthocyanins may scavenge the free radicals and ROS (Gould 2004). The anthocyanins diminish the oxidative trend in the leaf simply by filtering out the yellow-green light, because most of the reactive oxygen in plant cells is derived from excitation of the chlorophyll molecule (Neill and Gould 2003). Agreeing with this theory, in juvenile and senescing plants the regulation of photosynthetic apparatus functions is often impaired,

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making it less efficient in utilization of the absorbed light and therefore prone to the photodamage (Merzlyak et al. 2008a). As a general rule, these situations are accompanied by an increased generation of ROS causing photooxidative damage to the plant and, eventually, its death (Bukhov 2004). Under these conditions, the anthocyanins may afford a detoxifying sink for some ROS when the chloroplast, the first line of the antioxidative defense, is surpassed (Kytridis and Manetas 2006). It is not clear, however, whether the ROS scavenging occurs predominantly through the anthocyanins found inside the vacuole or through their counterparts located in the cytosol. Both anthocyanin forms have impressive antioxidant potentials (Neill and Gould 2003), but due to their proximity to the chloroplastic source of ROS it is more probable that anthocyanins located in the cytosol (mesophyll tissue) than in the vacuole (epidermal tissue) provide the major contribution to antioxidant defense (Kytridis and Manetas 2006). In a similar trend, recent evidences suggest that flavonoids may scavenge the ROS within or near sites of its generation (Schmitz-ho*rner and Weissenböck 2003; Tattini et al. 2005; Agati et al. 2007). Interestingly, equal effectiveness as antioxidant molecules of other colorless phenolics suggests that the putative photooxidative protection afforded by the anthocyanins should be unrelated to their ability to quench oxidants. Noteworthy the accumulation of anthocyanins in terrestrial plants has always been a contentious issue of the special interest. They often appear in juvenile plants but mature plants usually lack them or display transiently levels under stressful conditions (Merzlyak et al. 2008a). Obviously, upon maturation of the photosynthetic apparatus or its acclimation to stressors the photoprotective screen of anthocyanins is no longer required and the juvenile reddish pigmentation disappears. However, unfavorable environmental conditions such as low temperatures, heavy metals, drought, wounding, and pollutants can also predispose the photosynthetic apparatus to photoinhibition and photooxidation, and then the plants may increase, although not necessarily ascribed to, the accumulation of anthocyanins in vegetative organs

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

(Gould 2004). Accordingly, the production of anthocyanins would fit neatly into the definition of Leshem and Kuiper’s (1996) general adaptation syndrome (GAS). The GAS indicates that different types of stress evoke similar adaptation responses. In this context, along with compounds such as tocopherols, flavonoids, glutathione, and ascorbate, the anthocyanins may function as general mitigators of the oxidative damage. However, it should be addressed that there is no direct evidence that terrestrial plants benefit from the antioxidant properties of anthocyanins yet (Neill and Gould 2003). Although anthocyanins are of special importance for the photoprotection in senescing leaves, it seems not to be the only function of anthocyanins. It has been suggested, for example, that the red color may also deter aphids from laying their eggs or from feeding on the sugar-rich sap in the phloem. Noteworthy, despite that the autumnal color may be an extravagancy without a vital function. This phenomenon that enchants so many tourists each year may hold a vital key to the survival of deciduous trees (Archetti et al. 2009). Also the anthocyanins are involved in the photoprotection of ripening fruits, for example, the chlorophyll in apple fruit peel with high anthocyanin content showed a very high resistance to the photobleaching as compared with the anthocyanin-free zones of the same fruit (Merzlyak et al. 2008b). Despite its function as photoprotective molecules, the anthocyanins may instead serve to decrease the leaf osmotic potential. The resulting depression of leaf water potential could increase the water uptake and/or reduce transpirational losses. This phenomenon may allow to the anthocyanincontaining leaves to tolerate suboptimal water levels. The often transitory nature of the foliar anthocyanin accumulation may allow plants to respond quickly and temporarily to environmental variability rather than through more permanent anatomical or morphological modifications (Chalker-Scott 2002). Interestingly, the anthocyanins also fulfill the less common but important function of avoiding the photodegradation of sensitive molecules. The Ambrosia chamissonis, for example, hold strands of laticifers surrounded by an anthocyanin sheath. These laticifers contain

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thiarubrines, toxic chemicals that are believed to deter herbivory and prevent both fungal and bacterial infections. The thiarubrines are photolabile molecules and are degraded by both visible and UV light giving thiophenes that are less toxic. Page and Towers (2002) have shown that the anthocyanin sheath, by absorbing a proportion of the rays that would otherwise strike the laticifers, protects these light-sensitive defensive chemicals from degradation, and thus provides a mechanism for the antiherbivory under conditions of strong sunlight. However, although the role of anthocyanins in protecting plant tissues under stress conditions, including the photodamage mediated by both UV-B and visible light, as well as in the pollinator attractiveness and seed dispersion seems to be important, it is clearly evident that the adaptive significance of anthocyanins is still not fully understood (Close and Beadle 2003). Meanwhile two poorly explored areas became interesting: (a) how the increase of anthocyanin production is integrated to tissue responses to UV-B; (b) how the UV-B-induced anthocyanins contribute to the plant survival.

7.3

Morphogenic Responses

Studies carried out in greenhouses or in growth chambers using ultraviolet lamps and filters to simulate different solar UV-B enhancements have been conducted on a variety of terrestrial plants, including economically important crops (Santos et al. 2004) and wild plant species (Zu et al. 2010). Overall these studies showed that the UV-B enhancement besides physiological effects induces a range of morphological changes including: (a) increase/decrease of the leaf area and leaf thickness (González et al. 2002; Hilal et al. 2004); (b) reduction of the plant height (Santos et al. 2004) and increase/decrease of the shoot/root ratio (Furness and Upadhyaya 2002); (c) axillary branching (Kakani et al. 2003); (d) increase of the leaf glandular and uniseriate trichome density (Liakopoulos et al. 2006); (e) deposition of the waxy surface structures (f*ckuda et al. 2008); (f) opening of the cotyledon curling (Boccalandro

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et al. 2001; Barnes et al. 2005); (g) inhibition of the hypocotyl and stem elongation (Shinkle et al. 2004; Gerhardt et al. 2005); (h) premature leaf senescence (Pradhan et al. 2006). The effects of UV-B also include changes (increase/decrease) in the number and size of flowers as well as in the size of seeds (Kakani et al. 2003; Qaderi and Reid 2005). While some of the UV-B responses constitute a stimulation of the growth (e.g., axillary branching, leaf thickening), others reflect a growth inhibition (e.g., reduced hypocotyl elongation). However, in these experimental setups, frequently unrealistic balances between UV-B/ UV-A/PAR are obtained, and in some cases the plants have been exposed to relatively high shortterm doses of UV-B, which lack the ecological relevance (Newsham and Robinson 2009). Additionally, the levels of UV-A or PAR as well as other experimental conditions also affect the morphogenic responses, making it difficult to compare the results from different indoor studies. In addition, it is clear that not all the plant species respond in the same way to UV-B exposure (Pliura et al. 2008). In general, the monocots are more morphologically responsive to UV-B than the dicots (Pal et al. 1997). Closely related species or ecotypes, especially when occupy different habitats, also differ with respect to their morphogenic responses (Hofmann et al. 2003). Plant species also differ in the use of PAR and UV-B radiation; while some species use the PAR to trigger responses others use the UV-B radiation. Then the plants responding mainly to PAR radiation will probably be more sensitive to UV-B radiation than the UV-responding ones (Rozema et al. 2005). A critical factor in the UV-B studies is the visible light irradiance, which in growth chambers and greenhouses can be quite different to the natural sunlight (Flint et al. 2009). Indeed, it has been shown that as a result of the insufficient visible wavelengths and, therefore, of unrealistically high UV-B/PAR ratios in indoor studies, the morphogenic effects of the UV-B radiation are magnified (Musil et al. 2002a, b). In fact, even if realistic levels of the UV-B radiation in simulating ozone reductions are used the indoor responses of plants to UV-B radiation may be quite variable and exaggerated in relation to

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the field. Microclimatic conditions and the interactions of different abiotic and biotic environmental factors additionally contribute to inconsistency between the results obtained in growth chambers or greenhouses with those obtained under the field conditions (Flint et al. 2003; Caldwell et al. 2007). Furthermore, the plant responses to above ambient UV-B radiation (e.g., from stratospheric ozone depletion) have rarely been assessed in the broader context of the possible effects emerging from variations in the UV-B radiation within the ambient range. Also there is a significant knowledge gap between field and laboratory studies, which has two major components: (a) the occurrence of certain effects of the UV-B radiation under laboratory conditions has not yet been demonstrated in the field studies; (b) although some indoor responses are known to occur in the field, their functional implications are still unclear. Therefore, the obvious corollary from greenhouses or growth chambers studies is: study methodologies are as varied as results. In fact, from the field grown plants, the consensus that effects of artificially changed spectral UV-B irradiances are less pronounced (Searles et al. 2001). While under UV-B enhancement among other changes, leaf thickness, reduced leaf area, decreased plant height, changes in plant architecture, and biomass/yield reduction have been observed (Searles et al. 2001; Flint et al. 2003; Barnes et al. 2005). Nevertheless, the more recent studies have suggested that in the field, primary effects of the most realistic solar UV-B enhancements are subtle morphological and chemical changes with altered carbon partitioning and allocation, but doubt reveals such changes show significant effects on both plant growth and biomass accumulation (Gilbert et al. 2009; González et al. 2009; Morales et al. 2010; Ren et al. 2010; Zu et al. 2010). The morphogenic effects of the realistic UV-B enhancements are not usually considered as primary ecological factors influencing both species abundance and species distribution in relation to other abiotic environmental factors (e.g., drought, temperature, salinity). There are, however, situations where the UV-B-induced morphogenic effects can be ecologically important, giving changes in the

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

competitive ability with a significant impact on the composition of the plant community (Flint et al. 2003). The UV-B enhancement alters the leaf angle and differential transmission, and absorbance of the UV-B radiation through stands of erectophilous or planophilous plant species may have an important consequence on terrestrial plant responses to the UV-B radiation (Rozema 2000). In a model study, it was predicted that a more planophilous leaf angle in erectophilous species would reduce the UV-B/PAR ratio and therefore the UV-B damage. Of course, this effect may affect the competitive relations among species and also the ecosystem composition (Deckmyn 1996 in Rozema et al. 1997). The morphogenic effects often can be pronounced on different organisms at other trophic levels (Bassman 2004). The UV-B radiation also affects the decomposition of plant materials into ecosystems. Plants grown under the enhanced solar UV-B showed a reduced rate of the litter decomposition when compared to control plants grown under the ambient solar UV-B. The accumulation of UV-B-induced lignin and/or tannin accounts for the reduced litter decomposition rate (Cybulski et al. 2000). Nevertheless, the reduced rate of the litter decomposition can be produced as consequence of detrimental effects of the enhanced UV-B radiation on decomposing fungi and other decomposer organisms (Pancotto et al. 2003). In opposite trend, the plant litter material exposed to the enhanced solar UV-B can be decomposed by photodegradation more rapidly than under the ambient solar UV-B (Gallo et al. 2006). Moreover, it has also been demonstrated that the species growing for several generations under enhanced UV-B radiation show accumulation and exacerbation of the UV-B effects and likelihood they might be heritable (Mpoloka et al. 2007). Much of the UV-B research on terrestrial plants has concentrated on vegetative plant parts, but fitness of the organisms depend mainly on their successful reproduction. Of particular concern is the detrimental effect of UV-B on the pollen quality observed for some species (Koti et al. 2004; He et al. 2007). This finding suggests that pollination may be an ecologically critical developmental stage vulnerable to the UV-B

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damage, even in the UV-B-tolerant species. The pollen surface of some species may transmit up to 20% of the incident UV-B radiation (Stadler and Uber 1942 in He et al. 2007), despite the presence of a variety of UV-B-absorbing pigments (Rozema et al. 2001). Thus, the mature pollen grains are potentially susceptible to the UV-B damage during a short period between the dehiscence of anthers and the penetration of the pollen tube into the stigmatic tissue (Koti et al. 2004). This fact may lead to both reduced pollen quality and altered patterns of competition among species affecting the composition of the ecosystem. Furthermore, the UV-B enhancement can alter the production and/or the temporal availability of flowers so as to make the plant a less attractive host for the pollinators and impinge upon competition of plants for the pollinator service, as well as on the reproductive success of the plant/pollinator system (Sampson and Cane 1999).

8

UV-B Radiation and Signaling Pathways

The UV-B radiation triggers diverse responses involving a differential regulation of the genes and participate in several protective pathways including the DNA repair, detoxification of ROS, and production of secondary metabolites as well as in photomorphogenic events (Agrawal et al. 2009). The UV-B responses can be elicited with either high fluence (HF-UV-B, over 15 kJ m−2), intermediate fluence (IF-UV-B, 5–7 kJ m−2), low fluence (LF-UV-B, 1–3 kJ m−2), or very low fluence (VLF-UV-B, less than 1 kJ m−2) (Brosché et al. 2002). However, the exposure of plants to low fluence UV-B promotes the expression of varying genes involved in the UV-B protection, and genes responsible for the production of flavonoids and several phenolic compounds, while as the low fluence photomorphogenic responses seem to be initiated by photoreceptors and no alternative UV-B-absorbing molecules seem to mediate the photomorphogenic UV-B responses (Ulm and Nagy 2005). Also, many components of the protective pathways which lead to the changes of the gene expression in response to

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both UV-B radiation and pathogens are similar or identical (upregulation of PDF1.2), except the pathways which are distinct (upregulation of PR-1), signifies the effects of the high fluence UV-B radiation on the gene expression are unlikely to be due to nonspecific damage and a yet unidentified UV-B photoreceptor (Brosché et al. 2002). The response of plants to UV-B radiation depends upon the adaptation and acclimation to UV-B irradiances, as well as of the interactions with other environmental factors. Moreover, studies carried out with Arabidopsis plant suggest that some genes are differentially responsive to UV-B in both 280–290 nm and 300–310 nm ranges, hence could be multiple UV-B photoreception mechanisms (Kalbina et al. 2008). Consequently, the exposure of plants to UV-B radiation can cause multiple responses on the primary and secondary metabolisms as well as different changes on the growth and overall performances. Although other light-dependent photoreceptors (e.g., phytochrome, cryptochrome) have been described (Carvalho et al. 2010), presently a UV-B-specific photoreceptor has still not been described and therefore the basic mechanism of UV-B perception and the signal transduction remain still poorly understood. Nevertheless, the chromophores that could act as photoreceptors to absorb the UV-B radiation exist. Pterins or flavins in their reduced forms are candidates where some experimental work supports the involvement of the perception of UV-B radiation (Galland and Senger 1988a, b in Jenkins 2009). For example, the compounds that antagonize the flavins and pterins impair the UV-B induced anthocyanin synthesis in maize (Jenkins 2009) along with UV-B suppression of the hypocotyl elongation in tomato plants (Ballaré et al. 1995). In addition, other possible chromophore can be a phenolic molecule, with this assumption the p-coumaric acid chromophore present in the photoactive yellow protein (PYP), a photoreceptor found in the purple photosynthetic bacteria Ectothiorhodospira halophila, enables to absorb in UV-A and blue regions of the solar radiation spectrum (Imamoto and Kataoka 2007). Moreover, an alternative possibility is that the UV-B radiation be sensed through some form of direct activation of a cel-

F.E. Prado et al.

lular component. Whether or not the UV-B photoreceptor exists the responses to UV-B radiation could be mediated by nonspecific signaling pathways involving the DNA damage, ROS production, hormone synthesis (e.g., salicylic acid, ethylene, jasmonic acid), and wound/defense signaling molecules (e.g., flavonoids, phenolics) (Apel and Hirt 2004; Demkura et al. 2010) ; or by specific UV-B signaling pathways mediated by the UV-B-specific component UV RESISTANCE LOCUS8 (UVR8) (Cloix and Jenkins 2008.). The UVR8 acts specifically to mediate the UV-B response, together with the expression of genes to establish the UV-B protection (Jenkins 2009). Moreover, the UVR8 also mediates the expression of genes activated at low UV-B fluence level, showing consistency with their involvement in the photomorphogenic UV-B signaling pathway (Brown and Jenkins 2008). No other component is known to act specifically in the photomorphogenic UV-B responses. The transcriptome analysis revealed that a set of approximately 70 indentified genes are stimulated by UV-B under control of the UVR8. Among these several genes are known to have key roles in the UV-B protection mechanism, including those encoding principal enzymes of the flavonoid biosynthetic pathway, as well as DNA photolyases and enzymes involved in amelioration of the photooxidative damage (Jenkins 2009). The findings demonstrate that the Arabidopsis UVR8 mutant shows severe necrosis under exposure to UV-B levels found in the bright sunlight, whereas it is indistinguishable from the wild type in the absence of UV-B (Brown and Jenkins 2008). The UVR8 regulates the expression of both ELONGATED HYPOCOTYL5 (HY5) and HY5 hom*oLOG (HYH) transcription factors at low UV-B fluence levels. The transcriptome analysis shows approximately the half of genes regulated by the UVR8 is also regulated by the HY5 transcriptor factor, but this is an underestimate and does not take into account the functional redundancy between HY5 and HYH (Brown et al. 2005). Further analysis, however, suggests that the HY5 and HYH transcription factors may regulate all the genes of the UVR8 component, and therefore are pivotal downstream

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

effectors of the UVR8 signaling pathway. In fact, the HY5 is evidently a very important regulator of the UV-B responses because the HY5 mutant, similar to the UVR8 mutant, is very sensitive to UV-B, while the HYH mutant is less sensitive indicating that it has a subsidiary role (Brown and Jenkins 2008). These findings demonstrate that the UVR8 is a key regulator of the UV-B protection and therefore helps to promote the survival of terrestrial plants exposed to UV-B radiation (Jenkins 2009). Another important component of the low UV-B signaling pathway is the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) (Oravecz et al. 2006). Contrarily to UVR8, the COP1 represses the expression of photomorphogenic genes and the plant development in the darkness. The COP1 acts as an E3 ubiquitin ligase, destroys HY5 and other positive regulators of the expression of photomorphogenic genes (Yi and Deng 2005). Following illumination, the COP1 is inactivated and moves slowly out of the nucleus, enabling the HY5 and other transcription factors to accumulate and promote the photomorphogenesis. In contrast to this function, the COP1 is a positive regulator of the UV-B responses such as the accumulation of flavonoids. Nevertheless, nearly half of genes regulated by the COP1 are controlled by HY5, indicating HY5 a key effector of the COP1 pathway. Both COP1 and HY5 transcriptor factors must act together in the nucleus to evoke the UV-B responses. Furthermore, the positive role of the COP1 seems not to be specific to UV-B because some evidences show a comparable function in several responses to the red light that require the involvement of the phytochrome B. With regard to this theory, the COP1 is required for the nuclear accumulation of the transcriptor factor PHYTOCHROME INTERACTING FACTOR3 (PIF3) in the darkness, although it does not mediate its destruction following the red and far-red illumination (Oravecz et al. 2006). Moreover, a more recent study showed that exposure to supplemental far-red (FR) light compared to red (R) light under UV-B radiation leads to a fast elongation growth and a phenolic accumulation in leaves of the silver birch seedlings (Tegelberg et al. 2004). Whether the COP1

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acts positively in other light responses, however, still remains unknown (Jenkins 2009). On the other hand, both UVR8 and COP1 regulate many of the same genes and are required for the low fluence UV-B induction of the HY5 transcriptor factor expression which plays a central role in the regulation of genes involved in the photomorphogenic UV-B responses (Brown and Jenkins 2008). Although both UVR8 and COP1 seem to function in the same pathway, little information is available to explain their functional relationships. Since UVR8 is a UV-B specific component, it may have a direct action of the COP1 in UV-B responses. To explain this fact one possibility is that the UVR8 regulates the nuclear accumulation of the COP1 or vice versa, while another can be that the UVR8 recruits the COP1 into a complex involved in the regulation of the transcription by UV-B. The last hypothesis is supported by a recent study demonstrating that the UVR8 colocalizes with the COP1 and directly interacts with a UV-B-dependent manner (Favory et al. 2009). Besides these, the expression of some genes at low UV-B fluence levels occurs independently of the action of both UVR8 and COP1 components (Jenkins 2009). According to data of A-H-Mackerness et al. (2001), the expression of genes by intermediate UV-B fluence levels (IF-UV-B) may be regulated partly by the enzymatic ROS formation after the specific UV-B induction, whereas the changes in mRNA levels of the high fluence (HF-UV-B) genes could be due to the formation of ROS as a result of the nonspecific damage to plant cells. However, it seems unlikely that sufficient ROS would be generated by the exposure of plants to the current ambient solar UV-B to cause the activation of the signaling pathway leading to biosynthetic responses; thus, presumably, the activation by ROS would not be UV-B specific (Jenkins 2009). Nevertheless, evidence exists for the involvement of ROS in some morphological changes and gene expression responses initiated by the UV-B radiation. Furthermore, the Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1) transcriptor factor is also involved in the UV-B signaling pathway. Interestingly, the expression of RCD1 genes is

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not significantly changed by the UV-B radiation. Previous study has shown that the SALT TOLERANCE (STO) protein is interacting with RCD1 in vitro being the mRNA level of the STO (SALT TOLERANCE) gene greatly increased in the Arabidopsis rcd1-1 mutant after UV-B irradiation. However, the expression of UV-Binduced HY5 and CHS (CHALCONE SYNTHASE) genes is partially inhibited in the STO mutant, subsequently, seems to be the RCD1, together with the STO, involved in the Arabidopsis UV-B signaling (Jiang et al. 2009). The cotyledon curling in Brassica napus stimulated by both UV-B and H2O2 is also inhibited by ascorbate (Gerhardt et al. 2005). In addition, the exposure to relatively high fluence rates of UV-B decreases the abundance of transcripts of the Arabidopsis LHCB1 gene that encodes the major chlorophyll binding protein of the chloroplast, and this response is inhibited by ascorbate as well as by a scavenger of superoxide radicals (A-H-Mackerness et al. 1999). Interestingly, supplementation of the ambient UV-B under greenhouse conditions increased the formation of CPDs and reduced the leaf area in G. magellanica, however did not cause lipid peroxidation being the modulation of the ascorbate content that counters the oxidative stress (Giordano et al. 2004). The exposure to UV-B stimulates the expression of a set of genes normally induced in response to the pathogen attack, insect predation, and wounding (Stratmann 2003; Ulm and Nagy 2005). It also reduces the level of insect herbivory in a range of species, probably because of increased production of the secondary metabolites, proteinase inhibitors, and other molecules that deter the herbivorous insects (Ibañez et al. 2008). An explanation for the overlap in responses to UV-B, wounding, and pathogens is that the UV-B stimulates the accumulation of ROS and other signaling molecules (e.g., jasmonic acid, ethylene, salicylic acid) that mediate the wounding defense responses (Izaguirre et al. 2007; Demkura et al. 2010). Molecules mediating the responses of some UV-Bregulated genes have been reported (Izaguirre et al. 2003). Alterations in the induction of defenserelated genes by the UV-B radiation have been observed in both ethylene ETR-1 and jasmonic

F.E. Prado et al.

acid JAR1 Arabidopsis insensitive mutants (A-HMackerness et al. 1999; Jenkins 2009). Also a transgenic Arabidopsis plant expressing the salicylate hydroxylase was unable to accumulate salicylic acid and showed a reduced UV-B induction of the several PR genes (Surplus et al. 1998). The expression of genes related to the pathogenesisrelated proteins (PR) and class I Endo-b-1,3glucanases (IbGlus I) are also induced by the UV-B radiation (Kucera et al. 2003). The PR genes have been grouped as: (a) intermediate UV-B level genes (PR-5); (b) high UV-B level genes (like PR-1). The IbGlus I are also assigned to PR proteins and constitute the PR-2 family. In addition, it has been demonstrated that the UV-B-induced DNA damage seems to be related to induction of the IbGlus I genes, but not with the synthesis of flavonoids under high levels of UV-B radiation (Kucera et al. 2003). The involvement of ROS in defense signaling is well established and evidences show that superoxide generated by the plasma membrane NADPH oxidase seems to be involved in UV-B-induced regulation of some defense genes, either directly or through the production of H2O2 (A-H-Mackerness et al. 2001). In agreement with this assumption, it has been reported that both redox activity of the plasma membrane and cytosolic-free Ca2+ homeostasis are involved in the induction of gene expressions by UV-B and blue/ UV-A wavelengths in Arabidopsis plants (Long and Jenkins 1998). Although the available data supports that ROS might be used by plants to modulate the expression of different genes in response to varying levels of UV-B, little information is available regarding the scope and nature of the ROS production and function in response to the solar UV-B radiation under natural growth conditions. However, the ROS are very dangerous to the cellular integrity and must be eliminated (Agrawal et al. 2009). Most plants scavenge the excessive amounts of ROS using a combination of enzymatic scavengers such as superoxide dismutase, ascorbate peroxidase and glutathione reductase, and nonenzymatic scavengers such as ascorbate, glutathione, carotenoids, tocopherols, and secondary metabolites (mainly flavonoids, hydroxycinnamic acids derivatives, and anthocyanins) (Xu et al. 2008).

3 UV-B Radiation, Its Effects and Defense Mechanisms in Terrestrial Plants

Although it is clear that the UV-B radiation stimulates both defense and wound signaling, there is little information on how the UV-B activates components of the signaling pathways. Experiments in tomato indicate that the UV-B radiation initiates similar signaling processes to systemin, an 18-aminoacid peptide that stimulates the wound responses (Ulm and Nagy 2005). Although data suggests that the tomato systemin receptor might also function as a brassinosteroid receptor (Wang and He 2004), more recent evidences indicate otherwise (Holton et al. 2007). In fact, a better knowledge of the mechanisms involved in the signaling pathways can help to understand the functional roles of the solar UV-B radiation in the resistance of plants to environmental factors into the terrestrial ecosystems.

9

Reduced Solar UV-B: A Future Scenario?

Despite the influence that the ambient level of solar UV-B radiation can exert on the plant life, recent findings have shown that the increases of UV-absorbing tropospheric gases (e.g., ozone, SO2, NO2) and aerosols can reduce the amount of solar UV-B radiation reaching the Earth’s surface (McKenzie et al. 2001). Thus, a further attention is needed to understand how the reduced solar UV-B can have an effect on the dynamics of ecosystems. Similar cues can occur when species originated from highland regions such as mountains (Ren et al. 2010), the Bolivian Altiplano (González et al. 2009) and the Tibet Plateau (Yang et al. 2008) or those from the equatorial regions that naturally receive high UV-B irradiances, are grown in lowland areas and/or high latitudes (Turunen and Latola 2005). So far the ecological cost of terrestrial plant responses to the solar UV-B radiation has not been studied in detail. Clearly, some morphological and physiological responses must have a cost in terms of resources that in absence of the UV-B could have been allocated elsewhere (Snell et al. 2009). Under this perspective seems to be an interesting point to study the question how the terrestrial plants respond to the solar UV-B

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radiation growing under UV-B-reduced irradiances, because till now data on this topic are very scarce. Like morphological features, emerging findings revealed that the UV-absorbing compounds could be affected by the reduced solar UV-B (Ibañez et al. 2008; González et al. 2009). In this context, the removal of UV-B from the natural solar radiation causes large increases of the growth in Glycine max and Cyamopsis plants and a marginal increase in the Vigna radiata but did not affect the growth of Vigna mungo plants (Varalakshmi et al. 2003; Amudha et al. 2010). In addition, it has also been demonstrated that the photomorphogenic regulatory mechanisms, rather than the photosynthesis seems to play key roles in the observed metabolic changes upon exposure to the reduced solar UV-B (Kadur et al. 2007). In agreement with these findings, the prolonged treatment with chronic low doses of UV-B caused changes in the morphology, gene expression, and biomass redistribution without cessation of the growth and in absence of the stress symptoms (Hectors et al. 2007). In fact, the reduced solar UV-B induces probably a plethora of key enzymes into the metabolic pathways transmitting a general plant response, which under a future long-time solar UV-B-reduced scenario will probably affect the plant productivity, species competition, trophic interactions, and ultimately the structure of ecosystems.

10

Conclusion and Future Perspective

The enhanced UV-B radiation produced important physiological and morphological effects on the terrestrial plants, but most of these studies were carried out in greenhouses and growth chambers. Then the extrapolation and quantification of the observed indoor effects to field experiments is very complex, because they can reflect exacerbated responses of the plants and confuse the interpretation of physiological and morphogenic responses, as well as the molecular analysis at both individual species and ecosystem composition. In fact, much remains to be done to define and establish the effects of both increased and

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reduced solar UV-B, as well as the signaling pathways to understand how they may be integrated to terrestrial plants growing in the natural environment. However, although in the solar UV-B alchemy, each successive understanding produces a larger doubt, God does not play dice with the universe! (Einstein). Acknowledgments We are grateful to anonymous reviewers for their useful comments on the manuscript. We are also grateful for the financial support provided by the Consejo Nacional de Investigaciones de la Universidad Nacional de Tucumán (CIUNT, grants 26/G423 and 26/ G437). FEP and MR are members of the Career of Investigator from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).

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Flint SD, Ryel RJ, Caldwell MM (2003) Ecosystem UV-B experiments in terrestrial communities: a review of recent findings and methodologies. Agric For Meteorol 120:177–189 Flint SD, Ryel RJ, Hudelson TJ, Caldwell MM (2009) Serious complications in experiments in which UV doses are affected by using different lamp heights. J Photochem Photobiol B Biol 97:48–53 f*ckuda S, Satoh A, Kasahara H, Matsuyama H, Takeuchi Y (2008) Effects of ultraviolet-B irradiation on the cuticular wax of cucumber (Cucumis sativus) cotyledons. J Plant Res 121:179–189 Furness NH, Upadhyaya MK (2002) Differential susceptibility of agricultural weeds to ultraviolet-B radiation. Can J Plant Sci 82:789–796 Gallo ME, Sinsabaugh RL, Cabaniss SE (2006) The role of ultraviolet radiation in litter decomposition in arid ecosystems. Appl Soil Ecol 34:82–91 Gerhardt KE, Wilson MI, Greenberg BM (2005) Ultraviolet wavelength dependence of photomorphological and photosynthetic responses in Brassica napus and Arabidopsis thaliana. Photochem Photobiol 81:1061–1068 Gilbert M, Pörs Y, Grover K, Weingart I, Skotnica J et al (2009) Intra- and interspecific differences of 10 barley and 10 tomato cultivars in response to short-time UV-B radiation: a study analysing thermoluminescence, fluorescence, gas-exchange and biochemical parameters. Environ Pollut 157:1603–1612 Giordano CV, Galatro A, Puntarulo S, Ballaré CL (2004) The inhibitory effects of UV-B radiation (280–315 nm) on Gunnera magellanica growth correlate with increased DNA damage but not with oxidative damage to lipids. Plant Cell Environ 27:1415–1423 González JA, Liberman-Cruz M, Boero C, Gallardo M, Prado FE (2002) Leaf thickness, protective and photosynthetic pigments and carbohydrate content in leaves of the world’s highest elevation tree Polylepis tarapacana (Rosaceae). Phyton 71:41–53 González JA, Gallardo MG, Boero C, Liberman-Cruz M, Prado FE (2007) Altitudinal and seasonal variation of protective and photosynthetic pigments in leaves of the world’s highest elevation trees Polylepis tarapacana (Rosaceae). Acta Oecol 32:36–41 González JA, Rosa MA, Parrado MF, Hilal M, Prado FE (2009) Morphological and physiological responses of two varieties of a highland species (Chenopodium quinoa Willd.) growing under near-ambient and strongly reduced solar UV-B in a lowland location. J Photochem Photobiol B Biol 96:144–151 Gould KS (2004) Nature’s swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 5:314–320 Hada H, Hidema J, Maekawa M, Kumagai T (2003) Higher amounts of anthocyanins and UV-absorbing compounds effectively lowered CPD photorepair in purple rice (Oryza sativa L.). Plant Cell Environ 26:1691–1701 He JM, Bai XL, Wang RB, Cao B, She XP (2007) The involvement of nitric oxide in ultraviolet-B-inhibited

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82 Rozema J, van de Staaij JWM, Tosserams M (1997) Effects of UV-B radiation on plants from agro- and natural ecosystems. In: Lumsden P (ed) Plants and UV-B. Responses to environmental change. Cambridge University Press, Cambridge, pp 213–232 Rozema J, Noordijk AJ, Broekman RA, van Beem A, Meijkamp BM et al (2001) (Poly)phenolic compounds in pollen and spores of Antarctic plants as indicators of solar UV-B. Plant Ecol 154:11–26 Rozema J, Björn LO, Bornman JF, Gaberščik A, Häder DP et al (2002) The role of UV-B radiation in aquatic and terrestrial ecosystems–an experimental and functional analysis of the evolution of UV-absorbing compounds. J Photochem Photobiol B Biol 66:2–12 Rozema J, Boelen P, Blokker P (2005) Depletion of stratospheric ozone over the Antarctic and Arctic: responses of plants of polar terrestrial ecosystems to enhanced UV-B, an overview. Environ Pollut 137:428–442 Rozema J, Boelen P, Solheim B, Zielke M, Buskens A et al (2006) Stratospheric ozone depletion: high arctic tundra plant growth on Svalbard is not affected by enhanced UV-B after 7 years of UV-B supplementation in the field. Plant Ecol 182:121–135 Ryan KG, Hunt JE (2005) The effects of UVB radiation on temperate southern hemisphere forests. Environ Pollut 137:415–427 Sampson BJ, Cane JH (1999) Impact of enhanced ultraviolet-B radiation on flower, pollen, and nectar production. Am J Bot 86:108–114 Santos I, Fidalgo F, Almeida JM, Salema R (2004) Biochemical and ultrastructural changes in leaves of potato plants grown under supplementary UV B radiation. Plant Sci 167:925–935 Sarma AD, Sharma R (1999) Anthocyanin–DNA copigmentation complex: mutual protection against oxidative damage. Phytochemistry 52:1313–1318 Schmitz-ho*rner R, Weissenböck G (2003) Contribution of phenolic compounds to the UV-B screening capacity of developing barley primary leaves in relation to DNA damage and repair under elevated UV-B levels. Phytochemistry 64:243–245 Searles PS, Flint SD, Caldwell MM (2001) A meta-analysis of plant field studies simulating stratospheric ozone depletion. Oecologia 127:1–10 Searles PS, Flint SD, Diaz SB, Rousseaux MC, Ballaré CL, Caldwell MM (2002) Plant response to solar ultraviolet-B radiation in a southern South American Sphagnum peatland. J Ecol 90:704–713 Semerdjieva SI, Sheffield E, Phoenix GK, Gwynn-Jones D, Callaghan TV, Johnson GN (2003) Contrasting strategies for UV-B screening in sub-Arctic dwarf shrubs. Plant Cell Environ 26:957–964 Shinkle JR, Atkins AK, Humphrey EE, Rodgers CW, Wheeler SL, Barnes PW (2004) Growth and morphological responses to different UV wavebands in cucumber (Cucumis sativum) and other dicotyledonous seedlings. Physiol Plant 120:240–248 Shulski MD, Walter-Shea EA, Hubbard KG, Yuen GY, Horst G (2004) Penetration of photosynthetically

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K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants Manuel Nieves-Cordones, Fernando Alemán, Mario Fon, Vicente Martínez, and Francisco Rubio

Abstract

The increasing world population makes high yield crop production a necessity in future agriculture. However, the negative effects of the emission of greenhouse gases into Earth’s atmosphere and the resulting climate change may impede reaching this goal. New stresses will appear and the existing ones will be exacerbated. Important plant processes such as the acquisition of K+, which is an essential macronutrient for plants, will be negatively affected. The development of new crop varieties with enhanced capacities in the acquisition of K+, especially under the future environmental conditions, is an important challenge. One of the first steps may be the identification of the K+ uptake systems operating in the roots, which may be later improved to enhance K+ acquisition under stress conditions. Some gene families encoding K+ transporters, that is, the HAK1-type, and channels, that is, the AKT1-type, key pieces for root K+ uptake, have been identified. Members of other families of transport systems, such as the cation proton antiporter (CPA) family, or the cyclic nucleotidegated channel (CNGC) family may also participate in that process. The use of T-DNA insertion lines in the model species Arabidopsis thaliana has allowed the demonstration of the role of some of these transport systems and, in some cases, the results obtained in the model plant also apply to crop species. Important points of the regulation of these transport systems are found at the transcriptional and the translational level. Internal K+ concentrations, plasma membrane potential, reactive oxygen species (ROS), hormones, kinases, and phosphatases are involved in their regulation. The knowledge accumulated to date and that to be obtained in the future could be used in biotechnological approaches to produce more efficient K+ transporters that endow plants with enhanced performance to face the future environmental challenges. Genetic engineering, natural variability, and the development of -omic technologies are valuable tools for the achievement of these objectives. F. Rubio () • M. Nieves-Cordones • F. Alemán • M. Fon • V. Martínez Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus de Espinardo, Murcia 30100, Spain e-mail: [emailprotected] P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_4, © Springer Science+Business Media, LLC 2012

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Keywords

Potassium • Transporters: Signaling molecules • Abiotic stress • Genetic engineering

1

Introduction

The increasing world population makes high yield crop production a necessity in future agriculture. One important factor that will affect agriculture worldwide and that prominent climate scientists have been warning of is the dangerous effects of the continual emission of greenhouse gases into Earth’s atmosphere. Atmospheric CO2 concentration, average temperature, and tropospheric ozone concentration will be higher in the near future. Droughts will be more frequent and severe, more intense precipitation events will lead to increased flooding, some soils will degrade, and climatic extremes will be more likely to occur. Existing abiotic stresses such as salinity will be exacerbated. All these changes will produce an environmental stress for plants and will have many important implications for plant physiology and crop yield. Among the processes that will be altered, K+ nutrition deserves special attention. K+ is an essential macronutrient that is absorbed from the soil solution by the roots. Environmental stress may reduce K+ availability and at the same time may produce physiological alterations that impair K+ acquisition. The development of plant varieties with increased capacity to absorb K+ may contribute to reducing the negative effects of the abiotic stress conditions derived from climate change. Characterizing the systems involved in the process of K+ absorption and how they are regulated and the effect that abiotic stresses will have on their functioning will help to obtain the required new plant varieties (Fedoroff et al. 2010).

K+ functions can be classified into those that rely on high and relatively stable concentrations of the nutrient in certain cellular compartments or tissues and those that rely on its movement between different compartments, cells, or tissues (Amtmann et al. 2004). The first class of functions includes enzyme activation, stabilisation of protein synthesis, neutralization of negative charges on proteins, and maintenance of cytoplasmic pH homeostasis (Marschner 1995). Other roles of K+ are linked to its high mobility. This is particularly evident where K+ movement is the driving force for osmotic changes – as, for example, in stomatal movement, light-driven and seismonastic movements of organs, and phloem transport. In other cases, K+ movement provides a charge balancing counter-flux essential for sustaining the movement of other ions. Thus, sugar, amino acid, and NO3− transport can be accompanied by a flux of K+ (Marschner 1995). The most general phenomenon that requires directed movement of K+ is growth. Accumulation of K+ (together with an anion) in plant vacuoles creates the required osmotic potential for cell expansion which relies on its high mobility, and therefore only few other inorganic ions can replace K+ in this role (Reckmann et al. 1990). Once cell growth has come to a halt, maintenance of osmotic potentials can be carried out by less mobile sugars, and K+ ions can be partially recovered from vacuoles (Marschner 1995; Poffenroth et al. 1992).

2.1

Range of K+ Levels Inside the Plant

Role of K+ in Plants

2 +

K is an essential macronutrient for plants, composing up to 10% of the total plant dry weight (Taiz and Zeiger 1991) and fulfilling important functions in metabolism, growth, and stress adaptation.

As for other nutrients, three ranges in internal K+ concentrations can be distinguished (Marschner 1995): a deficient, an adequate, and a toxic range. K+ internal levels found in plants may vary depending on the tissue considered and the

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

nutritional status of the plant, but they are always in the millimolar range. An optimum K+ concentration of around 50–100 mM in the cytosol is required for the performing of the functions mentioned above (Jones 1983). Many of the K+activated enzymes require K+ at concentrations between 10 and 50 mM to achieve their maximum activity and plant cells keep the cytosolic K+ concentration around this value (Walker et al. 1996). In contrast, vacuolar concentrations are more flexible and mirror the external supply of K+. Under K+ limiting conditions, cytosolic K+ activity can be efficiently maintained around its optimum value due to the release of vacuolar K+ ions into the cytosol. Once the electrochemical gradient from the vacuole reaches its limit, cytosolic K+ activity starts to decrease (Nieves-Cordones et al. 2008; Walker et al. 1996, 1998). K+ homeostasis is not necessarily effective in all cell types. In barley epidermal leaf cells, cytoplasmic K+ levels as low as 15 mM have been reported under saline conditions (Cuin et al. 2003). These cells were still alive but they probably showed very low metabolic activity. Indeed, low epidermal K+ concentrations reflect selective tissue allocation of K+ within leaves, which allows the plant to protect metabolically active mesophyll cells against K+ deficiency.

K+ deficiency entails important effects on plant physiology such as limited cellular expansion and reduction of photosynthesis which ultimately results in growth reduction and development impairment. When external K+ is limiting, its translocation from mature leaves and stem is activated. Under conditions of severe deficiency, these organs become chlorotic and even necrotic. At the cellular and metabolic levels, important negative effects are observed. It has been reported that in barley, K+-limiting conditions give rise to a decrease in K+ activity and pH in the cytosol of root cells (Walker et al. 1996). These reductions positively correlated with a decrease in protein synthesis and a subsequent decline in growth (Walker et al. 1998). At the metabolic level, increase in sugars and depletion of pyruvate has been described in roots and shoots (Armengaud et al. 2009). Other changes also take place to (1) maintain carbon flux into amino acids and proteins, (2) decrease negative metabolic charge, and (3) increase the nitrogen–carbon ratio in amino acids. In addition, K+-deficient plants are more susceptible to abiotic and biotic stresses such as drought, cold, salinity, and fungal attacks (Marschner 1995).

3 2.2

Problems Related to K+ Deficiency

Although current agriculture is based on high levels of fertilization, soils of some intensively cultured lands may became K+ deficient (Dobermann et al. 1998; Pal et al. 2001; Rengel and Damon 2008; Yang et al. 2004) because of the withdrawal of K+ during crop harvests (Pal et al. 2001) or due to K+ losses as a result of lixiviation in sandy soil (Kayser and Isselstein 2005). In other cases, the importance of K+ has sometimes been overlooked, and financial constraints have forced farmers to prioritize applications of nitrogen (N) over K+ (Armengaud et al. 2009). As a result, a considerable area of farmland has become K+ deficient (Andrist-Rangel et al. 2007; Dobermann et al. 1998; Hoa et al. 2006).

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K+ Uptake Systems

Root K+ uptake occurs through specialized epidermal cells (root hairs) which increase the surface in contact with the soil solution as well as through cortical cells. K+ must get through root tissues to reach the stele where the xylem vessels will distribute K+ to the rest of the plant. There are two possible parallel pathways that + K can follow to reach the stele: the apoplastic and the symplastic pathways. The apoplastic pathway is blocked by the Casparian strip that limits K+ flux into the stele. Therefore, K+ needs to enter into the symplast to reach the xylem vessels by crossing the plasma membrane of an epidermal or cortical root cell and through K+ transport systems of different capacities, with the latter constituting crucial pieces in the control of K+ acquisition.

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3.1

High- and Low-Affinity K+ Uptake

In the 1950s, of last century Epstein reported that in barley roots, K+ uptake exhibited biphasic kinetics in response to increasing external K+ concentrations (Epstein and Hagen 1952; Epstein et al. 1963). The first system showed high-affinity for K+ (Km for K+ of 21 mM) and was not inhibited by Na+. The second system showed low-affinity for K+ (Km of 11.4 mM) and was inhibited by Na+. In maize roots, the low-affinity component was linear (Kochian and Lucas 1982), and traditionally, it has been thought that ion channels are mostly responsible for the low-affinity component (Maathuis and Sanders 1996). The high-affinity system has been thought to be mediated by transporters because with the reported data on plasma membrane potentials and gradients for K+ concentrations for barley and Arabidopsis (Maathuis and Sanders 1993, 1994; Walker et al. 1996), a channel would not be operative for K+ influx. A K+:H+ symport with a 1:1 stoichiometry has been suggested as the transport mechanism. In Neurospora crassa, a fungus which exhibited high-affinity K+ uptake similar to that present in plants, the high-affinity K+ uptake was shown to be mediated by a K+:H+ symport (Rodriguez-Navarro et al. 1986). In plant cells, as in fungal cells, the membrane potential is sustained by the H+-ATPase, supporting the idea of the plant K+:H+ symport. Other features observed in the high-affinity K+ uptake in plants are the upregulation by K+ starvation, the lack of discrimination between K+ and Rb+ and the inhibition by NH4+ (Kochian and Lucas 1988; Maathuis and Sanders 1996; Rodríguez-Navarro 2000; Rodríguez-Navarro and Rubio 2006).

3.2

Molecular Entities Mediating Low- and High-Affinity K+ Uptake

3.2.1

Shaker Channels

The identification of the genes involved in K+ uptake began in the 1990s by using the functional complementation of Saccharomyces cerevisiae

mutants defective in the endogenous K+ uptake systems TRK1 and TRK2. In 1992, the Arabidopsis Shaker K+ channels AKT1 and KAT1 were isolated (Schachtman 1992; Sentenac et al. 1992). Both AKT1 and KAT1 did not exhibit inactivation through time which suggested that they mediated long-term K+ supply (Gaymard et al. 1996; Schachtman 1992). AtAKT1 expression was preferentially localized in the peripheral cell layers of the root mature regions, which was consistent with a role of AtAKT1 in root K+ uptake (Lagarde et al. 1996). However, AtKAT1 is mainly expressed in leaves discarding its participation in this process (Szyroki et al. 2001). After the cloning of AtAKT1, several cDNAs encoding K+ channels with hom*ology to AKT1 were obtained from other species. For instance, SKT1 from potato (Zimmermann et al. 1998), LKT1 from tomato (Hartje et al. 2000), TaAKT1 from wheat (Buschmann et al. 2000), OsAKT1 from rice (Golldack et al. 2003), DKT1 from carrot (Formentin et al. 2004), CaAKT1 from pepper (Martínez-Cordero et al. 2005), NKT1 from tobacco (Sano et al. 2007), and VvK1.1 from grapevine (Cuellar et al. 2010). Intriguingly, another cDNA from Arabidopsis, AtKC1, encoding a protein with high hom*ology to K+ channels was cloned, but it never exhibited ion currents when expressed alone in heterologous expression systems (Reintanz et al. 2002). Later, it was shown that AtKC1 together with the syntaxin SYP121, regulated AtAKT1 function by forming a ternary complex (Honsbein et al. 2009) (see below). hom*ologues of AtKC1 have also been found in other plant species (Downey et al. 2000; Wang et al. 2002).

3.2.2 HAK/KT/KUP Transporters High-affinity K+ uptake in plants resembles that mediated by SoHAK1, a high-affinity K+ transporter from the yeast Schwanniomyces occidentals. By using an RT-PCR approach, a plant hom*olog, HvHAK1, was isolated from barley (Santa-María et al. 1997). Yeast expression of HvHAK1 demonstrated that it was a high-affinity K+ transporter that did not discriminate between K+ and Rb+ and was inhibited by Na+ and NH4+, in agreement with the characteristics of high-affinity

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

K+ uptake previously described in barley roots. hom*ologous transporters were later isolated in Arabidopsis (Fu and Luan 1998; Kim et al. 1998; Quintero and Blatt 1997). One of them, AtHAK5, showed a high hom*ology to HvHAK1 and no discrimination between K+ and Rb+ (Rubio et al. 2000). Phylogenetic analyses show that the HAK family (also named KT or KUP family) of transporters is composed of four clusters and that all high-affinity K+ transporters from this family characterized so far belong to Cluster I. These Cluster I transporters are encoded by genes that are rapidly upregulated in root cells when the supply of the external K+ is removed (Rodríguez-Navarro and Rubio 2006). When expressed in yeast they mediate K+ uptake of similar characteristics to plant high-affinity K+ uptake such as low Km values for K+ or sensitivity to NH4+, suggesting that they are major components of this system (Rodríguez-Navarro and Rubio 2006). After AtHAK5 cloning, numerous genes encoding K+ transporters of the HAK family were identified in other plant species: 17 genes in rice (Bañuelos et al. 2002), GsKT1 in cotton (Ruan et al. 2001), LeHAK5 in tomato (Wang et al. 2001, 2002), CnHAK1 and CnHAK2 in Cymodocea nodosa (Garciadeblas et al. 2002), four genes in Mesembrianthemum cristallinum (Su et al. 2002), LjKUP in Lotus japonica (Desbrosses et al. 2004), VvKUP1 and VvKUP2 in Vitis vinifera (Davies et al. 2006), AlHAK in Aeluropus littoralis (Su et al. 2007), five genes in Phragmites australis (Takahashi et al. 2007b, c), CaHAK1 in pepper (Martínez-Cordero et al. 2004), and ThHAK5 in Thellungiella halophila (Alemán et al. 2009b).

3.2.3 HKT Transporters In 1994, a wheat cDNA, TaHKT1, was isolated, and it encoded a protein with hom*ology to the TRK transporters from yeast (Schachtman and Schroeder 1994). Its heterologous expression showed that TaHKT1-mediated high-affinity K+ uptake coupled with Na+ symport in addition to low-affinity Na+ uptake (Rubio et al. 1995). Because Na+-activated high-affinity K+ uptake has never been demonstrated in higher plants, the

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involvement of this system in high-affinity K+ uptake seems unlikely. Later studies have involved HKT transporters from different plant species to Na+ uptake from the external solution and Na+ movements within the plant through loading and unloading at the xylem and the phloem (Berthomieu et al. 2003; Garciadeblas et al. 2003; Golldack et al. 2002; Haro et al. 2005; Horie et al. 2007; Laurie et al. 2002; Rus et al. 2001, 2004; Uozumi et al. 2000). This has focused the research on the physiological roles of plant HKT towards adaptation to saline environments rather than K+ nutrition.

3.2.4

Cation Proton Antiporters

This superfamily of antiporters is composed of three families: the monovalent cation:proton antiporter-1 (CPA1) family (eight members), the monovalent cation:proton antiporter-2 (CPA2) family also referred to as the CHX family (28 members), and the NhaD family (two members) (Maser et al. 2001; Saier et al. 1999). In plants, Na+/H+ and K+/H+ antiporters belonging to the first family (CPA1) are likely critical determinants of salt tolerance and K+ homeostasis either by compartmentalization in cellular organelles, for instance NHX transporters, or by extrusion from the cell, for example SOS1 transporters (Apse et al. 1999; Leidi et al. 2010; Pardo et al. 2006; Rodriguez-Rosales et al. 2008; Shi et al. 2003). On the other hand, some members of the CPA2 family have been related to K+ acquisition. KEA5, AtCHX17, and AtCHX13 are upregulated in K+-starved roots (Cellier et al. 2004; Shin and Schachtman 2004; Zhao et al. 2008). AtCHX13 was indeed shown to complement a K+ uptake deficient yeast mutant showing a Km K+(Rb+) of 136 mM (Zhao et al. 2008), although its expression in the root tip suggested a role in sensing external K+ rather than mediating K+ uptake.

3.2.5 Cyclic Nucleotide-Gated Channels Cyclic nucleotide-gated channels (CNGCs) share structural hom*ology with Shaker channels, although they lack a GYGD motif (Szczerba et al. 2009; Talke et al. 2003). Published data has shown that their activation is cGMP- and/or

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cAMP-dependent and that most CNGCs do not discriminate among monovalent cations, have a limited Ca2+ permeability, and are blocked by NH4+ and external Mg2+ (Balague et al. 2003; Demidchik and Maathuis 2007; Leng et al. 2002). A notable exception is AtCNGC2 that exhibited a high degree of K+ selectivity as opposed to Na+ (Hua et al. 2003), a feature that is unknown in animal CNGCs. In particular, some CNGCs have shown features related to K+ uptake. For instance, AtCNGC10 was shown to rescue K+ channel mutants of Escherichia coli (LB650), yeast (CY162), and atakt1 Arabidopsis plants (Kaplan et al. 2007). In heterologous systems, AtCNGC3 can function as a Na+ and K+ uptake pathways (Gobert et al. 2006). Moreover, promoter-driven GUS activity data has shown that, in seedlings, AtCNGC3 is mainly expressed in epidermal and cortical root tissues, something consistent with a role in K+ uptake.

3.3

Advances in the Characterization of Entities Mediating Root K+ Uptake

Although the characterization in heterologous systems together with gene expression patterns strongly suggested that many of the aforementioned entities participated in root K+ uptake, a clear demonstration was still pending. It was necessary to demonstrate the transport activity of such systems in vivo, in their native environment. Furthermore, a subcellular localization compatible with K+ uptake (plasma membrane localization) was also required. The first successful study on this topic demonstrated that an Arabidopsis T-DNA knock-out mutant in AKT1, atakt1-1, had reduced plant growth in the presence of 2 mM NH4+ when external K+ was below 1 mM (Hirsch et al. 1998). These results suggested that K+ channels could also mediate K+ uptake in the high-affinity range of concentrations. Further studies showed the presence of two components of K+ uptake: an NH4+-insensitive AKT1-mediated component and an NH4+-sensitive component (Spalding et al. 1999). A later study showed that AKT1 fused to

GFP localized to the plasma membrane in tobacco mesophyll protoplasts (Hosy et al. 2005). AtAKT1 function is regulated by the AtKC1 subunit, whose gene is strongly expressed in roots (Reintanz et al. 2002) and upregulated by salt stress in the shoot (Pilot et al. 2003). When AtKC1 is coexpressed together with AtAKT1 in heterologous systems, a shift in the activation threshold toward more negative values is observed (Duby et al. 2008; Geiger et al. 2009), probably preventing AKT1 from mediating K+ efflux. Recently, it has been shown that AtKC1 forms a ternary complex with AtAKT1 and a syntaxin (SYP121) that mediates K+ uptake in Arabidopsis roots (Honsbein et al. 2009). When expressed in Xenopus oocytes, the characteristics observed in this ternary complex are more similar to those observed in native Arabidopsis roots than those recorded without the syntaxin (Honsbein et al. 2009), indicating that in planta, the functional unit is the ternary complex. As for HAK transporters, experiments performed with T-DNA insertion mutants in Arabidopsis demonstrated that root high-affinity K+ uptake was impaired in the athak5-1, athak5-2, and athak5-3 mutants which lacked a functional AtHAK5, (Gierth et al. 2005; Rubio et al. 2008). Moreover, in the athak5 plants, NH4+ did not inhibit Rb+ uptake, while this cation did so in WT plants, indicating that AtHAK5 mediates the NH4+-sensitive high-affinity K+ uptake in plants, in agreement with the sensitivity to NH4+ that AtHAK5 shows when expressed in yeast (Rubio et al. 2000). Recently, it has been shown that adult athak5-3 plants display lower plant biomass due to reduced K+ uptake when they are grown for several weeks at 10 mM K+ (Nieves-Cordones et al. 2010), evidencing that this transporter supports growth at very low-external K+ concentrations. These results are in agreement with another study performed in agarose plates in which AtHAK5 mutants seedlings exhibited reduced root growth when grown below 10 mM K+ (Qi et al. 2008). Moreover, in this study, it was also reported that AtHAK5 fused to an epitope of the human influenza virus hemaglutinin protein (HA epitope) localizes to the plasma membrane.

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

Fig. 4.1 Molecular entities participating in K+ uptake in Arabidopsis roots. The graphic shows the external K+ concentrations at which the high- and the low-affinity K+ uptake systems operate. In the high-affinity range, AtHAK5 is the only system mediating K+ uptake when the external concentration is below 10 mM. At higher concentrations, AtHAK5, AtAKT1, and AtCHX13 may be the main systems participating in this process. In the low-affinity range of K+ concentrations, AtAKT1, AtCHX13, unidentified members of the CNGC or unknown systems could contribute to K+ uptake. Dashed horizontal lines depict the range of external K+ concentrations in which the system just above plays its important role in root K+ uptake

As stated above, AtAKT1 also contributes to K+ uptake in the high-affinity range of concentrations. Recent studies with single athak5, atakt1 and double athak5, atakt1 mutants in combination with NH4+ and Ba2+ that inhibit AtHAK5 and AtAKT1, respectively, have allowed the establishment of the relative contributions of each of these two systems to K+ uptake from diluted solutions (Rubio et al. 2010). AtHAK5 is the only system-mediating K+ uptake at concentrations below 10 mM. Between 10 and 50 mM K+, AtHAK5 and AtAKT1 have been demonstrated to be the only systems contributing to K+ uptake. Above 50 mM K+, both systems are thought to act, and at concentrations higher than 200 mM, the contribution AtHAK5 decreases and the only system operating is probably AtAKT1, although the contribution of other unknown systems cannot be ruled out (Fig. 4.1). Recent research has demonstrated that members of the HKT transporter/channel family

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mediate important Na+-tolerance mechanisms in plants mainly by improving K+/Na+ homeostasis. Previous work based on the identification of a quantitative trait locus (QTL) determining salt tolerance showed that Kna1 in Triticum aestivum controls the selectivity of Na+ and K+ transport to shoots, resulting in a high K+:Na+ ratio in leaves (Dubcovsky et al. 1996; Gorham et al. 1987, 1990; Luo et al. 1996). Other loci identified by QTL analyses in durum wheat, Nax1 and Nax2, also contributed to salt tolerance (Lindsay et al. 2004; Munns et al. 2003). Furthermore, the presence of Nax1 and Nax2 was shown to enhance K+ accumulation in leaf blades and sheaths, leading to the model that Nax1 and Nax2 mediate K+loading into the xylem (James et al. 2006). These QTLs are attributable to polymorphisms in copies of wheat HKT genes, TmHKT1;4-A1 and TmHKT1;4-A2 (also named TmHKT7-A1 and -A2) for Nax1 and TmHKT1;5 and TaHKT1;5 for Nax2 and Kna1 (Byrt et al. 2007; Huang et al. 2006). Independent analysis of another QTL in rice, named SKC1 (shoot K+ content), resulted in an identical model for the function of the rice gene OsHKT1;5 (Ren 2005). The SKC1 QTL was due to point mutations in OsHKT1;5 that replace several amino acid residues in the salttolerant cultivar NonaBokra (Ren 2005). Studies performed in different null mutant types of the class I HKT transporter AtHKT1 has shed light into the mechanisms by which this entity controls Na+/K+ homeostasis under salt stress. According to these studies, AtHKT1 may be involved in Na+ recirculation in plants by operating in Na+ loading in the phloem (Berthomieu et al. 2003) or removal of Na+ from the xylem preventing the accumulation of Na+ in the leaves (Davenport et al. 2007; Horie et al. 2006; Sunarpi et al. 2005). In agreement with this model, AtHKT1 overexpression in the root pericycle improves salt tolerance (Moller et al. 2009). Concerning K+ homeostasis, mutations in OsHKT1;5 and AtHKT1 have also been found to reduce K+ and enhance Na+ accumulation in shoots during salt exposure, contributing to enhanced salinity stress (Ren 2005; Sunarpi et al. 2005). Interestingly, the disruption of AtHKT1 in mutants of the salt overly sensitive (SOS) pathway

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prevented the K+-deficiency symptoms observed in sos mutants when grown at low K+ concentrations and improved cellular K+/Na+ ratios when compared with single sos mutants under saline conditions (Rus et al. 2001, 2004). All the results described above refer to HKT transporters belonging to the class I of this family. They have been shown to mediate Na+ transport and the effect upon K+ nutrition may be indirect. On the other hand, some members of the Class II of HKT transporters can operate as Na+–K+ symporters under some conditions (Haro et al. 2005; Jabnoune et al. 2009; Rubio et al. 1995), and a contribution to K+ uptake may be expected. However, mutations in the OsHKT2;1 gene do not have a strong impact on high-affinity K+ (Rb+) uptake into intact rice roots (Horie et al. 2007). Many of the presently characterized HKT class two genes are also induced by K+ starvation, including those in wheat, barley, and rice (Garciadeblas et al. 2003; Horie et al. 2001; Wang et al. 1998). Therefore, in addition to mediating K+ uptake, this system mediates Na+ uptake, allowing Na+ to act as a substitute nutrient for K+ in K+-starved rice plants under moderate Na+ concentrations (Horie et al. 2007), supporting the long-standing hypothesis that Na+ may substitute for K+ when this nutrient is scarce (Flowers et al. 1983; Mengel and Kirkby 1982). Regarding CHX transporters, it has been shown that disruption of AtCHX17 led to lower root K+ concentrations under saline and K+deprivation conditions, although a subcellular localization and a functional characterization for AtCHX17 remain to be assessed (Cellier et al. 2004). On the other hand, AtCHX13 was localized to the plasma membrane and AtCHX13 knock-out and over-expressing mutant plants showed impairment and enhancement of K+ uptake, respectively (Zhao et al. 2008). All these results suggested that this transporter may be involved in K+ uptake in planta. With respect to CNGCs, AtCNGC10 is targeted to the plasma membrane, transports both K+ and Na+ and partially rescues Arabidopsis akt1 mutant plants (Kaplan et al. 2007). Results obtained in antisense lines of this channel, which displayed lower expression than WT plants,

indicated a role in Na+/K+ homeostasis in roots by providing a pathway for Na+ influx and K+ efflux at the root/soil interface (Guo et al. 2008). Similar results were obtained for AtCNGC3. A null mutation in this channel altered both short-term Na+ influx and K+ uptake at high-external K+ conditions, suggesting an alternate role in nonselective monovalent cation uptake at the plasma membrane level (Gobert et al. 2006).

4

Regulation of the Transport Systems

4.1

Transcriptional Regulation

In general terms, genes encoding AKT1-like K+ channels do not show great differences in expression levels in response to the external supply of K+ as it has been observed for AtAKT1 and CaAKT1 (Lagarde et al. 1996; Martínez-Cordero et al. 2005; Pilot et al. 2003). However, K+ withdrawal from the growth solution increased TaAKT1 transcript levels (Buschmann et al. 2000), and NaCl treatments or hormonal addition to the external medium produce changes in AtAKT1 expression (Kaddour et al. 2009; Pilot et al. 2003). Substantial differences are found in the expression pattern of the genes encoding HAK1-type K+ transporters in comparison to that observed in the aforementioned genes for K+ channels. It seems that an important point in the regulation of this type of transporters resides in the control of gene expression. K+-starvation is a common inducer in the gene expression of the HAK1-type K+ transporters. This induction has been observed in HvHAK1 (Santa-María et al. 1997), AtHAK5 (Ahn et al. 2004; Armengaud et al. 2004; Gierth et al. 2005; Qi et al. 2008; Shin and Schachtman 2004), OsHAK1 (Bañuelos et al. 2002), LeHAK5 (Nieves-Cordones et al. 2007; Wang et al. 2002), CaHAK1 (Martínez-Cordero et al. 2004), and ThHAK5 (Alemán et al. 2009b). The reduction in root K+ concentration below a threshold level has been proposed as the stimulus that could trigger the increase in the transcription of these genes (Martínez-Cordero et al. 2005).

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

Some other factors have been shown to considerably modify gene transcription of the HAK1type genes. CaHAK1 expression in K+-starved plants was reduced, if half of the nitrogen of the growth solution was supplied as NH4+ (MartínezCordero et al. 2005). Similarly, AtHAK5 promoter activity was diminished in K+-starved plants after exposure to NH4+ (Qi et al. 2008). Conversely, HvHAK1 transcript levels were increased by the presence of NH4+ in the growth solution in K+-sufficient plants (Fulgenzi et al. 2008). Intriguingly, LeHAK5 was downregulated when NO3− was supplied again after a withdrawal period (Wang et al. 2001). Another case in the regulation of HAK transporters was the increase in AtHAK5 mRNA levels after exposing plants to sucrose in the absence of light (Lejay et al. 2008). Similar results were found previously in Arabidopsis when plants were grown in the sucrose-containing media MS (Rubio et al. 2000): It was observed that in K+-sufficient plants AtHAK5 expression was high; in this medium, K+ starvation did not further increase the expression levels, probably because K+ was substituted for NH4+ in these experiments. Another important factor affecting the expression of these genes seems to be the presence of salinity. This was illustrated, for example, in the decrease of LeHAK5 (Nieves-Cordones et al. 2007), AtHAK5, and ThHAK5 (Alemán et al. 2009b) transcripts when plants were starved of K+ in the presence of Na+. Importantly, salinity decreased, to a lesser extent, the levels of ThHAK5 mRNA in T. halophila than those of AtHAK5 transcripts in Arabidopsis. There is not much information about the signal transduction elements involved in the induction of HAK-type K+ transporters expression. In 2004, the important role of reactive oxygen species (ROS) in the signaling events after removing K+ from the growth solution was described for Arabidopsis (Shin and Schachtman 2004). Recently, it has been proposed that ethylene signaling acts upstream the increase of ROS during K+ deprivation (Jung et al. 2009). In tomato plants, LeHAK5 expression levels correlated with steady plasma membrane potentials registered in root cells (Nieves-Cordones

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et al. 2008) and high-affinity K+ uptake (NievesCordones et al. 2007). Changes in plasma membrane potentials are one of the first signals that root cells sense after a stress is applied (Wang and Wu 2010). In tomato roots, the recorded plasma membrane potentials were importantly affected by long-term changes in the composition of the growth solution. For instance, the presence of NH4+ and K+ starvation hyperpolarized and Na+ depolarized plasma membrane potentials, which produced an increase and a decrease in the LeHAK5 mRNA levels, respectively. Short-term exposure of depolarizing agents such as CCCP or Vanadate to K+-starved roots also decreased LeHAK5 expression. These changes in LeHAK5 expression at both long- and short-term denoted a tight regulation at the transcription level and they also indicated that LeHAK5 contribution to K+ uptake could be limited to some specific conditions even if K+ deficiency was still present (Nieves-Cordones et al. 2008).

4.2

Post-translational Regulation

Recent studies have gained insights into AtAKT1 regulation. By analyzing mutants sensitive to low K+ stress, a CBL-interacting protein kinase, CIPK23, turned out to be essential in the activation of AtAKT1, therefore permitting Arabidopsis plants to grow under low K+ conditions (Xu et al. 2006). Two positive regulators of CIPK23, CBL1, and CBL9, which are two Calcineurin B-like proteins, were also found. Both CBL’s could phosphorylate CIPK23 which became active after this phosphorylation (Fig. 4.2). Activation of the CBL’s depended on Ca2+ as demonstrated by patch-clamp recordings (Li et al. 2006). Later on, it was shown that the network was more complex, being CIPK6 and CIPK16, in addition to CIPK23, able to interact with CBL1, CBL2 and CBL3 and CBL9 (Lee et al. 2007). Furthermore, a PP2C phosphatase (AIP1) inactivated AKT1 by dephosphorylating the latter. AIP1 bound AKT1 through AKT1’s ankyrin domain. In addition, formation of heterotetramers of AKT1 subunits with AtKC1 also serves as another mode of regulation in which such heterotetramers display different

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changes in the K+ status or to direct/indirect regulation of K+ uptake, suggesting a role in this process. In some cases, significant advances in the mechanisms by which some of these molecules regulate K+ nutrition have been reported (Fig. 4.3).

Fig. 4.2 Regulation of the activity of AtAKT1 by the CBL–CIPK complex and by the phosphatase AIP1. (a) Low K+ stress provokes an increase in ROS levels that can possibly be translated into a cytosolic Ca2+ wave. This wave mainly activates CBL1, CBL9, and CIPK23. The activated CBL/CIPK complex phosphorylates the AtAKT1 channel, resulting in its activation (channel open). (b) At high-external K+, the AIP1 phosphatase is thought to dephosphorylate AtAKT1, resulting in the inactivation of the channel (channel closed)

voltage dependence and sensitivity to external K+ (Duby et al. 2008; Geiger et al. 2009). The only report in which the regulation of HAK1-like K+ transporters at the protein level was discussed revealed that HvHAK1-mediated Rb+ uptake in yeast cells was modulated by the HAL4/5 kinases and the PPZ1 phosphatase (Fulgenzi et al. 2008). Both types of enzymes seemed to negatively regulate HvHAK1 activity in K+-starved yeast cells.

4.3.1 ROS and Ethylene ROS have been shown to accumulate in a discrete region of roots active in K+ uptake and they are translocated during low K+ stress (Shin and Schachtman 2004). Knock-out of an NADPH oxidase (rhd2 plants) prevented upregulation of genes that are normally induced by K+ starvation, such as AtHAK5, but the high-affinity K+ uptake remained unchanged. Application of H2O2 restored the expression of those genes induced by K+ deficiency in rhd2 plants. Both ethylene production and the transcription of genes involved in ethylene biosynthesis also increased when plants were deprived of K+ (Jung et al. 2009; Shin and Schachtman 2004). However, in the ethylene insensitive2-1 (ein2-1) mutant, the ethylenemediated low K+ responses were not completely eliminated, suggesting that some K+ deprivationinduced responses are either ethylene independent or EIN2 independent. Ethylene signaling stimulated the production of ROS and thereby it seems to constitute an earlier step in low K+ response. Nevertheless, elements acting upstream ethylene signaling in the onset of low K+ responses are still unknown. These results suggested that K+-dependent regulation of AtHAK5 mRNA levels relied, at least to some extent, on this ethylene-ROS pathway. 4.3.2

4.3

Signaling Molecules: ROS and Hormones

ROS and hormones represent two of the most important signaling mechanisms in plants. They are produced when stimuli act upon certain types of cells and they constitute an effective means of communication between cells, adjusting plant to the new environmental conditions. In recent years, several hormonal activities and ROS have been related to responses that take place after

Abscissic Acid

Abscissic acid (ABA)-mediated control of ROS levels has been invoked to explain its function in protecting plants against oxidative conditions caused by many stress conditions, including nutritional deficiencies (Rubio et al. 2009). For instance, K+ deprivation increases ABA levels both in the root and in the shoot (Kim et al. 2009) that would produce, among the responses of this hormone, a decrease in plant transpiration. Moreover, exposure of Arabidopsis roots to ABA evoked a dramatic reduction in the transcript levels of SKOR, the

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Fig. 4.3 Hormonal responses in relation to K+ uptake. Root apex is an important source of cytokinins and when it is removed, rapid efflux of K+ is observed. Such efflux can be reversed by adding cytokinins. K+ transport through TRH1 greatly contributes to auxin gradients in the root and therefore supporting auxin responses such as gravitropic growth and root hair development. Low-external K+ concentrations trigger stress-related pathways by increasing ethylene, abscisic acid (ABA), and Jasmonic acid

levels. Ethylene activates reactive oxygen species (ROS) production, which mediates AtHAK5 upregulation and, in turn, high-affinity K+ uptake. The ABA increase reduces stomatal aperture in leaves and in roots, it induces SKOR downregulation and thereby decreases K+ xylem load. Jasmonic acid production activates several pathways which aim at enhancing nutrient recycling, storage, and allocation processes

outward-rectifier K+ channel involved in K+ release in the xylem (Gaymard et al. 1998). Interestingly, important reductions in SKOR mRNA levels in the roots were also observed due to K+-starvation (Pilot et al. 2003), suggesting that this reduction could be related to the ABA increase under low K+ growth conditions.

to these plants downregulated the expression of 19 JA biosynthesis-related genes. Interestingly, these transcriptional responses were observed mainly in the shoot, but not in the root, indicating that there is organ specificity in the K+mediated control of JA accumulation. This specificity could be related to the increased nutrient recycling observed in senescent leaves of K+-deprived plants, a process that is triggered by JA (He et al. 2002). If we consider the exact position of ethylene and jasmonate signals within the K+ starvation response, there is still a gap to be filled.

4.3.3

Jasmonic Acid

Upregulation of the genes controlling the first three steps of jasmonic acid (JA) biosynthesis has been observed in K+-deprived plants (Armengaud et al. 2004). In contrast, K+ resupply

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4.3.4 Auxins Both physiological and molecular evidences show a relation between K+ transport and polar auxin transport. For example, it was observed that Arabidopsis mutant plants that lack activity of the K+ transporter TRH1 (AtKUP4/AtKT3) displayed agravitropic root growth and impaired root hair development and that these defects correlated with altered auxin transport or perception, as shown by the fact that addition of auxins rescued these trh1 phenotypes (Vicente-Agullo et al. 2004). K+ status may also affect auxin accumulation, as observed by reduced expression of genes controlling auxin biosynthesis, such as CYP79B2 and CYP79B3, when K+-deprived Arabidopsis plants were resupplied with this nutrient (Armengaud et al. 2004). Conversely, a role for auxins in the control of K+ homeostasis is supported by the fact that treatment of maize plants with auxins increased expression of the gene encoding the K+ transporter ZMK1 (Philippar et al. 1999). Such transcriptional induction is in agreement with the increase in K+ transport observed in maize coleoptiles protoplasts upon auxin application. These results contrast with those found in Arabidopsis in which exposure to the synthetic auxin 2,4 D drastically reduced AKT1, AtKC1, and SKOR root mRNA levels (Pilot et al. 2003). 4.3.5 Cytokinins Cytokinins have long been implicated in the regulation of K+ transport in plants (Abutalybov et al. 1980; Abutalybov and Akhundova 1982; Alizade et al. 1988; Green and Muir 1979; Shabala et al. 2009). It has been described that K+ release into the xylem (as measured by 86Rb+ release into root exudates) is inhibited by micromolar concentrations of kinetin (Collins and Kerrigan 1974; Hong and Sucoff 1976; Rains 1969). These observations are in agreement with the important decrease in the SKOR root transcript levels after exposure to benzyladenine (Pilot et al. 2003). In another study (Albacete et al. 2009), it was shown that xylem K+ (but not Na+) concentration was strongly correlated with leaf size and maintenance of the photosynthetic apparatus in tomato under salt stress, as was leaf xylem zeatin concentration, highlighting another interesting cytokinin–K+ interaction.

At the root level, cytokinins seemed to play a role in the regulation of K+ fluxes in root epidermis (Shabala et al. 2009). Removal of the root apex, an important source of cytokinins, evoked significant K+ efflux from root segments that was rapidly reversed by the addition of exogenous kinetin. Regarding regulation of K+ transport systems, exposure of roots to the synthetic cytokinin, benzyladenine, produced a rapid decrease in the mRNA levels of the Shaker subunits AKT1 and AtKC1 in addition to the one previously mentioned of SKOR (Pilot et al. 2003). This effect of benzyladenine mirrors that observed after the addition of exogenous auxins. Nonetheless, studies revealing the underlying mechanisms of this interaction cytokinins–K+ are lacking.

5

The Arabidopsis Model

5.1

Integration of Uptake Systems

Presently, root K+ uptake systems from Arabidopsis are the best characterized and it is possible to integrate all the information available to generate a comprehensive model. Plants show a high concentrative capacity for K+ (Martínez-Cordero et al. 2005; Nieves-Cordones et al. 2007), mediated by an active transport mechanism (Maathuis and Sanders 1994) through HAK transporters. As described in a previous section, the disruption of AtHAK5 led to lower K+ uptake rates and growth impairment at 10 mM K+ and lower K+ concentrations (Nieves-Cordones et al. 2010). Together with the micromolar Km obtained when AtHAK5 was expressed in yeast (Rubio et al. 2000), this suggested that at the lowest K+ concentrations, AtHAK5 is the only system mediating root K+ uptake, in agreement with the higher Km values in athak5 plants (Gierth et al. 2005). AtHAK5mediated uptake probably occurs via H+–K+ symport, as membrane potentials registered in root cells were not negative enough to drive passive flux of K+ into root cells. Currently, the mechanism of transport through AtHAK5 remains elusive. When external K+ concentrations are above 10 mM K+, AtAKT1 becomes an active player. In such conditions, the electrochemical gradient is

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

compatible with K+ fluxes through channels and therefore AtAKT1 could mediate K+ uptake. Indeed, this idea is further supported by the fact that athak5 mutants did not display a defective phenotype at 20 mM K+ (Pyo et al. 2010), exhibited Rb+ uptake above that K+ concentration (Rubio et al. 2008) and only double mutants atakt1,athak5 lacked any of these features (Pyo et al. 2010; Rubio et al. 2010). These results strongly suggested that the channel was mediating K+/Rb+ uptake in this context. It is worth noting that in this range of K+ concentrations, there is an overlap in the K+ uptake capacities between AtHAK5 and AtAKT1 which made the phenotypic study of their corresponding single mutants difficult. AtAKT1 contribution to K+ uptake starts in the low micromolar range, but the upper limit is not well defined. At 1.4 mM K+, atakt1 plants exhibited lower plant biomass and lower tissue K+ concentrations, including an upregulation of AtHAK5 that reflects clear symptoms of K+ deficiency stress (Rubio et al. 2008). Therefore, with growth solutions containing around 1 mM K+, the AtAKT1 function was not compensated by other systems such as AtHAK5, and thus AtAKT1mediated uptake seems to be unique. At higher concentrations, for example, 10 mM K+, atakt1 mutant plants exhibited normal growth and tissue K+ concentrations, denoting that other systems were indeed able to support growth under these conditions (Nieves-Cordones et al. 2010; Rubio et al. 2010). Similar results were obtained in the athak5,atakt1 double mutants showing that AtHAK5 was not important in this range of concentrations. Interestingly, a complete inhibition of K+ uptake and plant growth were not observed in the aforementioned double mutant at 0.5 mM K+ (Rubio et al. 2010), suggesting that unknown systems are mediating K+ nutrition in this athak5,atakt1 line. Candidate systems thought to mediate K+ uptake in this mutant line are AtCHX13 (Zhao et al. 2008) or non-selective channels regulated by cyclic nucleotides (CNGC’s) (Demidchik and Maathuis 2007; Li et al. 2005).

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Usefulness of This Model to Understand Other Species

Arabidopsis has been proven to be a useful plant model for the understanding of physiological processes in other plant species such as tomato, pepper, and T. halophila (Alemán et al. 2009b; Martínez-Cordero et al. 2005; NievesCordones et al. 2007; Rubio et al. 2008). With this in mind, it has been shown that lower upregulation of HAK transporters due to salt stress when plants are deprived of K+ is a common feature observed in barley, tomato, T. halophila, and A. thaliana. In contrast, regulation by NH4+ of HAK transcript levels was found to be different in Arabidopsis with respect to tomato but similar to barley and pepper. Moreover, CIPK–CBL pathways from Arabidopsis (like that regulating K+ uptake through AKT1) are conserved among species and can interact with other Shaker channels from other species such as VvK1.1 from grapevine and ZmK1 from maize (Cuellar et al. 2010; Geiger et al. 2009). This interaction has allowed the production and characterization of functional channels in Xenopus oocytes. On the other hand, comparing Arabidopsis with monocots species becomes more difficult. For instance, genome complexity in monocots implies that some K+ transport systems are found as different isoforms (e.g., HvHAK1A B) that could exhibit differential regulation and contribution, something not yet observed in Arabidopsis. Moreover, HKT transporters belonging to subfamily II are present in monocots and absent in dicots such as Arabidopsis. Therefore, although some general assumptions regarding K+ uptake could be valid for monocots, for example, occurrence of high- and low-affinity components with similar properties or regulation of HAK transporters by K+, NH4+, and Na+, indepth studies addressing the contribution of the different transport systems deserves a monocot model.

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6

Future Environmental Stresses Affecting K+ Acquisition

Current agriculture faces many environmental abiotic stresses such as drought, salinity, or high temperatures. In the future, climate change will worsen the effects of these stresses. In addition, the continuous over-fertilization of crops to maintain production and the irrigation with low-quality water will also produce negative effects. All these abiotic stress conditions will have an important impact on K+ acquisition by the plant (Fig. 4.4).

6.1

High CO2

Stomatal movements and, in turn, plant transpiration are regulated by CO2 levels in the atmosphere. These elevated atmospheric CO2 concentrations could induce stomatal closure and thereby reduce plant transpiration. Implications of low transpiration rates in K+ uptake were studied in short-term experiments (hours) and it was found that the accumulation of K+ by the cells of the root was unaffected by water flux, whereas the passage through the root to the shoot via the

Fig. 4.4 Predicted effects of global warming and consequences over K+ nutrition. An increase in CO2, O3, and temperature is expected, enhancing stomatal closure, and reducing transpiration. Irrigation with low-quality water

vessels was linearly related to it (Bowling and Weatherley 1964). Similarly, K+ transport to the shoot was shown to be increasingly reduced at low transpiration rates (high relative humidity) in comparison to normal conditions, whereas K+ absorption by the root was less affected by such changes in transpiration (Hooymans 1969). A long-term study concluded that, in response to sustained exposure to elevated CO2 concentration, biomass is enhanced by 47% in C3 plants, 21% in CAM plants, and 11% in C4 plants (Poorter and Navas 2003). Generally, elevated CO2 alters root architecture and fine-root turnover (Tingey et al. 2000) and increases the proportions of fine roots and secondary roots, implying an expansion of the rhizosphere (Curtis et al. 1994; Norby 1994; Pregitzer et al. 1995). Several studies have demonstrated that elevated CO2 increases the root to shoot ratio (Norby 1994; Rogers et al. 1996; Rogers et al. 1994; Stulen and den Hertog 1993), thereby improving the capacity of the root system to acquire nutrients from the soil. In fact, it was described that after 2 years of exposure to elevated concentrations of CO2 (around 700 ppm), an increase in the K+, Pi, and N accumulation was observed in every organ in Larix kaempferi, together with an increase in dry matter (Shinano et al. 2007). The enhanced

may cause secondary salinization. Drought and flooding periods may also alter soil moisture triggering nutrient imbalance

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

uptake of nutrients in plants exposed to elevated CO2 resulted from increased root biomass rather than increased root activity. Although, as stated above, elevated CO2 may have a positive effect on biomass production, important adverse effects also take place. Some plant species could respond to high CO2 by a downregulation of net photosynthesis rate, which may be attributed to an end-product inhibition by elevated carbohydrate concentration in the leaf, combined N and P deficiency. In addition, photoinhibition, possibly due to the damage of PS core complexes, may also occur (Reddy 2010).

6.2

High Temperature

High temperatures may be the main factor in global change that could decrease crop yield (Battisti and Naylor 2009). In general, increased soil temperature appears to have a more consistent effect on root nutrient uptake kinetics than elevated CO2 (Bassirirad 2000). A number of studies have demonstrated that changes in soil temperature can directly affect root transport properties for K+ (Siddiqi et al. 1984), NH4+ (Chapin et al. 1986), NO3− (Bassirirad et al. 1991, 1993; Cumbus and Nye 1982), and PO43− (Chapin 1974, 1977; Cumbus and Nye 1985). Temperature sensitive processes, such as root respiration, which affects ion movement across the root, could partially explain the observed changes in nutrient uptake with increasing soil temperature. However, in maize roots, it was observed that the absorption rates for K+ increased with temperature but they reached a maximum between 25 and 35°C, whereas root respiration did not reach its optimum even at 40°C (Bravo and Uribe 1981). Interestingly, the enhanced uptake with temperature was observed in both high- and low-affinity components of K+ uptake, revealing a general improvement in K+ uptake capacity by the root.

6.3

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may lead to lower rainfall values in some lands, which would ultimately produce drought and episodes of water logging and floods in other areas. These opposite water disorders evoke a series of stress responses in plants. First, drought leads plants to a stand-by state in which general physiological processes such as transpiration and growth are greatly diminished to prevent further water loss. K+ homeostasis can also be affected, as K+ deficiency symptoms often appear on drought stressed maize, grain sorghum, and soybeans (Wiebold and Scharf 2003). These symptoms may occur even if the soil tests high for K+. If root growth is inhibited by dry soil, K+ uptake decreases and deficiency symptoms are induced. There are additional problems derived from low water potentials in the soil, like salinity. Water loss from the soil increases salt concentrations (for instance, sodium salts) leading to toxic levels of these salts in the plant and further impairing nutrient homeostasis. Conversely, soil flooding causes oxygen deficiency (hypoxia), leading to a reduction in adequate soil conditions for plant growth (Ponnamperuma 1972), and adversely affecting nutrient uptake in plants (Pezeshki et al. 1999). The mineral nutrition of plants in response to flooding depends on plant species and soil type (Kozlowski 1984; Pezeshki 2001). In floodintolerant species, the concentrations of N, P, and K+ in foliages are often, but not always reduced by flooding (Kozlowski 1984; Pezeshki 1995). Furthermore, flooding and drought together with extreme temperatures could modify chemical and physical properties of the soil, which can lead to reduced nutrients mobility and absorbance or leaching of some individual nutrients. The final outcome resulting from the interaction of these climate changes described above on K+ uptake and nutrition requires further investigation. Nevertheless, it clearly indicates that K+ uptake is going to be negatively affected and special attention is needed in this issue to ensure plant performance in the field.

High and Low Soil Moisture

Variations in moisture levels are mainly driven by precipitation, and are also affected by temperature and other factors that determine evapotranspiration rates. As for water supply by rain, climate change

6.4

Salinity

Most crops are glycophytes (Greenway and Munns 1980) and have been grown in soils with

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low salinity. Thus, the mechanisms developed to absorb, transport, and manage nutrients may not operate as efficiently under salinity as they would under non-saline conditions. Salinity negatively affects plant development and growth and, therefore, reduces crop yield and quality (Laüchli and Epstein 1990). Over 800 million hectares of land worldwide are affected by salinity (Munns 2005), comprising nearly 7% of the world’s total land area and approximately 5% of cultivated land (Amtmann et al. 2004). The expected drought periods that global warming is predicted to cause will entail more water used by farmers. As water resources are becoming more scarce, secondary salinization will be increased as the water quality is likely to be reduced. There are two types of effects of salt stress on plants: osmotic effect and the specific effect. The latter includes the toxic effect, the nutritional imbalance and the oxidative stress. The osmotic effect results from the reduction of the soil water potential due to salt accumulation. Plant cells respond with osmotic adjustment by synthesizing compatible organic solutes and by accumulating ions from the external environment (Niu et al. 1995). By doing this, plants can revert water flow and permit growth (Kurt et al. 1986). The specific effect depends on the salt species present. The most abundant salt under salinity conditions is NaCl and therefore, the specific effect is mainly derived from the high Na+ and Cl− concentrations. Na+ specifically affects K+ nutrition because Na+ and K+ share physicochemical properties and Na+ competes for the K+ binding sites that are essential for the cellular function. More than 50 enzymes are activated by K+ and Na+ cannot replace its function (Bhandal and Malik 1988). Therefore, K+ nutrition under salt stress is greatly impaired.

6.4.1 Salt Stress Affects K+ Nutrition One of the key physiological processes disrupted by high Na+ concentrations is the maintenance of cellular and whole-plant K+ homeostasis (Cakmak 2005; Flowers et al. 1983; Gaxiola et al. 1992; Kader and Lindberg 2008; Kronzucker et al. 2006; Peng et al. 2004; Rains and Epstein 1967; SantaMaría and Epstein 2001; Takahashi et al. 2007a; Zhu et al. 1998). Reductions in K+ tissue

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concentrations can be a consequence of the inhibition of K+ uptake by Na+ (Alemán et al. 2009a; Kochian et al. 1985; Kronzucker et al. 2006), stimulation of root K+ efflux (Lynch and Lauchli 1984; Nieves-Cordones et al. 2010; Shabala et al. 2006) (Cramer et al. 1985; Nassery and Baker 1975), and differential allocation between organs (Graifenberg et al. 1995; Naidoo and Rughunanan 1990). Inhibition of K+ uptake by high-external Na+ concentrations is a consequence of the competition between K+ and Na+ for the K+ uptake systems. The characteristics of such an inhibition may depend on the conditions and species. Thus, the addition of 3 mM Na+ to the growth media resulted in a 50% suppression of K+ influx in maize plants (Kochian et al. 1985), and this effect was limited to the lowaffinity transport range for K+. In barley, highexternal Na+ was shown to inhibit both high- and low-affinity K+ uptake (Kronzucker et al. 2006, 2008) and the same has been observed in many other studies (Botella et al. 1997; Fu and Luan 1998; Martínez-Cordero et al. 2005; Rains and Epstein 1967; Rubio et al. 2000; Santa-María et al. 1997). Differences in Na+ effects may originate from differences in experimental conditions, but in some cases they are due to differences between species. For example, the inhibition of K+ uptake by Na+ has been observed in Arabidopsis and in its salt-tolerant relative T. halophila when grown under several saline conditions. However, the inhibition was less intense in T. halophila in comparison to Arabidopsis (Alemán et al. 2009a). The lower reduction of K+ uptake in T. halophila was concomitant to lower Na+ absorption rates, whereas the contrary was observed in Arabidopsis. It could be concluded that plant species that differ in salt tolerance may have different K+ uptake systems that results in different sensitivities to high-external Na+ which may constitute important tools for improving plant K+ nutrition efficiency under salinity conditions. K+ efflux from salinity-induced plants is another process that contributes to the reduction of tissue K+ concentration. The strong membrane depolarization caused by high Na+ uptake favors K+ efflux via depolarization-activated outwardrectifying K+ channels (Shabala et al. 2006). This is a Na+-specific effect because isotonic mannitol solution causes significant membrane

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

hyperpolarization, resulting in increased K+ uptake (Chen et al. 2005; Cuin and Shabala 2007; Shabala et al. 2000; Shabala and Lew 2002). In agreement with this, net K+ balances in Arabidopsis roots grown for 14 days in the presence of 30 mM NaCl and low-micromolar K+ concentrations reflected a prominent K+ loss (Nieves-Cordones et al. 2010), although other studies have shown that under steady-state conditions high-external Na+ reduced K+ efflux (Kronzucker et al. 2008). Salinity also interferes with K+ sensing because high Na+ suppresses the induction of high-affinity K+ uptake by low K+ (Alemán et al. 2009b; NievesCordones et al. 2007, 2010) leading to lower K+ uptake capacity under these conditions. The reduction in tissue K+ concentrations due to salinity also leads to a redistribution of this macronutrient to maintain its concentration buffered in metabolic active cells. For example, it has been observed that in leaves of plants grown at 50 mM external NaCl, K+ concentrations decreased preferentially in mesophyll cells, whereas at higher salt levels, K+ concentrations decreased only in epidermal cells (Fricke et al. 1996). Since K+ is more compatible with cellular (i.e., cytoplasmic) processes than Na+, K+ is preferentially retained in metabolically active (i.e., mesophyll) compared with metabolically less active (i.e., epidermis) tissues or cell compartments (Cuin et al. 2003).

6.5

Effect of Other Nutrients

The increasing world population makes high yield crop production a necessity in agriculture. The use of fertilizers has raised crop yield considerably (Stewart et al. 2005). The expansion of agriculture has lead to an important increase in global K+ consumption [4.4% per year between 1999 and 2005] and K+ fertilization to maintain crop production is a regular cultural practice. Global K+ consumption reached 33.9 Mt K2O in 2008. In some cases, over-fertilization occurs, which implies a financial and an environmental cost. Moreover, the high input of fertilizers to crops may lead to the inhibition of K+ acquisition because of the presence of high concentrations of other

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nutrients. It is, therefore, important to optimize the efficiency of fertilizer usage. Cultivating crops that acquire and/or utilize K+ more efficiently can reduce the use of K+-fertilizers which would be environment and economic friendly (White and Brown 2010). Efforts to minimize fertilizer input and to develop nutrient-efficient, high-quality crops rely on detailed understanding of the exact interaction among the nutrients for uptake and the possible deficiency effects of a given nutrient caused by the supply of other nutrients (Amtmann and Armengaud 2009).

6.5.1 NH4+ Interaction between NH4+ and K+ has been studied since long. Several studies have shown that external NH4+ decreases K+ uptake (Deanedrummond and Glass 1983; Kirkby 1968; Martínez-Cordero et al. 2004; Pyo et al. 2010; Rubio et al. 2010), which is probably due to the fact that NH4+ and K+ share some features such as charge value, hydrated ion diameter, and their effect on membrane electric potentials (Wang et al. 1994). In agreement with this, amelioration of NH4+ toxicity by K+ supply has been shown (Cao et al. 1993). Since the description showing that NH4+ specifically inhibits the non-AKT1 pathway of root K+ transport (Santa-María et al. 1997; Spalding et al. 1999), some advances have been made in the understanding of the NH4+–K+ interaction in K+ uptake. The use of Arabidopsis mutants lead to the demonstration that the non-AKT1, NH4+-sensitive component of high-affinity was exclusively mediated by AtHAK5 (Pyo et al. 2010; Rubio et al. 2010). Cross-Talk in the Responses to K+, NH4+, NO3−, and Pi The development of molecular biology techniques and the availability of plant genome sequences have accelerated the identification of transport systems for all plant nutrients. Now the research focus is moving towards the understanding of the regulation of these systems and cross-talk between them (Ohkama-Ohtsu and Wasaki 2010). A relationship between K+ supply and regulation of NO3− and NH4+ transporters seems to exist. NO3− transporters are downregulated in K+-starved plants (Armengaud et al. 2004) and NH4+ 6.5.2

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transporters are upregulated by K+ deprivation (Maathuis et al. 2003). Short-term (6 h) K+ deprivation in maize plants leads to the rapid upregulation of a maize NO3− transporter NRT2 hom*olog (Schachtman and Shin 2007), suggesting that factors other than changes in NO3− metabolism may act on NO3−-sensing mechanisms. Moreover, it has been recently described that K+ and NO3− sensing share a protein kinase in the signaling pathway. As mentioned previously CIPK23 is known to be involved in K+ signaling through phosphorylation of the AKT1 channel (Krouk et al. 2010), and CIPK23 is also an NO3−-inducible gene which is downregulated in chl1 (nrt1.1) mutants. Regulation of Pi and K+ transporters may also share signaling elements. Sucrose supply induce the Pi transporters, PHT1;4 and PHT3;1, as well as the K+ transporter HAK5 upstream of hexokinase (HXK) sugar sensing pathways (Lejay et al. 2008). Other NO3−, SO42−, and K+ transporters are also upregulated by sucrose but downstream of the HXK sugar sensing pathway. On the other hand, genes encoding an MAP kinases, transcription factors, and nutrient transporters are induced by K+ and Pi deprivation (Wang et al. 2002). ROS may be a common signaling element of the plant response to the deficiency of several nutrients. ROS in roots is a common feature in response to NO3−, Pi, K+, and SO42− deprivation. Although this response occurs because of the deprivation of several macronutrients, it appears that there are some differences in localization as well as differences in the molecules that produce the ROS (Schachtman and Shin 2007), thus allowing some specificity.

7

Biotechnological Perspectives

To improve crop productivity, it is necessary to understand the mechanisms of plant responses to environmental changes with the ultimate goal being the increase of food availability. There are concerns about our ability to increase, or even maintain, crop yield and production in the context of global environmental change and its associated abiotic stresses (Tester and Langridge 2010). Furthermore, the current increment in biofuels production adds more doubts about our capacity to produce enough food.

The correlation between the increased frequency of extreme environmental events and global warming, underlie an urgent need for protective measures including the development and introduction of new crop cultivars with enhanced tolerance to environmental stresses (Etterson and Shaw 2001; Mittler 2006). Different strategies could be carried out for this purpose, and each of them will present certain advantages and inconveniences that need to be taken into consideration. Two of these strategies, natural variability exploitation and genetic engineering, that could be used for the improvement of the K+ uptake systems in plant roots, will be discussed below.

7.1

Natural Variability and QTL Mapping

Natural variability offers a large resource of polymorphism, which is often explored to identify traits with environmental adaptive value or quality properties. Responses to environmental conditions depend on numerous genes and are typically controlled by QTLs. Genomic mapping of such QTLs may lead to the identification and cloning of important regulatory genes or allelic variants. It could also provide genetic markers for molecular breeding and/or cloned genes for genetic engineering for the improvement of stress tolerance in plants (Papdi et al. 2009). Natural variability is often based on minor genetic changes, generating small quantitative alterations in responses to environmental conditions. One single nucleotide change, so-called single nucleotide polymorphism (SNP), could lead to different plant yield (Fleury et al. 2010; Papdi et al. 2009; Rafalski 2002; Xing and Zhang 2010). The identification of genetic variability which affects stress responses requires phenotypic screenings capable of distinguishing between plants with different stress tolerance. Although previously tedious and time-consuming, nextgeneration sequencing of natural accessions can reveal sequence variability at the genome scale and will facilitate the large-scale identification of SNPs in different ecotypes and varieties. This is one of the goals of the “1001 Genomes Project,” spearheaded by Magnus Nordborg, Joe Ecker,

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Fig. 4.5 Proposed points of genetic engineering for obtaining plants with improved K+ nutrition: Upregulation of known root K+ uptake transporters (HAK5) and channels (AKT1); selective upregulation

of Na+/K+ transporters (HKT); upregulation of unknown K+ transporters (CPAs, CNGCs). In addition, protein activity and selectivity may be also modified to enhance K+ uptake

and Detlef Weigel among others, http:// 1001genomes.org/where 617 accessions have been committed as of 2010-6-2. There are many examples of successful uses of genetic markers and QTL mapping. ERECTA was the first Arabidopsis gene that was mapped as a main QTL, and it regulates transpiration efficiency by controlling leaf photosynthesis efficiency and stomatal conductance (Masle et al. 2005). Freezing tolerance is controlled by seven QTLs in Arabidopsis. QTL mapping revealed that the C-repeat binding factor (CBF) locus is the most important component in cold acclimation (Alonso-Blanco et al. 2005). As mentioned above, the identification of QTLs for determining salt tolerance or K+ accumulation have highlighted the importance of HKT transporters in these processes. Highthroughput ionomic coupled with genomic analysis allowed the identification of the genetic alteration that drives the natural variation in shoot Na+ accumulation in Arabidopsis populations (Rus et al. 2006). Polymorphism of the AtHKT1 gene and sensitive wild populations of Arabidopsis illustrate the importance of this transporter in salt tolerance. Other examples are the already mentioned SKC1, a rice HKT-type Na+-selective transporter involved in unloading Na+ from the xylem, characterized as a QTL for salt tolerance (Ren 2005); or the durum wheat Nax1 and Nax2 loci, linked to Na+ exclusion which correspond to the Na+ transporters HKT1;4 (HKT7) and

HKT1;5 (HKT8), respectively (Byrt et al. 2007; Huang et al. 2006). Recently, RAS1 (Response to ABA and Salt 1) were found by using QTL mapping of a recombinant inbred population derived from Landsberg erecta (Ler; salt and ABA sensitive) × Shakdara (Sha; salt and ABA resistant). This transcription factor have been shown to play an important role in salt tolerance and ABA sensitivity (Ren et al. 2010). As we can see, the analysis of natural variation in crop plants and Arabidopsis has provided an unprecedented amount of information on the genetic and molecular mechanisms that determine intraspecific variation and adaptation. It can be anticipated that this trend will continue in the next decade, especially with the broad implementation of “-omics” technologies for the precise analysis of natural variation at different levels (Alonso-Blanco et al. 2009).

7.2

Genetic Engineering

Two main strategies can be developed by researchers to attain biotechnologically improved plants: Modifying the protein quantity of interest or modifying the protein quality to make it more selective or active. These two strategies may be achieved with modifications at the nucleic acid level that will encode the protein. These strategies may be applied to K+ uptake systems to improve K+ nutrition (Fig. 4.5).

M. Nieves-Cordones et al.

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7.2.1 Modification of Gene Expression Although all cells in an organism contain essentially the same DNA, cell types and cell functions vary because of qualitative and quantitative differences in their gene expression. Overexpression and downregulation of key genes will provide plants with the necessary resources to grow properly in the global warming challenge. The nutrition of K+, one of the most important macronutrients presently found in most commercial fertilizers, could be improved in this way. Thus, enhanced expression of K+ transporters and channels could result in an increase in plant K+ uptake which could lead to high-yield production. At the same time, improved K+ nutrition could lead to enhanced salt tolerance, because of the aforementioned competition between K+ and Na+, or to alleviation of drought stress as K+ is a known osmolite (Amtmann et al. 2004; Römheld and Kirkby 2010). To our knowledge, only two plasma membrane K+ transporters or channels have been overexpressed. Overexpression of the barley high-affinity K+ transporter HvHAK1 in Arabidopsis transgenic plants has been characterized (Fulgenzi et al. 2008). They showed increased K+ uptake when plants were deprived of K+ but not in K+-sufficient conditions. Also a correlation between HvHAK1 transcript level and K+ uptake was absent. The cation:proton antiporter AtCHX13 has also been overexpressed (Zhao et al. 2008). Driven by the 35S promoter, AtCHX13 increases K+ (Rb+) uptake in intact plants with a Km of 196 mM, although it is not clear whether AtCHX13 localizes to the plasma membrane of the root cortex in native tissue. More K+ transporters and/or channels have to be upregulated to unravel their biotechnological potential. Biotechnological success has been recently reported by cell-specific overexpression of an HKT transporter. Specific expression of AtHKT1 in the mature root stele leads to a reduction of root-to-shoot transfer of Na+ and increased salinity tolerance (Moller et al. 2009). Interestingly, not only Arabidopsis but also rice plants exhibit salinity tolerance with the specific expression of AtHKT1 in root cortex cells (Plett et al. 2010).

7.2.2 Modification of Protein Activity Future modification of K+ transporters and channels may contribute to K+ nutrition under adverse conditions such as high-external concentrations of K+ uptake inhibitors. The finding of transporters and channels with higher affinity for K+ (lower Km) and/or higher transport capacity (higher Vmax) could lead to obtaining plants better suited to cope with the problems derived from climate change. Some advances have been made for this purpose and several random and sitedirected mutagenesis have been developed. S. cerevisiae, as a screening system model, has allowed the identification of modifications in the gene sequence encoding the transporters that confer salt tolerance or enhanced K+ uptake (Garciadeblas et al. 2007; Mangano et al. 2008; Rubio et al. 1995, 1999). None of these studies have shown whether the enhanced yeast growth was due to a higher Vmax or to an increase of transporter quantity at the plasma membrane. Only Garciadeblas and colleagues (2007) have reported on a mutated transporter of the moss Physcomitrela patens (PpHAK1), with a lower Km for K+, but still high for high-affinity K+ uptake (200 mM). Although good candidates for increasing K+ uptake in salt environments have been described for some K+ transporters, no biotechnological advances in plants have been made to date (or to our knowledge). Promising candidates would finally confirm in planta the hypothesis that the K+/Na+ ratio and not absolute Na+ concentration in the cytoplasm is critical for stress tolerance (Amtmann et al. 2004; Maathuis and Amtmann 1999) and that by modifying root K+ uptake systems salt tolerance can be increased. In addition, the possibility that improved K+ uptake ameliorates the plant response to other stresses such as drought and cold should be also considered.

8

Conclusion and Future Perspective

The development of new crop cultivars with enhanced tolerance to environmental stresses such as salinity, heat, etc., is a necessity for modern

4 K+ Nutrition, Uptake, and Its Role in Environmental Stress in Plants

agriculture, as these stresses will be exacerbated as a consequence of future climate change. One of the targets for improvement is the plant’s capacity to maintain/control its mineral nutrition. As regards to the acquisition of K+, the research developed in the past decades has allowed the identification of key pieces to this process, namely the identification of genes encoding the transport systems, the regulation of these genes and the elements involved in modulating the activities of these transport systems. Relevant transcription factors and promoter regions will hopefully be identified in the near future, which in turn will constitute objectives and targets of further research. Another set of targets in need of study are composed by the regulatory proteins that have been shown to interact with structural proteins involved in K+ acquisition. In this sense, elements of Ca2+ signaling pathways such as kinases, phosphatases, and Ca2+ binding proteins are of special importance. In addition, key players involved in hormone and ROS homeostasis should be also considered. The development of genetic engineering tools, the -omics approaches and the exploitation of natural variability should produce advances in the understanding of the process of K+ acquisition that could lead to the development of improved crop varieties better suited for facing future challenges. Acknowledgements This work was supported by grants 08696/PI/08 from Fundación Séneca of Region de Murcia (Spain) and AGL2009-08140 form MICINN of Spain to FR.

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5

Temperature Stress and Responses of Plants Anna Źróbek-Sokolnik

Abstract

Among the abiotic environmental factors temperature is the most important factor which significantly affects life processes of all organisms. Temperature stresses experienced by plants are usually classified into three types: (a) chilling stress (occurring at temperatures below freezing), (b) freezing stress (occurring at low temperatures above freezing), and (c) high temperature stress. This chapter shows the influence of low and high temperature to physiological and metabolic processes in plants. The consequences of chilling and freezing or heat stresses are presented as well as mechanisms of plant resistance to low or high temperature and adaptation or/and acclimatization possibilities is reported in this chapter. Keywords

Temperature • Vernalization • Stratification • Metabolism • Freezing • Acclimatization • Adaptation

1

Introduction

Temperature is an abiotic environmental factor that significantly affects life processes in all organisms by modifying membrane properties, enzyme activity levels, the rate of chemical reactions and diffusion, viscosity of vacuole

A. Źróbek-Sokolnik () Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Plac Łódzki 1, 10-727 Olsztyn, Poland e-mail: [emailprotected]

solution and the cytoplasm, phloem, and xylem solutions in plants (Sung et al. 2003). Living organisms can be classified into three groups, subject to the preferred temperature of growth (Fig. 5.1). This chapter analyzes the impact of temperature on plant growth with emphasis on plant response to temperature stress. It is believed that land plants evolved in a tropical climate. This evolution process was spurred not so much by a warm climate, but by the stability of ambient temperature. Plants gradually migrated into temperate regions both north and south of the equator as they developed mechanisms that allowed them to accommodate

P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_5, © Springer Science+Business Media, LLC 2012

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Fig. 5.1 Classification of the living organisms, subject to their preferred temperature of growth

wider variations in temperature on both a daily and a seasonal basis (Fitter and Hay 2002). The growth and development of plants involves a countless number of biochemical reactions that are sensitive to temperature. Plant life is generally limited by the freezing point of water at the low end of the temperature scale and the irreversible denaturation of proteins at the high end. Temperature is a critical factor in the plant environment, and it may play a significant role in growth and development. Growth is defined as an increase in dry weight, while development is the increase in the number and/or dimension of organs by cell division and/or expansion: leaves, branches, spikelets, florets, root apices, etc., including those present in seed embryos. It also seems that the rate of plant development tends to be controlled primarily by temperature, and it is less sensitive to other environmental factors. The development of vegetation is determined by a broad variety of environmental factors that exert combined effects. Plant organisms are rarely affected by individual factors, and temperature stress is usually accompanied by water stress and, in consequence, oxidative stress (Fitter and Hay 2002). Temperature can also play a part in controlling the pattern and timing of plant development, and this accounts for the below phenomena.

1.1

Vernalization

In some plant species, a period of low temperatures is required to induce flowering, while in other plants, low temperatures only accelerate flowering or have no effect at all. Plants with a vernalization requirement experience a period of low temperatures in late fall and/or winter at the stage of seed imbibition or young seedlings (annual winter crops) or upon reaching vegetative maturity (biennial and perennial plants) (Kim et al. 2009). Flowering is induced in the temperature range of 0 to +10°C. The duration of the vernalization period, that is, the required number of days with low temperatures, varies subject to species, and it usually reaches from 2 weeks to several weeks (Dennis et al. 1996; Amasino 2006). In seeds, temperature stimuli are perceived by the embryo, while in seedlings and matured plants, this signal is sensed by apical meristems. A vernalized meristem retains competence following the reception of the inductive signal. When the signal is absent for a longer period of time, the plant is de-vernalized, and a similar effect can be achieved by exposing the plant to higher temperatures (around 40°C for 1–2 days) (Tretyn et al. 2003). The mechanisms underlying vernalization have not yet been fully explained. It is believed that low temperatures lead to changes in the permeability

5

Temperature Stress and Responses of Plants

of cell membranes and/or the level of expression of “vernalization” genes. Phytohormones, in particular gibberellin, significantly contribute to this process (Sheldon et al. 2000; Amasino 2005).

1.2

Stratification

Stratification is a popular method of breaking seed dormancy that has been used for centuries. This technique involves the storage of seeds in a moist and well-ventilated environment at relatively low temperatures in the range of 1–10°C. Stratification is generally defined as the process of subjecting seeds to cold or warm and cold conditions in a moist and ventilated environment to break the dormancy stage. Low temperature, high moisture content, and oxygen supply during the treatment induce deep physiological and biochemical changes in seeds. Stratification leads to the decomposition of germination inhibitors in seeds, and it induces the production of growth stimulators: cytokinin, gibberellin, and auxin. At various stages of the dormancy breaking period, changes are noted in the quantitative ratio of various stimulators which modify the seeds’ sensitivity to light and temperature and support dormancy breaking in various dormancy mechanisms (e.g., Baskin and Baskin 1998; Opik and Rolfe 2005; Wróbel et al. 2005).

1.3

The Effect of Temperature on Membranes, Enzymes, and Metabolic Processes

An increase or a decrease in temperature changes the kinetic energy of particles, accelerating their motion and weakening hydrogen bonds in macromolecules. All of the reactions contributing to growth are catalyzed by enzymes whose activity depends on their precise, three-dimensional, tertiary structures, to which the reacting molecules must bind exactly for each reaction to proceed. As the temperature rises, tertiary structures are damaged, reducing enzyme activity and reaction rates (Price and Stevens 1999). The asymmetry of response curves, such as Fig. 5.2a, b, is the net

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result of an exponential increase in the reaction rate, caused by increased collision frequency, and increasingly modified by the thermal denaturation of macromolecules (Fitter and Hay 2002). The effect of temperature on enzyme activity is not a simple correlation. Activity levels rise with an increase in temperature, but only within a temperature range that guarantees the enzyme’s stability (Cornish-Bowden 2004). When the critical temperature is exceeded, enzymes undergo thermal denaturation, and their activity drops rapidly. The average rate of enzymatic reactions increases twofold with every 10°C increase in temperature within the range that does not cause enzyme denaturation (Fig. 5.3). The correlation between temperature and the increase in enzymatic activity is described by temperature coefficient Q10 which illustrates changes in reaction rate when the temperature increases by 10°C: Q10 =

v( t +10) vt

.

Parameter Q10 applies only in a nondenaturing range of temperatures, it is enzyme specific and determined by the activation energy of the catalyzed reaction. Enzyme activity reaches the highest level at optimal temperature. The representative values of temperature coefficients (Q10) for selected plant processes measured at varying intervals within the range 0–30°C are determined at 1–2.3 (e.g., light reactions of photosynthesis ~1; diffusion of small molecules in water: 1.2–1.5; water flow through seed coat: 1.3–1.6; water flow into germinating seeds: 1.5–1.8; hydrolysis reactions catalyzed by enzymes: 1.5–2.3; root axis extension: 2.3). Coefficient value reaches 2–3 for dark reactions of photosynthesis, 0.8–3 for phosphate ion uptake into storage tissue, and 2–5 for potassium ion uptake into seedlings. Grass leaf extension is characterized by Q10 of 3.2, and the relative growth rate is marked by coefficient value of 7.2 (Fitter and Hay 2002). The observed optimal temperature is the product of two processes: an increase in the reaction rate related to an increase in kinetic energy and an increase in the rate of thermal denaturation of an enzyme above a critical temperature point. When the

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Fig. 5.2 Schematic illustrations of plant responses to temperature (adapted from Fitter and Hay 2002). (a) The response of plant growth rate; (b) the influence of temperature on the rate of photosynthesis and respiration

second parameter is higher, a drop in activity levels is noted. For most enzymes, the optimal temperature falls within the range of 30–45°C. Enzymes are irreversibly denatured and inactivated at temperatures higher than 60°C. The enzymes of thermophilous organisms (such as thermal spring bacteria) remain active and attain maximum reaction rates at higher temperatures. The highest temperature at which an enzyme is not thermally inactivated under given conditions determines the enzyme’s thermal stability.

An alternative approach involves the application of the Arrhenius equation (from chemical kinetics) to plant processes: k = A exp( − E a / RT ), where k is the rate constant, Ea is the activation energy for the process, A is the constant, R is the gas constant, and T is the temperature expressed on the absolute temperature scale. Arrhenius constants (Ea/R for the process) can be useful in biochemical comparisons between

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Temperature Stress and Responses of Plants

Fig. 5.3 The influence of temperature on enzyme activity

species (Criddle et al. 1994; Levine 2005) and in analyses of plant membrane changes during cooling and freezing. Higher temperatures increase the liquidity of membrane lipid layers. A temperature drop has the opposite effect: biological membranes become more rigid and the activation energy of membrane enzymes increases. The above phenomenon is as the result of thermotropic changes in the lipid phase. Temperature modifies the organization of fatty acid residues in phospholipids and galactolipids, the components of various membranes. The configuration of polyunsaturated fatty acid residues is more difficult to reorganize at lower temperatures than that of saturated fatty acids, but polyunsaturated fatty acids residues “melt” more easily at higher temperatures. Temperature-induced changes in the liquidity of the cell membrane or its selected domains that modify the structure and function of membrane proteins. Cell membrane’s response to temperature variations may also be determined by its sterol content or interactions with other nonlipid organic compounds (Sung et al. 2003; Alberts et al. 2004). Temperature-induced changes in membrane properties also significantly affect water regulation in cells, and secondary water stress may occur when the rate of water uptake by the roots is slower than leaf transpiration. At temperatures below 0°C, liquid water changes into solid ice. Ice crystals are formed inside the protoplast which could lead to structural damage. Extracellular formation of ice may cause cell dehydration. The component processes of plant

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growth do not all respond to temperature in the same way. For example, in most crop species, gross photosynthesis ceases at temperatures just below 0°C (minimum) and above 40°C (maximum), with the highest rates being achieved in the range of 20–35°C. In contrast, rates of respiration tend to be low below 20°C but, owing to the thermal disruption of metabolic controls and compartmentation at higher temperatures, they rise sharply up to the compensation temperature, at which the rate of respiration equals the rate of gross photosynthesis, and there can be no net photosynthesis (Wilkinson 2000; Fitter and Hay 2002; Jenks and Hasegawa 2005; Wahid et al. 2007). Temperature stress in plants has been broadly researched, and the problem has been widely addressed by review articles (Wang et al. 2003; Wahid et al. 2007; Jan et al. 2009), books discussing various types of stress (Wilkinson 2000; Fitter and Hay 2002; Jenks and Hasegawa 2005), studies investigating the negative effects of extreme temperatures (Iba 2002; Sung et al. 2003), etc. It should be noted that unlike homeothermic animals, plants are unable to maintain their cells and tissues at a constant optimum temperature, therefore, their metabolism, growth, and development are profoundly affected by changes in environmental temperature. This suggests that as sessile organisms, plants must be able to sense transient fluctuations as well as seasonal changes in temperature and respond to these changes by actively adjusting their biology to fit the subsequent temperature regime. Temperature is a major environmental factor that changes from season to season and undergoes daily fluctuations and short, erratic lows and highs. For this reason, the stress-inducing role of temperature is difficult to define unambiguously since the response to various temperatures is determined by the plants’ ability to adapt to different climate regimes. Vegetation occurs in climate zones characterized by extreme temperatures of −50 to +50°C, that is, within a range of 100°C. The margin of thermal tolerance that conditions the stability of life processes in most plants is relatively wide, ranging from several degrees above zero to around 35°C, and it is genetically determined. Many genotypes specific

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Table 5.1 Factors which determinate temperature of above- or under-ground organs (adapted from Fitter and Hay 2002) Leaf/above-ground organs

Roots/under-ground organs

– – – – – – – – – – – – –

The amount of solar radiation intercepted The potential for energy exchange with the environment Time of day (regular diurnal variation of solar elevation) Month (typical seasonal variation) Cloud cover Wind force and origin of air mass (irregular, short-term variation) Position in the canopy (e.g., “sun” or “shade” leaf) Altitude above soil surface Canopy characteristics, including leaf shape, dimensions and surface properties Seasonal and diurnal variations in energy exchange Determine how much energy The interception of solar radiation by the canopy reaches the soil surface The account of the depth below the soil surface Soil properties which influence the energy balance at the soil surface, and the transfer of heat through the soil (e.g., moisture content, bulk density, color/albedo, and the vegetative or litter cover)

to extreme climate conditions, from arctic to tropical, have a much wider tolerance margin. In principle, plants in the dormant state (dry germs and seed embryos, dehydrated dormant organs) are far less sensitive to temperature change, and they are able to survive through periods of extreme temperature unharmed. Metabolically active tissues have thermal activity limits which, when exceeded, lead to a reversible drop in the rate of life processes to a minimum level. Further temperature change (referred to as critical or lethal temperature) causes permanent damage to cell structures, it affects cell metabolism, impairs vital life processes, and kills the protoplasm. During evaluations of plant response to extreme temperatures, special attention should be paid to the temperature of the plant which often differs from ambient temperature. In the summer, leaf temperature often exceeds ambient temperature by up to several degrees. Higher differences are noted in plants whose leaves are positioned horizontally, such as apple trees. In the spring and autumn, the night temperature of leaves, in particular when the sky is clear, may be even several degrees lower than ambient temperature (Wilkinson 2000; Fitter and Hay 2002; Jenks and Hasegawa 2005). At a given moment, leaf temperature is determined by several factors (Table 5.1). Roots demonstrate a stronger growth

response to extreme temperatures than the above-ground parts of plants, and the above applies to both extreme cold and extreme heat (Fig. 5.4). During the evolution process, roots became adapted to more stable temperatures. Nonetheless, the temperature of both the roots and other under-ground organs is also determined by factors presented in Table 5.1. Plants can adapt to changes in the temperature regime through the evolution of genotypes with more appropriate morphologies, life histories, physiological and biochemical characteristics, or by plasticity. Plants also adapt to changing temperatures during the growing season by plastic responses.

2

Low Temperature

Periodic temperature drops below zero degrees are reported on around 64% of the Earth’s surface. The lowest temperatures are noted in Antarctica, reaching around −50°C in coastal areas and up to −90°C in the interior. The minimum temperature at which a given species can survive is one of the main criteria determining plant distribution on our planet. In a temperate climate, low-temperature stress eliminates or inhibits the growth and yield of valuable plants and crops (Xin and Browse

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Fig. 5.4 Schematic illustration of the influence on growth and morphology of roots and above-ground organs of potato seedlings (adapted from Marschner 1995)

2000; Jan et al. 2009). Plants indigenous to colder regions are usually well adapted to chilling temperatures and are, therefore, not significantly impaired by cold periods, apart from a general slowing down of the metabolic rate and growth. In a temperate climate, plants respond differently to freezing temperatures and the winter environment than other factors that occur irregularly. In the winter, chilling temperatures do not come as a surprise for plants that have adapted to the periodic, adverse vegetation factors in the course of evolution. Low temperatures are accompanied by short daytime and low radiation intensity. The adaptation to growth inhibiting factors is characteristic of the dormant state (Jan et al. 2009). There are two types of injuries a plant can sustain through exposure to low temperatures (Fig. 5.5). On the other hand, many plants that are native to cold climates can survive extremely low temperatures without injury (Levitt 1980). An analysis of freezing winter temperatures as an environmental stressor should also account for the impact of other adverse factors such as low light intensity and short daytime. The above conditions arrest the growth and development of vegetation (Hopkins 2006).

The plants’ ability to survive freezing and other adverse temperature changes differs from the remaining stressors. Levitt’s stress avoidance theory (1980) does not apply in this case. Plants are unable to avoid freezing temperatures, and they can only protect themselves from the negative consequences of cold by increasing their tolerance to chilling. Many plants enter the dormant state to survive harsh winter weather. This is a typical feature of adaptation to freezing which is a genetically inherited trait. Plants can be classified into three categories based on the range of lethal temperatures and the characteristics of mechanisms conditioning their resistance to low temperatures (Fig. 5.6).

2.1

Consequences of Chilling and Freezing Stress

There are two theories explaining the plants’ primary response to temperature stress. The first concept, formulated by Lyons (1973), states that low temperatures induce the phase transition of cell membranes where a liquid-crystal structure is transformed into a crystal (gel) phase.

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Fig. 5.5 Two types of injuries a plant can sustain through exposure to low temperatures (adapted from Stushnoff et al. 1984)

Fig. 5.6 Classification of the plants, subject to their range of lethal temperatures and the characteristics of mechanisms conditioning their resistance to low temperatures (adapted from Stushnoff et al. 1984)

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Temperature Stress and Responses of Plants

Thermotropic phase changes are the primary cause of membrane dysfunctions that lead to irreversible damage and cell death. The above may produce reactive oxygen species and the accompanying oxidative stress. According to recent research, the phospholipid which initiates the phase transition of the cell membrane is phosphatidylglycerol (PG). If a PG molecule contains fatty acids with a high melting point, that is, saturated fatty acids, then the phase transition of this lipid takes place relatively easily at low temperatures and this, in turn, induces the transformation of other phospholipids and galactolipids adjacent to PG (Los and Murata 2004; Wang et al. 2006). According to the second chilling injury theory, the primary cause of damage is the sudden increase in the concentration of free calcium ions in the cytosol (Minorsky 1989). Calcium ion concentrations increase as calcium channels in the plasmalemma become opened due to sudden depolarization (Lecourieux et al. 2006). In chilling-sensitive plants, calcium opens the stomata, and transpiration significantly exceeds water uptake by the roots (Liang et al. 2009). In many sensitive species, the first indication of cold stress is striking wilting of the leaves, despite optimal water supply in the soil (Mahajan and Tuteja 2005; Solanke and Sharma 2008). The release of calcium ions into the cytosol has many secondary effects, including induced gene expression which could result from changes in the content or distribution of cell hormones, mainly abscisic acid (ABA). This phenomenon is in particularly related to the acidification of the cytoplasm at low temperatures (and the corresponding alkalization of the vacuoles) which, at least in part, is actively controlled by H+-transport from the cytoplasm to the vacuole catalyzed by H+-ATPase located on the vacuolar membrane. The inactivation of this enzyme has been reported to occur much earlier than other symptoms of cell injury (Yoshida et al. 1999; Lindberg et al. 2005). Chilling affects the entire internal environment of each cell and each molecule within the cells (Kartsch and Wise 2000). The rate and extent of injury is determined by temperature, its duration as well as the chilling rate. Sudden temperature

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drops (thermal shock) have particularly damaging consequences. The lower the temperature and the longer its effect, the greater the extent of the sustained injury (Mahajan and Tuteja 2005; Solanke and Sharma 2008). Plant structures and physiological cell processes have varied sensitivity to chilling temperatures (Fig. 5.7). Most injuries are sustained in the cell membrane which may represent a potential site of perception and/or injury (Lindberg et al. 2005). There are changes in the viscosity and liquidity of the membrane, leading to an increase in diffusion resistance and, in many cases, enzyme inactivation. The reversibility of those effects is determined by the severity of damage. Changes in chemical composition may be observed as the result of lipid degradation, the release of fatty acids and changes in the activity of metabolizing enzymes, peroxidation, disintegration of lipid–protein bonds, and higher membrane permeability. The chemical composition of the cytoplasm and differences in lipid quality in various chilling-sensitive species determine the phase transition point, that is, the point at which the membrane is transformed from a liquid-crystal state into a gel state (Solanke and Sharma 2008; Jan et al. 2009). This change in the membrane’s physical state impairs its normal functioning. In most chilling-sensitive plants, the phase transition point is around 10°C. Chilling sensitivity is mostly related to a higher content of saturated fatty acid residues in lipids, while the cold-hardiness mechanism is explained by the desaturation of fatty acids which enables the plant to quickly acclimatize to low temperatures. The above is only one of the factors explaining variations in the plants’ response to temperature stress (Lindberg et al. 2005; Zhang and Tian 2010). Interactions between membrane components, including lipid–lipid and lipid–protein, are also believed to play an important role. Higher sterol concentrations increase membrane rigidity. The role of membrane proteins during chilling is also a source of controversy, but there is general agreement that conformational changes in protein–lipid systems may lead to membrane disintegration and dysfunction (Los and Murata 2004; Lindberg et al. 2005). Frost-induced

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Fig. 5.7 Functional disturbance occurred in chilling-sensitive plants, subjected to stress duration (adapted from Kacperska 1998)

changes may lead to inhibited protoplast movement, excessive protoplast vacuolization, damage to the endoplasmic reticulum, drop in turgidity, and higher membrane permeability. Cytoplasmic streaming and photosynthesis, including thylakoid functioning in chloroplasts (as demonstrated by enhanced in vivo chlorophyll fluorescence), are most susceptible to reversible disruptions. Irreversible damage, including injuries caused by stressors other than temperature, is also most likely to affect thylakoid membranes, mostly photosystem II. Chloroplast lipids undergo various metabolic changes in both chilling-sensitive and cold-hardy plants. Higher levels of galactolipase activity and, consequently, higher free fatty acid concentrations are noted in the chloroplasts of chilling-sensitive species (faba beans, beans, tomatoes, maize) than in cold-hardy plants (spinach, pea). Lower temperatures disrupt the maintenance of the proton gradient in thylakoid membranes conditioning ATP synthesis. Powerful radiation during or directly after chilling intensifies the relevant injuries and retards, or even disables, damage repair in both chilling-sensitive and cold-hardy plants. Long-term frost inhibits the synthesis of chlorophyll and starch (Muller

et al. 2005; Liang et al. 2009; Sun et al. 2010). Other membranes (plasmalemma and tonoplast) are damaged after relatively longer exposure, as demonstrated by membrane cells’ ability to plasmolyze and vital staining. Those injuries are irreversible. Other metabolic functions are marked by varied sensitivity to low temperatures which cause metabolic disorders and lead to toxin accumulation, for example, respiration efficiency may be higher or lower subject to environmental factors that accompany freezing temperatures. Chilling may also inhibit the activity of many oxidoreductive enzymes, such as catalase, leading to the accumulation of hydrogen peroxide and the production of free radicals (Suzuki and Mittler 2006; Liang et al. 2009; Sun et al. 2010). In sublethal cold stress, fruit ripening and seed germination are most severely inhibited (Kumar and Bhatla 2006). Frost leads to the appearance of stress which is linked not directly to low temperature, but to freezing (crystallization) of water in the plant (Mahajan and Tuteja 2005). Intracellular and extracellular crystallization produces different effects. Ice crystals are formed readily in those parts of the plant where temperature drops most

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Temperature Stress and Responses of Plants

rapidly and where water freezes most easily (due to high water potential), mostly vascular bundles and intracellular spaces in above-ground parts where water vapor undergoes condensation. Ice crystals spread quickly via vessels and other tissues with uniform structure. The presence of air-filled intercellular spaces as well as tissues with lignified or cutinized walls slows down crystallization. Ice formation is accelerated by icenucleation active bacteria of the genera Ervinia and Pseudomonas. The proteins formed on the outer bacterial cell wall react with water particles and facilitate the formation of ice crystals at temperatures just below 0°C. In the absence of icenucleation active bacteria on the surface of tissues and on the walls of intracellular spaces, ice formation would begin at temperatures several degrees lower due to the supercooling of water solutions. If tissue is supercooled rapidly (e.g., faster than 5 K min−1) and the cells have high water potential, or if cell water had been first deeply supercooled, ice may be formed in the protoplast. The above invariably leads to cytoplasm destruction and cell death (Fitter and Hay 2002; Rajashekar 2000; Jan et al. 2009; Janska et al. 2010). Water freezing in intracellular spaces is a less dangerous phenomenon. In nature, where temperature decline is generally slow (1–5 K min−1), crystallization usually takes place outside the protoplast in intracellular spaces and between the cell wall and the protoplast (partly due to the extracellular fluid having a higher freezing point, i.e., lower solute concentration, than intracellular fluid). The above leads to extracellular crystallization. Vapor pressure decreases in the spaces above ice, and a water potential gradient is created between the unfrozen interior of the cell and the extracellular environment. Water moves along this gradient into extracellular spaces where it is crystallized (Fitter and Hay 2002; Jan et al. 2009; Janska et al. 2010). Cells are dehydrated (secondary stress) and they contract due to desiccation. The lower the surrounding temperature, the longer it takes for an equilibrium to be reached between the water potential above ice and inside cells, and the greater the effect of cell dehydration (Solanke and Sharma 2008).

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Multiple forms of membrane damage can occur as a consequence of freeze-induced cellular dehydration including expansion-induced-lysis, lamellar-to-hexagonal-II chase transitions, and fracture jump lesions. The above leads to cell contraction and the associated changes in reactions between the plasmalemma and the cell wall, partial loss of plasmalemma due to exocytosis and endocytosis, changes in the structure of the plasmalemma and other cell membranes, and the creation of protein-deprived lipid areas in the membrane. The greatest damage is done to the plasmalemma. Dehydration also increases the concentration of solutions in the cytosol and the cell sap, leading to higher salinity (Mahajan and Tuteja 2005; Solanke and Sharma 2008; Jan et al. 2009). Conformational changes in proteins found in the plasmalemma and other membranes lead to changes in the activity of various membrane enzymes, including ATPases responsible for the movement of protons and other ions through membranes (Lindberg et al. 2005). Some ions, accumulated in cells by ion transporters (e.g., potassium ions), are diffused after thawing into intracellular spaces together with water, for example, in leaf tissue. Certain proteins, such as the thylakoid coupling factor, become dissociated in the process. The effect of chill injury on life processes is often visible when plants resume their normal growth after freezing temperatures subside. Even partial degradation of thylakoid membranes inhibits photosynthesis, and the process may be reversible. PS II activity may be partially or completely inhibited, and the balance between the light-dependent phase and CO2 assimilation may be upset. There is a rise in photorespiration intensity (Alam et al. 2005). Changes in the mitochondria and the respiration process are not as profound. In strongly dehydrated cells, the membrane undergoes lyotropic phase transitions, and hexagonal arrangements are formed in lipid bilayers of a single membrane or two layers of two adjoining membranes (e.g., plasmalemma and endoplasmic reticulum). The membranes’ primary structure is not always restored after thawing, and water is diffused into the extracellular environment together with ions through membrane

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channels. Cell dehydration caused by extracellular crystallization increases the concentrations of salt and organic acids in the protoplast which, in turn, may lead to protein denaturation and enzyme inactivation (Mahajan and Tuteja 2005; Solanke and Sharma 2008). Few enzymes remain active at below zero temperatures, but some of them are activated, such as phospholipase D which catalyzes the hydrolysis of phospholipids (Ruelland et al. 2002). The degradation of membrane lipids begins during freezing and after thawing, releasing unsaturated fatty acids which are peroxidized. Chlorophyll may be also be photooxidized in green tissues exposed to light (Sung et al. 2003). Chill injuries may occur not only during freezing, but also during the thawing of tissue. Plant survival is also determined by post-thawing environmental factors – rapid temperature growth and high light intensity may disturb metabolic pathways in cells and cause additional damage. During rapid melting of ice, the cell is rehydrated, and it quickly increases its volume. The above leads to tension and cracks in cell structures, mostly in the cytoplasm which is the site of primary cell injury. The above changes have less damaging consequences for dormant plants. In a temperate climate, winter frost is not a typical stressor for plants, but freezing temperatures could be a source of stress if they occurred in the spring or summer (Muller et al. 2005).

2.2

Resistance to low Temperature, Acclimatization

Resistance is related to frost tolerance, that is, the ability of the organism to survive low temperatures without damage. In regions characterized by seasonal climate change, plants’ resistance to freezing fluctuates periodically – it is the lowest during intensive elongation growth in the spring, and it rises significantly in the fall when growth is arrested by the direct effect of low temperature or the combined effect of shorter daytime and temperature drop (Li et al. 2005a, b). Frost resistance is usually achieved by preventing ice formation in the symplast. An important mechanism

preventing or delaying symplastic ice formation is frost plasmolysis. Poorly hydrated plants which are acclimatized to water stress usually show increased cold resistance, for example, plants which are extremely tolerant to drying out, for example, embryos of ripened seeds, can be conserved alive at −200°C without damage (Jan et al. 2009). Species-specific cold resistance is a genetically programmed trait that can be modified by both endogenous and exogenous factors. For a vast number of species, frost tolerance is not a static feature, but it is closely correlated with season, it fluctuates in various growing periods, and it is not identical for all organs (Rorat et al. 2006; Hekneby et al. 2006). The above-ground parts of wheat seedlings were acclimatized even to −20°C, but the roots’ sensitivity to frost did not change. Acclimatization can be accelerated by hardening the plants, that is, exposing them to increasingly lower temperatures on successive days, initially above zero, followed by insignificantly below zero (Li et al. 2005a; Zhang and Tian 2010). This process is continued for several weeks. Plants are characterized by the greatest frost resistance 1–3 weeks from the beginning of exposure to freezing temperatures. The period of deacclimatization, that is, dehardening, is much shorter, and it usually lasts several days. The higher the ambient temperature, the faster the deacclimatization process. After dehardening, repeated exposure to frost can severely damage many plants (Li et al. 2004; Burbulis et al. 2008). Plant organs are also marked by varied sensitivity to frost (Li et al. 2005a; Rorat et al. 2006). Roots are most susceptible to the damaging effects of freezing temperatures, shoots are less sensitive, while tree trunks and older branches are characterized by the highest frost resistance (Muller et al. 2005; Kato-Noguchi 2007). Snow cover minimizes the temperature drop in the soil, and it protects crops from freezing. The cold sensitivity of flowers is determined by the given species’ phenological growth stages (Thakur et al. 2009; Ohnishi et al. 2010). ABA stimulates and speeds-up plant hardening. According to Weiser (1970), acclimatization, and perhaps also hardening, is determined by modifications in gene expression. In this case,

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ABA can enhance cold resistance if it is able to induce the expression of the respective genes (Gusta et al. 2005). Gibberellins and auxins deliver an opposite effect. Substances that retard gibberellin synthesis accelerate hardening. Intensive nitrogen fertilization generally delays dormancy and increases susceptibility to freezing. Heavy potassium fertilization has the opposite effect by increasing the frost resistance of both herbaceous and arborescent plants. The concentrations of sugar and other osmoprotectors that protect the cell from dehydration increases in the cytosol and vacuoles (Liang et al. 2009). Fluid supercooling inside the cell is yet another factor that increases the plants’ cold resistance by delaying crystallization in the cell. The presence of substances dissolved in the vacuolar sap lowers crystallization temperature. In small, weakly vacuolated cells, water may undergo deep supercooling. In large and hydrated parenchymal cells and xylem vessels, the supercooled state is very unstable, and it rarely lasts longer than several hours. Supercooling provides temporary protection against freezing caused by, for example, strong ground frost. In tissues comprising small, densely packed and weakly vacuolated cells whose walls prevent ice crystals from spreading, a supercooled state may persist until the temperature drops below a threshold value. The accumulation of nonpolar lipids on the surface of the plasmalemma also prevents ice penetration from the apoplast to the cell interior. In herbaceous plants, the supercooling of water is observed at −1 to −15°C, and in arborescent plants at −30°C, and even −50°C. Such a high degree of supercooling is observed only in some living tissues, such as core parenchymal cells, meristematic tissue, leaf bud scales, and flower buds. When ambient temperature drops below the critical supercooling point, this meta-stable state is rapidly disrupted, and ice is formed inside the cells, ultimately leading to their death. In some extremely frost-resistant tree species, the protoplasm is able to vitrify. Vitrification is stimulated by a high concentration of sucrose and other sugars. In this relatively stable condition, it is possible to cool cells almost to absolute zero without destruction

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(Rajashekar 2000; Hopkins 2006; Jan et al. 2009). Membranes are restructured under exposure to cold before the temperature drops below zero. In these conditions, the water potential is gradually lowered with a simultaneous drop in the osmotic potential due to the accumulation of carbohydrates in vacuoles. ABA is accumulated, and it induces the synthesis of specific proteins. The next stage brings intensified changes in the cell membrane – degradation of phosphatidylcholine and phosphoinositol, accompanied by a continued increase in ABA levels and protein synthesis modifications (Gusta et al. 2005; Lindberg et al. 2005). Cryoprotectants, substances that directly protect the membrane from damage, are also synthesized at this stage. Rigid membranes are less likely to be deformed during frost-induced dehydration, and they protect cells against freezing more effectively. This parameter is largely dependent on the sterol content of cells (Hopkins 2006; Janska et al. 2010). In addition to membrane unsaturation, it appears that lipid asymmetry in the membrane also contributes to the physical structure of the membrane at low temperature (Gomès et al. 2000). The mechanism protecting chloroplast membranes enables the plant to begin photosynthesis as soon as ambient temperature increases. The cold resistance of plants is also determined by the following mechanisms: 1. Thermal insulation which delays and minimizes heat loss, for example, shoot apices are often covered with dense foliage (rosette plant habit) or they winter under a layer of leaves or litter (geophytes). Frost tender organs are often rejected before the onset of very low temperatures (deciduous plants shed leaves in the fall). In high mountainous regions of tropical zones, the leaves of large rosette plants close above the tip at night to protect the interior from freezing (Hopkins 2006). 2. Water freezing in intertissue spaces, for example, between the seed coat and the embryo or between bud scales, where extensive areas are covered with ice.

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3. Cell structures are protected against excessive dehydration with an accompanying increase in the effectiveness of barriers that prevent ice crystals from propagating from the apoplast inside the cell. The following mechanisms are involved: (a) Osmotic pressure increases to keep water inside the cell, and the water potential decreases due to the accumulation of osmotically active compounds (simple sugars and oligosaccharides, polyols, low-molecular-weight nitrogen compounds, such as selected amino acids) in vacuoles and hydrophilic proteins in the cytoplasm (Rorat 2006; Liang et al. 2009). The share of highly polar lipids in the membrane structure increases, such as phosphatidylcholine and phosphatidylethanolamine in the plasmalemma and cytoplasmic membranes or digalactosyldiacylglycerol in chloroplast membranes, which increases matrix interactions inside the cell. (b) The membrane is enriched with more stable lipids containing polyunsaturated fatty acid residues, selected sterols, and cryoprotectants are accumulated in the cytoplasm to protect cell structures against strong dehydration (Lindberg et al. 2005; Zhang and Tian 2010). These substances stabilize membrane structure and prevent conformational protein changes. They counteract the accumulation of salt ions and selected organic acids in the cell, and they protect proteins against denaturation. Small proteins, whose synthesis is enhanced or induced under exposure to low temperatures, play a protective role. Some of them show significant hom*ology to proteins synthesized in response to water stress, for example, to dehydrin (Rorat et al. 2006). The cell wall plays an important role in protecting the cell against the adverse consequences of dehydration, and it is the main barrier to ice penetration. In addition to mechanisms responsible for resistance to the primary consequences of frost, cold-resistant plants develop acclimatization

mechanisms that enable them to avoid secondary thermal stress at below zero temperatures, such as photoinhibition, draught, oxygen deficiency (under ice cover), or mechanical effects of ice load (Alcázar et al. 2011).

3

High Temperature

Heat stress occurs when a rise in temperature has negative consequences for a plant. It is a complex function of intensity (temperature in degrees), duration and the rate of temperature increase. For plants inhabiting very cold climates such as the Arctic, temperatures in the region of 15°C can already be a source of heat stress. In a temperate climate, heat stress takes place in the temperature range of 35–40°C. In scientific literature, heat stress denotes temperatures that exceed the optimum values by around 10–15°C (Larkindale et al. 2005). Plants can be divided into three groups, subject to their sensitivity to high temperature (Fig. 5.8). In geographic zones with a hot climate, in habitats marked by high fluctuations in daily temperature (soil surface, littoral zone, shallow waters) or seasonal fluctuations and in volcanic areas, temperature levels can be lethal for vascular land plants. High absorption of solar energy during windless weather can increase the temperature inside plant tissues in excess of the ambient temperature. Creeping grass shoots, the runners and tillers of young plants can also be subjected to heat stress. The lethal temperature range (thermal death point) is determined by the duration of tissue exposure to high temperature (Table 5.2). Only single-celled organisms can complete their life cycle during continued exposure to temperatures higher than 50°C, while only prokaryotic organisms can survive in temperatures higher than 60°C.

3.1

Consequences of Heat Exposure

At very high temperatures, severe cellular injury and even death may occur within minutes or even seconds (due to denaturation and/or aggregation of proteins), while at moderately high temperatures,

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Fig. 5.8 Classification of the plants, subject to their sensitivity to high temperature (adapted from Stushnoff et al. 1984)

Table 5.2 The lethal temperature range (thermal death point) characteristic for varied types of plants Type of plants Aquatic plants and plants growing in shaded habitats Temperate plants with hydrated and metabolically active organs Desert plants

Thermal death point 38–42°C following several hours of exposure 45–55°C following several hours of exposure

injuries or death may occur only after long-term exposure (due to disruptions in basic metabolic processes). The adverse effects of overheating are directly noticeable. The morphological symptoms of heat stress include scorching of leaves and twigs, sunburns on leaves, branches and stems, leaf senescence and abscission, shoot and root growth inhibition, fruit discoloration and damage, and reduced yield. Cell size reduction, closure of stomata, and curtailed water loss is observed at the tissue and cellular level. At the subcellular level, major modifications occur in chloroplasts (changing the structural organization of thylakoids, loss of grana stacking or its swelling) (Wahid et al. 2007; Mitra and Bhatia 2008). In vascular land plants, the negative consequences of elevated temperature are often related to secondary stress, namely, a negative water balance (leading to the perturbation of many physiological processes) due to intensive leaf transpiration during daytime.

Under field conditions, high temperature stress is frequently associated with reduced water availability (higher during daytime than at night). Heat stress may secondarily induce oxidative stress via the generation and the reactions of activated oxygen species (Xu et al. 2006; Almeselmani et al. 2006). Metabolic pathways and processes show varied sensitivity to temperature which may result in a deficit or an excess of selected metabolites. It is generally believed that the processes taking place in membranes are most sensitive to temperature change. A heat-induced increase in membrane liquidity (either by denaturation of proteins or an increase in unsaturated fatty acids) and changes in reactions between lipid and protein components impair membrane functions (Savchenko et al. 2002; Wahid et al. 2007), including the functioning of ion and water channels, ion transporters, metabolite transport, energy generation, and other processes. Ion leakage

Higher than 60–65°C for more than several hours per day

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from the cell is observed; photosynthesis and respiration are also impaired (Wahid et al. 2007; Wang et al. 2009). It has also been suggested that changing membrane fluidity plays a central role in sensing (plant thermometer) and influencing gene expression both under high and low temperatures (Plieth 1999). Photochemical reactions in thylakoid lamellae and carbon metabolism in the stroma of chloroplast have been suggested as the primary sites of injury at high temperatures (Yang et al. 2006a; Wang et al. 2009). Thylakoid membranes are particularly sensitive to high temperature, and this especially applies to photosystem II whose activity is greatly reduced or even partially stopped under high temperatures (Salvucci and Crafts-Brandner 2004; Camejo et al. 2005; Marchand et al. 2005). High temperature has a greater influence on the photosynthetic capacity of C3 plants than C4 plants (CraftsBrandner and Salvucci 2002). Heat shock reduces the amount of photosynthetic pigments (Wang et al. 2009), soluble proteins, rubisco binding proteins (RBP), large-subunits (LS), and smallsubunits (SS) of rubisco in darkness but increases them in light (Kepova et al. 2005). Moreover, heat stress greatly affects starch and sucrose synthesis, as demonstrated by the reduced activity of sucrose phosphate synthase, ADP-glucose pyrophosphorylase, and invertase (Wahid et al. 2007; Sumesh et al. 2008). In any plant species, the ability to sustain leaf gas exchange under heat stress is directly correlated with heat tolerance. During the vegetative stage, high daytime temperature can cause damage to compensated leaf photosynthesis, reducing CO2 assimilation rates (Crafts-Brander and Salvucci 2002; Morales et al. 2003). Photosynthesis is more sensitive to heat than dark respiration which could have additional consequences under prolonged stress, including the depletion of carbohydrate reserves and plant starvation (Sumesh et al. 2008). Heat stress rapidly increases selected phytohormone levels, including ABA, ethylene, and salicylic acid (SA), and it decreases cytokinin and gibberellin concentrations (Dat et al. 2000; Talanova et al. 2003; Larkindale and Huang 2004). The overlapping effects of the above changes in hormone levels speed-up plant aging.

3.2

Mechanism of Plant Resistance to High Temperature

Plants rely on two adaptation mechanisms to survive high temperatures: the ability to prevent excessive temperature growth in tissues or alleviate its effects and the heat tolerance of the protoplasm. Survival in hot, dry environments can be achieved in a variety of ways, by combinations of adaptations (Fitter and Hay 2002). Plants growing in a hot climate avoid heat stress by reducing the absorption of solar radiation. This ability is supported by the presence of small hairs (tomentose) that form a thick coat on the surface of the leaf as well as cuticles, protective waxy covering. In such plants, leaf blades often turn away from light and orient themselves parallel to sun rays (paraheliotropism). Solar radiation may also be reduced by rolling leaf blades. Plants with small leaves are also more likely to avoid heat stress: they evacuate heat to ambient more quickly due to smaller resistance of the air boundary layer in comparison with large leaves. Plants rely on the same anatomical and physiological adaptive mechanisms that are deployed in a water deficit to limit transpiration. In well-hydrated plants, intensive transpiration prevents leaves from heat stress, and leaf temperature may be 6 K or even 10–15 K lower than ambient temperature. Many species have evolved life histories which permit them to avoid the hottest period of the year. This can be achieved by leaf abscission, leaving heatresistant buds, or in desert annuals, by completing the entire reproductive cycle during the cooler months (Fitter and Hay 2002). Such morphological and phonological adaptations are commonly associated with biochemical adaptations favoring net photosynthesis at high temperatures (in particular C4 and CAM photosynthetic pathways), although C3 plants are common in desert floras (Fitter and Hay 2002). Heat tolerance is generally defined as the ability of the plant to grow and produce economic yield under high temperatures. This is a highly specific trait, and closely related species, even different organs and tissues of the same plant, may vary significantly in this respect. The above

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is affected by climate conditions and the species’ geographic origin. Plants native to cold regions (tundra, high mountain ranges) are much more sensitive to heat than temperate flora. The latter, in turn, are more susceptible to high temperatures than desert and tropical plants. The highest heat tolerance is demonstrated by selected sedge and grass species, mainly C4 plants. Heat tolerance is associated with greater enzyme thermostability and a higher share of saturated fatty acids in membrane lipids which increases the lipid phase transition (melting) temperature and prevents a heat-induced increase in the membrane’s liquidity. It is believed that PG is the phospholipid initiating phase transitions in thylakoid membranes. Heat tolerance leads to a rapid genome reaction even during short-term overheating. The biosynthesis of heat stress proteins (HSP) which prevent macroparticle denaturation is induced (Kotak et al. 2007; Al-Whaibi 2010). During exposure to high temperature, plants synthesize two groups of HSP: four high-molecular weight HSPs (HSP 100, HSP 90, HSP 70, HSP 60) and several lowmolecular weight HSPs (smHSPs). Those proteins remain stable over a certain period of time, and they are probably the main factor enabling plants to survive a temperature increase. HSPs are found in the cytoplasm and organelles such as the nucleus, mitochondria, chloroplasts, and endoplasmic reticulum. The tolerance conferred by HSPs results in improved physiological phenomena such as photosynthesis, assimilate partitioning, water and nutrient-use efficiency, and membrane stability. Those improvements make plant growth and development possible under heat stress (Wang et al. 2004). The HSPs/chaperones may be involved in stress signal transduction, gene activation, and the regulation of the cellular redox state. They also interact with other stress-response mechanisms such as the production of osmolytes and antioxidants (Kotak et al. 2007; Wahid et al. 2007; Al-Whaibi 2010). In heat-stressed plants, the induction of HSP synthesis inhibits the biosynthesis of other proteins. A plant’s resistance to heat is determined by protein synthesis in cells that are lost with age. For this reason, aging organs (and organisms) have impaired ability to acclimatize to high

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temperature. Smaller quantities of HSPs are also determined at optimal temperature, but in this environment, they play a different role than during and after stress. Under optimal conditions, HSPs regulate the formation of protein structures from newly emerged polypeptide strings to protect the cell from proteins that are nonfunctional due to synthesis “errors.” At excessively high temperatures, HSPs minimize cell injuries by protecting cell proteins from denaturation and creating chelate bonds with ions leaking from the vacuoles into the cytosol (Kotak et al. 2007; Wahid et al. 2007; Al-Whaibi 2010). An increased content of ABA mediates the acclimation/adaptation of plants to desiccation by modulating the up- or downregulation of numerous genes (Talanova et al. 2003; Wahid et al. 2007). It is suggested that the induction of several HSPs (e.g., HSP70) is regulated by ABA (Snyman and Cronje 2008). Increased ethylene secretion at high temperatures leads to the abscission of reproductive organs; this is accompanied by both reduced levels and transport capacity of auxins to reproductive organs (Wahid et al. 2007). Among other hormones, SA has been suggested to be an important component of signaling pathways in response to systemic acquired resistance (SAR) and the hypersensitive response (HR) during heat stress (Kawano et al. 1998; Wang and Li 2006). Gibberellins and cytokinins have an opposite effect on high temperature tolerance than ABA. The potential roles of other phytohormones in plant thermotolerance are yet unknown (Wahid et al. 2007). Under stress, different plant species may accumulate a variety of osmolytes such as sugars and sugar alcohols (polyols), proline, tertiary and quaternary ammonium compounds, and tertiary sulphonium compounds (Singh and Grover 2008). The accumulation of such solutes may contribute to enhanced stress tolerance of plants, for example, proline and glycinebetaine may buffer the cellular redox potential under heat and other environmental stresses (Wahid and Close 2007); gama-4-aminobutyric acid (GABA) has a physiological role in the mitigation of stress effects (Kinnersley and Turano 2000). Hightemperature stress induces the production of phenolic compounds such as flavonoids and

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phenylpropanoids. The heat-induced increase in the activity of phenylalanine ammonia-lyase (PAL) is considered to be the cell’s main acclamatory response to heat stress (Wahid and Ghazanfar 2006; Wahid 2007). Carotenoids of the xanthophylls family and selected terpenoids, such as isoprene or tocopherol, stabilize and photoprotect the lipid phase of thylakoid membranes during exposure to strong light and/or elevated temperatures (Wahid and Ghazanfar 2006; Wahid 2007). The expression of stress proteins is an important adaptive mechanism for environmental stress tolerance. Most stress proteins are soluble in water and, therefore, they contribute to stress tolerance, presumably by hydrating cellular structures (Wahid and Close 2007). Heat stress also induces the synthesis of other plant proteins, including ubiquitin (Sun and Callis 1997), cytosolic and chloroplasts Cu/Zn-SOD (Tang et al. 2006) and Mn-POD (Brown et al. 1993), cytosolic (Iba 2002) and chloroplasts APX (Tang et al. 2006), and other antioxidant enzymes (Sairam et al. 2000; Almeselmani et al. 2006), proteins of late embryogenesis abundant (LEA) (Goyal et al. 2005), and dehydrins. Their main function is to protect cellular and sub-cellular structures against oxidative damage and dehydrative forces.

3.3

Adaptation to High Temperature

Plants adapt to heat already after several hours of exposure to a temperature that evokes a stress response, but remains below the lethal temperature level (Xu et al. 2006). For most land plants, heat stress is triggered at temperatures slightly above 35°C, and in grasses at 38–40°C. The loss of resistance (dehardening) is a slower process that lasts several days in optimal growth conditions (Sung et al. 2003; Burke and Chen 2006). During acclimatization, the structure of the cell membrane changes by increasing the share of saturated fatty acids in the lipid layer. More unsaturated acyl residues are removed from the sn-2 position in a glycerolipid molecule by the respective hydrolases. They are replaced with saturated fatty acid residues (mostly 18-carbon chains) with the involvement of the respective

acetyltransferases and lipid transport proteins. At the current state of knowledge, it remains unknown whether a higher or a lower degree of membrane lipid saturation is beneficial for hightemperature tolerance (Klueva et al. 2001; Rahman et al. 2004). It is believed that the synthesis of HSP is also an effective mechanism protecting the plant from high temperature and other HSP synthesis-inducing stressors. In many plants, heat tolerance varies on a seasonal basis in view of their growth cycle and changes in seasonal temperature (Froux et al. 2004). During active growth, all plants are highly sensitive to temperature stress. Selected species of land plants increase their resistance to heat only in the summer, while others demonstrate the highest level of tolerance during winter dormancy. Dormant plants become resistant to stress upon reaching a developmental stage induced by factors other than high environmental temperature. In many land plant species, noticeable changes in heat tolerance are not observed. Due to the close correlation between drought and high temperature, the effects of each stressor on field-grown plants can be difficult to distinguish, and adaptations to arid environments can be effective only if they lead to avoidance or tolerance of both stresses (Fitter and Hay 2002).

4

Conclusion and Future Perspective

Temperature is a major environmental factor that changes from season to season and undergoes daily fluctuations. For this reason, the stressinducing role of temperature is difficult to define unambiguously since the response to various temperatures is determined by the plants’ ability to adapt to different climate regimes. Plants exhibit a variety of responses to different (high and low) temperatures, which are depicted by symptomatic and quantitative changes in growth and morphology. However, it must be remembered the ability of the plant to cope with or adjust to the temperature stress varies across and within species as well as at different developmental stages. Generally heat or low temperature

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stress induces structural changes in tissues and cell organelles, disorganization of cell membranes, disturbance of leaf water relations, and impedance of photosynthesis by effects on photochemical and biochemical reactions and photosynthetic membranes. In response to temperature stress, plants manifest numerous adaptive changes. Metabolic pathways and processes show varied sensitivity to temperature which may result in a deficit or an excess of selected metabolites (such as HSPS, osmoprotectants, antioxidative enzymes, etc.). According to this, it is important to discover the induction of signaling cascades leading to profound changes in specific gene expression is also considered an important temperature-stress adaptation. Molecular knowledge of response and tolerance mechanisms will pave the way for engineering plants that can tolerate high or low temperatures and could be the basis for production of crops which can produce economic yield under temperature-stress conditions (Iba 2002; Wahid et al. 2007). Although physiological mechanisms of heat and low temperature tolerance are relatively well understood, further studies are essential to determine physiological basis of assimilate partitioning from source to sink, plant phenotypic flexibility which leads to tolerance, and factors that modulate plant temperature-stress response. It is known that complex traits of abiotic stress phenomena in plants make genetic modification for efficient stress tolerance difficult to achieve. However, the modification of a single trait resulted in several cases in significant improvements in stress tolerance. By now, little is also known about the molecular mechanisms underlying signaling components during stress response and adaptation. It must be also remembered, alteration of further upstream molecules in the pathway often activates a much wider network of genes, other than stress-specific ones. The discovery and use of new stress-tolerance-associated genes, as well as heterologous genes, to confer plant stress tolerance (including those unique to extreme growth-environment organisms, e.g., halophytes, thermophilic organisms), has been the subject of ongoing efforts to obtain tolerant plants. It must be also remembered, plants receive

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various stresses from their surrounding environment, which affect them in a complex manner (plant is usually subjected to many abiotic and biotic stresses at the same time). That means, it is necessary to identify the key strategies that plants use to deal with complex stresses of both biotic and abiotic origin (Iba 2002; Wahid et al. 2007).

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6

Responses and Management of Heat Stress in Plants Abdul Wahid, Muhammad Farooq, Iqbal Hussain, Rizwan Rasheed, and Saddia Galani

Abstract

On the eve of global climate change, temperature increase, is the most evident phenomenon. This temperature increase is posing severe threat for sustainable crop production in many countries across the globe in the form of heat stress. Plants respond in many ways to the prevailing high temperature environment, and several inter- and intraspecific differences are reported. Heat stress produces quite tangible changes at cell, tissue, and organ levels. Photosynthetic acclimation to heat stress, synthesis and accumulation of primary and secondary metabolites, induction of stress proteins are among the major adaptive responses to heat stress. The important genes expressed in response to heat stress include heat shock protein (hsp) genes, dehydrins (dhn), senescence-associated (sag) genes, staygreen (sgr) genes. As mechanisms of heat stress tolerance, plants display the maintenance of membrane stability, scavenging of ROS, production of enzymatic and nonenzymatic antioxidants and adjustment of compatible solutes. Plant thermotolerance can be improved by various means; major being the mass screening and morphological and biochemical markersassisted selection, identification, and mapping of QTLs conferring heat resistance, conventional and molecular breeding, and exogenous use of

A. Wahid () • I. Hussain Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan e-mail: [emailprotected] M. Farooq Department of Agronomy, University of Agriculture, Faisalabad 38040, Pakistan R. Rasheed Biology Department, Foreman Christian College, Lahore, Pakistan S. Galani Khan Institute of Biotechnology and Genetic Engineering, University of Karachi, Karachi, Pakistan P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_6, © Springer Science+Business Media, LLC 2012

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osmoprotectants and stress-signaling agents. Although pretty well understood, more research efforts are required to understand novel aspects of heat tolerance including molecular cloning and characterization of genes/ proteins and understanding the basis of growth improvements with seed pretreatments and plant acclimations. In this chapter, we discuss the plant responses to high temperature stress and integrated approaches, such as genetics, breeding and management options to improve the resistance in plants against heat stress. Keywords

Heat stress • Metabolites • Molecular breeding • Osmolytes • Pretreatment strategies

1

Introduction

Plant growth and development throughout the globe is controlled, in one way or the other, by the prevailing environmental conditions. Abiotic stresses, including temperature extremes, salinity and drought, are serious intimidation to the sustainability and productivity of economic plants. Current climatic model predicts that global air temperature may increase by 1.1–6.4°C with doubling of atmospheric CO2 (Kim et al. 2007; Lobell and Field 2007). According to Intergovernmental Panel on Climatic Change (IPCC), there was an increase of 0.5°C during the past 100 years in ambient temperature, which is expected to rise by 0.2°C per decade for the next two decades and 1–3.4°C per decade warmer in the year 2100 (IPCC 2007). Such a rise in the global temperatures greatly influences the agricultural production, specifically in terms of aggravating the associated effects of salinity, drought, mineral toxicity stresses or as the case may be. Global warming is negatively affecting the agricultural activities. On exposure to high temperature, crop yield is decreased because of shortened life cycle and accelerated senescence in different agro-climatic zones (Porter 2005). Higher temperatures, either of days and nights or of soil and air, hamper plant growth or cause considerable pre- and postharvest losses (Hall 2001). Injuries occur due to short- and long-term exposure to high temperature. Severe heat stress for

short-terms can permanently damage to cells and tissues due to calamitous collapse of cellular organization (Schöffl et al. 1998). Long-term effects may be reduction in the size of tissues and organs and hampered morphological development (Gilani 2007; Rasheed 2009). Heat stress substantially affects yields of many economically important cereals such as wheat, rice, maize, etc., and effects are quite often registered at the reproductive stages. Photosynthesis is the fundamental basis for carbon accumulation, growth and biomass yield in plants. Photosynthetic response of terrestrial plants can potentially change ecosystem carbon balance and cycling under global warming (Gunderson et al. 2000). Increased ambient temperature affects plant productivity by damaging photosynthesis (Al-Khatib and Paulsen 1990). According to Berry and Bjorkman (1980), at moderately higher temperatures suppression of photosynthetic rate is reversible; nonetheless upon exposure to extremely high temperature, the whole system of photosynthesis may be permanently damaged. It may also decrease chlorophyll content, net photosynthetic rate, and stomatal conductance (Morales et al. 2003). Likewise, upon exposure to severe heat stress, net photosynthesis is substantially inhibited due to impaired supply of Rubulose-1, 5-biphosphate (RUBP) (Law and Crafts-Brandner 1999). Sudden heat stress may either denature the membrane proteins or increase the unsaturated fatty acids, leading to increased ion-leakage and thus the loss of cellular functions (Savchenko et al. 2002).

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Such damages occur due to production of activated (AOS) and reactive oxygen species (ROS) and dehydration-induced changes in phase transitions due to high temperature (Nishida and Murata 1996; Liu and Huang 2000). Both the AOS and ROS react with pigments, membranes, enzymes, and nucleic acids, and change their structure and functions (Smirnoff 1993; Scandalios 1993; Sairam et al. 2000). Electrolyte leakage, a measure of stress injury to membranes, varies in relation to membrane abilities to take up and retain solutes and reflect stress-induced changes in their potential. Studies indicated that finding genotypic variability for heat tolerance based on leaf electrolyte leakage might be more effective to screen plants for relatively hot areas (Li et al. 1991; Rahman et al. 2004; Wahid and Shabbir 2005). Heat stress decreases the activities of antioxidant enzymes leading to increase in injury to cell membranes by lipid peroxidation and leaf senescence (Liu and Huang 2000). Heat stress tolerance in plants is an intricate phenomenon involving a great variety of response, mechanisms, and management practices. Determination of responses and possible strategies to improve heat stress tolerance is important to grow crop plants in the heat-stressed areas of the world. In this perspective, this chapter presents explicit responses and some pragmatic management options to avert the high temperature stress effects in plants.

2

High Temperature Stress: Responses

Plants subjected to heat stress show a range of responses and manifest mechanisms to cope with its adversaries. These changes can be discerned at whole plant to subcellular and molecular levels. An individualistic account of all these responses is briefly described below.

2.1

Growth and Phenology

High temperature is a major determinant of agricultural production throughout the world and its effects are evident at all critical growth

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stages starting from seed germination to final yield harvested. An account of changes in the phenology of plants has been described in the following lines. Seeds put to germinate at supraoptimal temperatures show reduced or even inhibited germination. In soybean, heat stress changed the protein expression profiles and reduced the seed germination and seedling vigor, which appeared to determine the seed quality attributes (Egli et al. 2005; Ren et al. 2009). Seed germination, seedling emergence and its establishment is prone to increased temperature in most of the plant species (Grass and Burris 1995; Burke 2001; Ashraf and Hafeez 2004; Wahid et al. 2007). Columbo and Timmer (1992) demonstrated that black spruce plant seedlings are more susceptible to high temperature stress than adult plants. Maize shows optimal germination and growth at 20–30°C and 28–31°C, respectively (Hughes 1979; Medany et al. 2007; Farooq et al. 2008a, b, 2009). There are conflicting reports about the postemergence seedling growth in maize under heat stress. For instance, some studies show that maize coleoptile was more heat tolerant at all stages of seedling development (Venter et al. 1997; Momcilovic and Ristic 2007), while in some other studies on maize, upon exposure to 40°C, there was a substantial reduction in coleoptile growth and at 45°C growth was completely stopped (Weaich et al. 1996, Akman 2009). Heat stress lowered the activity of specific enzymes and thus reduced the protein synthesis in germinating maize embryos (Riley 1981). Likewise, seedling growth and development of cotton (Gossypium hirsutum L.) was also reduced under heat stress (Mahan and Mauget 2005). High temperature is a major environmental factor that determines the sustainability of crop growth and yield in some regions (Blum 1988; Al-Khatib and Paulsen 1999). Plants grown in warmer environments have much lower biomass than those grown at optimum or lower temperature (Kim et al. 2007). High temperature reduced the plant growth by affecting different mechanisms (Sibley et al. 1999; Wollenweber et al. 2003). For example, it decreased the dry weight,

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Table 6.1 Effect of heat stress on yield and yield components of the bread and durum wheat genotypes Genotype Grains per spike Bread wheat mean Golia 69 Sever Durum wheat mean Acalou TE 9306 Individual grain weight (mg) Bread wheat mean Golia 69 Sever Durum wheat mean Acalou TE 9306 Yield per spike (g) Bread wheat mean Golia 69 Sever Durum wheat mean Acalou TE 9306

Control

Hat stress

Genotype × temperature

70 ± 0.85 69 ± 1.31 a 71 ± 1.10 a 63 ± 0.75 66 ± 0.98 a 60 ± 0.99 b

70 ± 0.88 70 ± 1.43 a 71 ± 1.05 a 64 ± 0.74 65 ± 0.95 a 63 ± 1.15 a

ns ns ns ns ns ns

56.54 ± 1.08 47.11 ± 0.84 a 64.67 ± 1.03 b 72.06 ± 0.60 72.42 ± 0.70 a 71.74 ± 0.95 a

48.73 ± 0.90 43.75 ± 0.90 a 53.53 ± 1.23 b 59.97 ± 0.61 57.69 ± 0.86 a 62.55 ± 0.70 b

*** ** *** *** *** ***

3.97 ± 0.09 3.26 ± 0.08 a 4.58 ± 0.11 b 4.52 ± 0.06 4.76 ± 0.08 a 4.29 ± 0.09 b

3.43 ± 0.07 3.06 ± 0.09 a 3.78 ± 0.09 b 3.84 ± 0.06 3.77 ± 0.08 a 3.92 ± 0.08 a

** ns *** *** *** **

ns nonsignificant, significant at the **0.01, and ***0.001 levels of probability, respectively; for each Triticum species, different letters in the same column refer to significant differences between genotypes. Source: Dias and Lidon (2009)

growth and net assimilation rates of shoot (Wahid 2007 ) . Likewise, heat shock affected the meristematic activity and reduced the growth of various parts mainly the leaves (Salah and Tardieu 1996). Applied heat stress arrested the cell wall elongation and altered cell differentiation (Potters et al. 2007). Reproductive growth is more critically affected by the prevailing high temperature stress during anthesis and seed growth. Pollination is especially sensitive to heat stress. The mature pollens are more sensitive, and quite often fail to fertilize (Dupuis and Dumas 1990). Heat stress interferes with the development of pollen mother cell and microspore and causes male sterility (Sakata et al. 2000; Sato et al. 2006; Abiko et al. 2005). In the event of successful pollination, heat stress affected the kernel development in maize (Monjardino et al. 2005) and reduced the kernel density and reproductive growth in maize, wheat and Suneca during kernel development (Wilhelm et al. 1999; Maestri et al. 2002).

Dias and Lidon (2009) did not find any effect of heat stress on number of grains per spike in both durum and bread wheat; nonetheless upon exposure to heat stress during grain growth, grain size was substantially reduced in both bread and durum wheats (Table 6.1). Likewise, heat stress also reduced the grain yield in both wheats (Table 6.1). However, different genotypes responded variably in terms of grain size and grain yield and a strong relationship between genotypes and temperature has been observed (Table 6.1). High temperature also results in the boll and flower bud abortion in cotton, pea, and brassica, possibly owing to limited water supply and nutrients during reproductive development (Hall 1992; Guilioni et al. 1997; Young et al. 2004). During seed development, heat stress was found to affect seed storage process and kernel quality like starch and protein metabolism during grain filling in maize (Wilhelm et al. 1999; Maestri et al. 2002).

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Fig. 6.1 Hypocotyl elongation phenotype of different pea varieties for 36 h in the dark at 22°C after heat acclimation and stress [(a) local variety, (b) Shandong variety, (c) Taiwan variety]. Left is expressed as control (22°C); middle

is expressed as the germinated seeds were acclimated at 37°C for 1 h, followed at 22°C for 1 h, and then stressed at 48°C for 2 h. Right is expressed as the seeds were stressed at 48°C for 2 h. After Tian et al. (2009) with permission

2.2

round-shaped chloroplasts, swollen stroma lamellae, badly affected the antenna complex of photosystem (PS) II (Carpentier 1999), clump formation of vacuolar contents, disrupted cristae and deformed mitochondria (Zhang et al. 2005). Heat stress restricted the emergence and elongation of hypocotyls in three pea (Pisum sativum) varieties (Tian et al. 2009). Nonetheless, heat acclimation for 1 h at 37°C improved the germination and hypocotyl development (Fig. 6.1). In the sprouting buds of sugarcane, heat stress badly affected the differentiation of various cells and tissues (Rasheed 2009). Here the major changes were noted on mesophyll cell expansion and development of vascular connections (Fig. 6.2).

Anatomical and Developmental Responses

Like other abiotic stresses, heat stress brings about quite a few morphogenetic and histological modifications. At whole plant level, generally cell size is reduced (Santarius 1973; Berry and Bjorkman 1980). There may be several morpho-anatomical modifications in cells and tissues such as increased densities of stomata and trichomes and greater xylem vessels area of shoot and root in Lotus creticus seedlings (Banon et al. 2004). On exposure of grapes to heat stress, cell membrane permeability was substantially increased and mesophyll cells were severely damaged (Zhang et al. 2005). High temperature also causes various changes at subcellular level. For instance, in chloroplast, it changed the thylakoids structure in maize (Karim et al. 1997) and resulted in loss of swelling and stacking of grana (Gounaris et al. 1984). In grapes, heat stress damaged the mesophyll cells, which showed

2.3

Physiological and Metabolic Responses

In hot environments, plants exhibit various physiological and metabolic responses. The most

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Fig. 6.2 Sprouting bud of sugarcane under control condition (left). Effect of heat stress on the histological changes in sprouting buds of sugarcane after 36 h of exposure (middle) and role of proline in mitigating heat stress effect (right). Source: Rasheed (2009)

important of those may be the changes in carbon fixation, oxidative stress; tissue water status and metabolites accumulation. These responses are briefly discussed below.

2.3.1

Photosynthesis

Heat stress causes photosynthetic acclimation and alters the physiological processes directly and changes the developmental patterns indirectly (Downton and Slatyer 1972). All the steps, processes, and aspects of photosynthesis are prone to increased ambient temperature (Al-Khatib and Paulsen 1990). The photosynthesis in C3 plants is more affected by high temperature than C4 plants (Wahid and Rasul 2005). The maize seedling grown at 25°C and transferred to 35°C for 20 min led to 50% inhibition in photosynthesis (Sinsawat et al. 2004). Maize showed maximum net photosynthesis near 31°C, decreased at temperature above 37°C and was completely inhibited near 45°C (Crafts-brandner and Salvucci 2002). Heat stress diminished the net photosynthetic (Pn) and stomatal conductance substantially in many plant species (Ranney and Peet 1994; Crafts-Brandner and Salvucci 2002; Morales et al. 2003); in this regard, Pn in developed leaves was more sensitive than mature leaves (Karim et al. 1997, 1999). Photosynthetic apparatus are highly sensitive to heat temperature and inhibited when leaf temperature exceed 38°C in most plants (Edwards and Walker 1983). PS-II, water splitting and oxygen evolving complex (OEC) in photosynthesis are more heat-sensitive components of

photosynthesis (Havaux 1993; Pastenes and Horton 1996a; Heckathorn et al. 1998a). Extensive studies show that both PS-I and PS-II are damaged by increased temperature. In barley and potato, heat stress damaged PS-I and PS-II and affected electron transport (Havaux 1998; Szilvia et al. 2005). Heat stress damaged the antenna complex of PS-II and reduced photosynthetic behavior (Carpentier 1999; Rokka et al. 2000; Zhang et al. 2005). High temperature during greening led to the inactivation of PS-I and PS-II (Sasmita and Narendranath 2002). High temperature increased chlorophyll a:b ratio and decreased chlorophyll:carotenoid ratio in sugarcane (Wahid 2007). High temperature alters the energy sharing by changing the action of Calvin cycle and other metabolic processes such as photorespiration, synthesis and stability of the Rubisco enzyme (Holaday et al. 1992; Pastenes and Horton 1996b), disruption of electron transport activity and bound RUBP supply by heat stress (Ferrar et al. 1989). Extreme temperature reduced the activation state of Rubisco enzyme in the exposed leaf tissue and increased the RUBP (Feller et al. 1998; Crafts-Brandner and Law 2000), which inhibited photosynthesis as compared to control plants (Sharkey et al. 2001). High temperature enhances chlorophyllase activity and decreases the quantities of photosynthetic pigments (Todorov et al. 2003). The loss of chlorophyll is good indicator of heat tolerance in wheat (Ristic et al. 2007, 2008). High temperature modifies the activities of carbon metabolism enzymes,

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especially the Rubisco (Ferrar et al. 1989; Holaday et al. 1992; Pastenes and Horton 1996a, b). Moreover, activities of starch and sucrose synthesis enzymes are greatly influenced (Chaitanya et al. 2001; Vu et al. 2001).

2.3.2

Reactive Oxygen Species and Oxidative Damage Like other abiotic stresses, heat stress evokes the ROS generation including hydrogen peroxide (H2O2), superoxide radical (O2−), singlet oxygen (1O2) and hydroxyl radical (OH−), and induces oxidative stress (Mittler 2002; Taiz and Zeiger 2006; Potters et al. 2007). Chloroplast and mitochondria are the major sites where superoxide radicals are regularly produced, whereas some quantities are also produced in microbodies. Principally, ROS causes peroxidation of membrane lipids, destruction of pigments, and modification of membrane functions (Xu et al. 2006). The OH− appears to be more damaging than other ROS, which is formed with the combination of O2− and H2O2 in the presence of Fe2+ and Fe3+ in trace amounts in Haber–Weiss reaction (Apel and Hirt 2004). The OH− is greatly damaging to chlorophyll, proteins, lipids, DNA, and other important macromolecules (Sairam and Tyagi 2004). Tolerant plants have the tendency to protect themselves from the damaging effects of ROS with the synthesis of various antioxidant systems (Apel and Hirt 2004). This protection starts with the conversion of O2− by superoxide dismutase (SOD) into H2O2, with the help of ascorbate peroxidase (APX) or catalase (CAT). A number of physiological processes are affected by the overexpression of SOD in plants, including removal of H2O2, toxic reductants, biosynthesis and degradation of lignin in cell walls, auxin catabolism, etc. (Scandalios 1993). Activation of APX is due to the physiological injuries occurring in plants under heat stress (Mazorra et al. 2002). Increased levels of ROS under high temperature cause cellular injury due to reduced antioxidant activity in the stressed tissues (Fadzillah et al. 1996; Mittler et al. 2004). In order to increase the heat tolerance, the levels and activities of antioxidants must be increased to protect against high temperature-induced oxidative

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stress. Studies conducted in this regard revealed that heat acclimated turf grass showed reduced ROS production owing to enhanced ascorbate and glutathione synthesis (Xu et al. 2006). It is suggested that antioxidant capacity of cells can be increased by some signaling molecules (Gong et al. 1997; Dat et al. 1998). Nonetheless, research is imperative to add to the list of potential signaling molecules, which may enhance the antioxidant production in cells exposed to heat temperature stress (Wahid et al. 2007).

2.3.3

Water Relations

Heat stress drives the rapid loss of water from the plant surface, causes tissue and organ dehydration, and restricts growth in plant species, for example, sorghum (Machado and Paulsen 2001), tomato (Mazorra et al. 2002), and sugarcane (Wahid and Close 2007). Heat stress produces osmotic strain on the growing tissues due to diminished root hydraulic conductance and tissue water status (Jiang and Huang 2001; Morales et al. 2003). Likewise, it may result in substantial reduction in sorghum (Sorghum bicolor) leaf growth and leaf water content and water potential in wheat (Shah and Paulsen 2003). Heat stress also disrupts the uptake and translocation of water, ions, and organic solutes across the plant membranes, interferes with photosynthesis and respiration, increases evapo-transpiration rate, reduces the leaf osmotic potential and increases the chlorophyll fluorescence (Tsukaguchi et al. 2003; Huve et al. 2005; Taiz and Zeiger 2006). It results in stomatal closure and reduces the tissue water contents (Berry and Bjorkman 1980; Wahid et al. 2007). Heat stress-induced water stress thus is closely associated with reduction of soil water contents (Talwar et al. 1999).

2.3.4 Osmolytes Accumulation Accumulation of certain low molecular mass organic compounds, generally called compatible solutes or osmoprotectants, is an important adaptive mechanism in plants subjected to abiotic stresses including temperature extremes (Hare et al. 1998; Sakamoto et al. 1998). Several osmolytes, including sugars and sugar alcohols (polyols), proline, tertiary, and quaternary ammonium

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compounds and tertiary sulphonium compounds, are reported to accumulate in different plant species exposed to stress conditions (Sairam and Tyagi 2004; Wahid et al. 2007). Among different compatible solutes, enhanced synthesis of soluble sugars, free proline and glycinebetaine (GB) has been more frequently studied for their osmoregulatory and protective roles (Matysik et al. 2002; Bohnert et al. 2006; Wahid 2007; Wahid et al. 2008; Farooq et al. 2008a). GB plays a great role as osmoprotectant in plants under a range of abiotic stresses including high temperature (Sakamoto and Murata 2002). However, the ability of plants to synthesize GB under stressful conditions varies among species (Ashraf and Foolad 2007). For example, sugarcane under heat stress (Wahid and Close 2007) and maize under drought (Quan et al. 2004) and chilling (Farooq et al. 2008c) and rice under drought (Farooq et al. 2008a) are reported to accumulate large amounts of GB. Like GB, increased free proline accumulation in higher plants in response to abiotic stresses has also been reported (Kavi Kishore et al. 2005). Biosynthesis of GB or proline may buffer the cellular redox potential under heat and other abiotic stresses, suggesting their functional significance (Rontein et al. 2002). The accumulation of soluble sugars was greatly implicated for improved heat tolerance of sugarcane (Wahid and Close 2007). In view of the importance of osmoprotectants accumulation, more concerted efforts on engineering pathways for enhanced biosynthesis of osmolytes may be fruitful (Ashraf and Foolad 2007).

2.3.5 Metabolite Synthesis Heat stress leads to the accumulation of a range of primary and secondary metabolites. Primary metabolites are either direct products of carbon fixation (e.g., sugars, organic acids) or are synthesized after preliminary transformations of primary metabolites (e.g., amino acids, betaines alcohol sugars). Like other abiotic stresses, the accumulation of primary metabolites under heat stress has also been well documented (Iba 2002; Zhu 2003). Important primary metabolites showing accumulation under heat stress include free proline, GB, soluble sugars, etc., (Wahid 2007;

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Wahid and Close 2007; Wahid et al. 2008). In a recent study, using cluster and principal component analyses, it was revealed that out of 122 primary and secondary metabolites determined using advanced techniques like GC-MS and amino acids analyzer, only sucrose, quinate, trans-aconitate, guanine, g-amino butyric acid (GABA), and ethanolamine held relationships with the high temperature tolerance of sugarcane bud chips (Rasheed 2009). On the contrary, the synthesis and accumulation of secondary metabolites are less well understood under high temperature stress. Secondary metabolites are biosynthesized in plants from the intermediates of primary carbon metabolism via phenylpropanoic acid, shikmic acid, mevalonic acid, and methyl erythritol phosphate pathways (Taiz and Zeiger 2006). Recently, it is reported that heat stress induces production of secondary metabolites including phenolics, flavonoids, phenyl propanoids, and plant steroids (Bharti and Khurana 1997; Wahid 2007; Wahid et al. 2008). Carotenoids show a role in protecting cellular structures in various plant species under different stress types (Havaux 1998). Studies show that lipid layer of the thylakoid membranes are stabilized and photoprotected by various carotenoids and some terpenoids such as isoprene and a-tocopherol. Exposure of plants to strong light and high temperatures caused the partitioning of xanthophylls (violaxanthin, anthraxanthin, zeaxanthins, etc.) between the light-harvesting complexes and lipid phase of thylakoid membranes and increases membrane thermostability (Havaux 1993, 1998). Isoprenoids are low molecular weight volatile compounds, synthesized via mevalonic acid pathway (Taiz and Zeiger 2006); their emission from leaves confers their role in heat tolerance (Loreto et al. 1998; Sharkey 2005). Although their synthesis is cost intensive, they show compensatory benefits in terms of heat resistance (Funk et al. 2004). Plants, capable of emitting higher amounts of isoprene, photosynthesize better under heat stress, which indicates a relationship between isoprene emission and heat tolerance (Velikova et al. 2004). Isoprene emission protects PSII under high temperature (Sharkey 2005),

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whereas the endogenous production of isoprene protects the biological membranes by directly binding with singlet oxygen (1O2) by virtue of isoprene-conjugate double bond (Velikova et al. 2004). Phenolics are the largest class of secondary metabolites and include flavonoids, lignin, anthocyanin, etc. Accumulation of soluble phenolics under heat stress is accompanied with increased activity of phenyl ammonia lyase (PAL) but decreased activity of peroxidase polyphenyl lyase (Taiz and Zeiger 2006). Acclimation to heat stress is triggered by the biosynthesis of phenolic compounds induced by high temperature (Rivero et al. 2001). They act as efficient antioxidants in plant tissues under stressful conditions (Dixon and Paiva 1995; Sgherri et al. 2004). Levels of flavonoid (e.g., anthocyanins) are greatly altered in plant tissues under heat stress (Oren-Shamir and Nissim-Levi 1999; Sachray et al. 2002; Wahid et al. 2008). Plant steroids, a class of secondary metabolites, also influence a variety of functions under stressful conditions. Brassinosteroids (BRs) and ginsenosides are important plant steroids whose physiological importance to high temperature tolerance in plants has been explored. Studies confirm that BRs confer tolerance to high temperature stress in brassica and tomato seedlings, by inducing the biosynthesis of major heat shock proteins (Dhaubhadel et al. 1999). Production of ginsenosides, another important plant steroid, has been reported in all organs of Panax quinquefolius. It is recently reported that growing season had a great effect on the ginsenosides biosynthesis P. quinquefolius plants grown at high temperatures had 49% higher concentrations of storage root ginsenosides than respective control plants (Jochum et al. 2007; Wahid and Tariq 2008).

2.4

Molecular Responses

Transcritional regulation plays an important role in plant defense from heat stress (Singh et al. 2002). Heat stress induces numerous genes encoding transcriptional factors, which are involved in heat stress response and tolerance

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(Chen and Zhu 2004; Kotak et al. 2007). Different studies revealed that several genes are up- and downregulated by abiotic stresses (Kawasaki et al. 2001; Provart et al. 2003; Nogueira et al. 2003). Elevated temperature affects the gene expression in storage protein synthesis and starch metabolism during grain filling stage in rice (Yamakawa et al. 2007). Heat stress changes the pattern of gene expression, which is important for thermotolerance (Yang et al. 2006). An account of various genes and proteins showing expression under heat stress is given below.

2.4.1 Heat Shock Genes and Proteins Several transcriptome studies have identified many stress-responsive genes and encoding transcriptional factors during environmental stresses. Recent transgenic approaches suggest that heat tolerance is a multigenic character. Heat shock induces many genes, which are attributed to heat shock elements (HSE).These HSE are situated in the promoter region of hsp genes (Hubel and Schöffl 1994). Transgenic approach confirmed that Heat-shock transcription factor (HSF) binding to pentameric nucleotides (5¢nGAAn-3¢) of HSE sequences (Perisic et al. 1989; Sung et al. 2003). This HSF–HSE interaction and transcriptional activation is quite conserved in nature. This multigenic phenomenon modifying the expression pattern of transcription factors motivate a series of genes (Dong et al. 2003). Studies show that Hot1-Hot4 genes of Arabidopsis may function to improve heat tolerance; Hot1 is identified as Hsp101 in Arabidopsis thaliana (Hong and Vierling 2000). HsfA1 acts as master regulator of thermotolerance in tomato by reducing the expression of heat shock genes in co-suppression lines (Mishra et al. 2002). Hsfs are essential for gene expression in response to high temperature (Nover et al. 2001). Various studies show that distinctive hsp genes are not expressed in germinating pollen. Only hsp18 and hsp70 genes are transcribed in response to heat stress (Wahid et al. 2007). A defective heat shock response of mature maize pollen was due to inefficient induction of heat shock gene transcription (Hopf et al. 1992). Enhanced expression of HSP70 assisted in translocation,

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proteolysis, protein translation, protein folding, aggregation, and refolding of denatured proteins (Zhang et al. 2005; Iba 2002; Weggle et al. 2004; Gorantla et al. 2007). Recent studies revealed that a-amylase genes in seeds of rice reduced the seed weight and chalkiness during ripening under heat stress (Asatsuma et al. 2006; Yamakawa et al. 2007). The synthesis and accumulation of heat shock proteins (HSPs) through heat shock factors (HSFs) network play great role in plant responses to heat stress (Wang et al. 2004; Kotak et al. 2007). Amounts of specific mRNA synthesis, mRNA stability, translation efficiency and alteration in protein activity increase in plants as a result of gene expression (Sullivan and Green 1993). All organisms synthesize HSPs upon exposure to high temperature. Heat stress altered gene expression in reproductive organ of plant (Dupuis and Dumas 1990; Oshino et al. 2007). Abortion of development and demarcation of pollen mother cell due to heat shock is due to tissue specific alterations in gene expression (Sakata et al. 2000; Abiko et al. 2005). In plants, a heat shock of 8–10°C above ambient temperatures induces the synthesis of both high (60–110 kDa) and low (15–30 kDa) molecular weight HSPs (Vierling 1991; Waters et al. 1996; Sun et al. 2002). These HSPs were induced either to protect the plant from injury or to help repair the injury caused by the heat stress (Leshem and Kuiper 1996). The synthesis of HSPs occurred in different plant species when they were exposed 10–15°C above growing temperatures (Dubey 1999). Their synthesis is extremely fast, diverse, and intensive in a variety of organisms (Parsell and Lindquist 1993; Wahid et al. 2007). Both cytosolic and organelle synthesis of HSPs has been well studied. Some HSPs that accumulate in the cytosol at 27°C and in the chloroplast at 43°C and 37°C respectively, appeared to play a role in photosynthesis and thermotolerance (Heckathorn et al. 1998b). In maize, high temperature induced the synthesis and accumulation of chloroplast protein elongation factor EF-TU, which defended the chloroplasts proteins from heat-induced damage (Ristic

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et al. 2004; Momcilovic and Ristic 2007). Maize EF-TU is a 45–46 kDa HSP confined to chloroplast stroma is involved in development at heat tolerance in maize (Ristic and Cass 1992; Bhadula et al. 2001; Moriarty et al. 2002). In maize, heat shock of 40°C induces the synthesis of HSPs18 (Nieto-Sotelo et al. 2002). Interaction of HSPs 22 kDa with the Chenopodium album and common bean chloroplast membranes affects the composition of membrane and decreases its fluidity; thus increasing the efficiency of ATP transport (Barua et al. 2003; Simões-Araújo et al. 2003). Mitochondrial HSPs have been isolated from pumpkin (Cucurbita pepo) cotyledons under high temperature stress (Tsugeki et al. 1992; Kuzmin et al. 2004). They act as molecular chaperones in vitro (Schöffl et al. 1998; Guo et al. 2001; Kim and Schöffl 2002), prevent aggregation of denatured proteins (Sheffield et al. 1990), aid in folding of nascent polypeptides and refolding of denatured proteins (Lee et al. 1994; Goloubinoff et al. 1999). They also resolubilize the denatured aggregated proteins (Parsell et al. 1994). HSP68 synthesis was restricted to mitochondria as a precursor protein, but its synthesis increased during heat shock in cell (Neumann et al. 1993). When wheat, maize, and rye seedling were exposed at 42°C, five mitochondrial LMW HSPs (19, 20, 22, 23, and 28 kDa) were induced in maize and only one (20 kDa) in rye and wheat mitochondria each; the tolerance of maize was higher than wheat and rye (Korotaeva et al. 2001). The specific nucleusencoded HSPs have been identified in potato, maize, soybean, barley, and tomato (Neumann et al. 1993; Nautiyal and Shono 2010), peas (Ko et al. 1992; Watts et al. 1992) under heat stress. Although with less certainty, some putative functions have been assigned to HSPs when produced under normal or high temperature conditions. The rapid accumulation of HSPs may play a significant role in the safety of metabolic apparatus of the cell. Some HSPs are produced in some developing cells under control condition (Hopf et al. 1992) during embryogenesis, germination,

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Fig. 6.3 Effect of heat acclimation and stress on the expression of HSP70 in hypocotyls of different pea varieties. After Tian et al. (2009) with permission

pollen formation, fruit set and its maturation (Vierling 1991; Sun et al. 2002 Prasinos et al. 2005; Wahid et al. 2007). For instance, HSPs were produced in greater amounts in etiolated maize seedling after 5-h exposure to high temperature stress (Lund et al. 1998). Acquired thermotolerance depends upon the synthesis of HSPs and their cellular localization (Heckathorn et al. 1999; Korotaeva et al. 2001). In arid and semi arid areas, plants may accumulate significant amount of HSPs in response to high leaf temperatures. In 2-day-old soybean seedlings, HSPs appeared to maintain the conformation of other proteins, as an aid for the acquired thermotolerance (Jinn et al. 1997). The wide diversity and abundance of HSPs is important for altering the plant response to high temperature stress (Waters et al. 1996). The mature pollen was susceptible to high temperature and pollen viability was extremely reduced due to nonproduction of HSPs. A distinct set of HSPs was induced in male tissues of maize under heat stress (Dupuis and Dumas 1990). HSPs (64 and 72 kDa) were induced in germinating pollens under heat stress (Frova et al. 1989). In a recent study, Tian et al. (2009) reported the improved heat tolerance of

young pea seedlings due to enhanced synthesis of HSP70 (Fig. 6.3).

2.4.2 Dehydrins Dehydrins (DHNs), belonging to subclass of LEA group II (Dure et al. 1989), are produced at the later stages of seed development in various plant species under drought, salinity, low temperature, heat stress, nutrients deficiency, and ABA application (Close 1996; Campbell and Close 1997; Svensson et al. 2002; Wahid and Close 2007; Pulla et al. 2007; Rurek 2010). D-11 from cotton (Baker et al. 1988), RAB16 (responsive to ABA) in rice (Mundy and Chua 1988) and RAB17 in maize (Vilardell et al. 1990) were cloned and characterized as DHN genes (Campbell and Close 1997; Ismail and Hall 1999 Koag et al. 2003). Immunological evidence indicated that DHNs are expressed in cyanobacteria (Close and Lammers 1993), brown algae (Li et al. 1997), liverworts (Hellwege et al. 1994), ferns (Reynolds and Bewley 1993), ginkgo (Close and Lammers 1993), and conifers (Jarvis et al. 1996). Using immunological studies, DHNs were detected in the nucleus, cytoplasm, mitochondria, chloroplasts, and vacuole (Close 1996; Campbell

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Fig. 6.4 Immunohistochemical expression of dehydrins in the leaf of sugarcane clone HSF-240 under control and heat stress. The dehydrins were found to associate to stele,

lower and upper epidermis, and buliform cells during heat stress, as evident from golden brown color in staining. Source: Gilani (2007)

and Close 1997; Wahid et al. 2007) and found to be associated with cytoplasmic membranes system under abiotic stresses (Koag et al. 2003). Immuno-histolocalization studies revealed that the DHNs are associated with the mesophyll, vascular, and dermal tissues of heat-stressed sugarcane (Gilani 2007, Fig. 6.4). In maize, all parts of mature embryos show dehydrin accumulation (Godoy et al. 1994). In recent studies, three low molecular weight dehydrins were reported to be expressed in sugarcane leaves in response to heat stress (Wahid and Close 2007).

1994) and aspartic proteinase in Brassica (Buchanan-Wollaston and Ainsworth 1997) are associated with leaf senescence. A large number of SAGs and defense genes has been reported to express during leaf senescence in maize (Smart et al. 1995), barley (Kleber-Janke and Krupinska 1997), rice (Lee et al. 2001), A. thaliana (Lohman et al. 1994; Oh et al. 1996; Gepstein et al. 2003), tomato (John et al. 1997; Drake et al. 1996), radish (Azumi and Watanabe 1991), and Brassica napus (Buchanan-Wollaston and Ainsworth 1997). Heat stress accelerates the senescence and results in decreased assimilation partitioning to grains (Spano et al. 2003). For instance, high temperature induced the expression of dehydration responsive genes (ERD1), which is known as SAG15. This gene also protects the cells from injury (Weaver et al. 1999). A combine effect of heat-shock and drought induced a senescenceassociated gene (SAG12), at least in Nicotiana tabacum, which improved the stress tolerance in plants (Rizhsky et al. 2002). Heat shock (40°C) induced tmr genes in Agrobacterium, which delays the senescence. This was achieved by an inducible promoter such as HS6871 from soybean (Smart et al. 1991). Chen et al. (2002) identified 18 transcription factors such as WRKY

2.4.3

Senescence-Associated Genes

Temperature, pathogenic infection, drought, and nutrient deficiency; wounding and shading may increase leaf senescence (He et al. 2001). Thus about 183 senescence-associated genes (SAGs) are involved in energy metabolism, gene expression regulations, protein biosynthesis regulations, pathogenicity, stress and flower development (Liu et al. 2008). QTLs for some senescencerelated traits have been mapped on chromosome 2A, 3A, 3B, 6A, 6B, and 7A in winter wheat subjected to heat stress (Vijayalakshmi et al. 2010). A number of encoding SAGs for proteinases such as serine proteinase in parsley (Jiang et al. 1999), cysteine proteinase in Arabidopsis (Lohman et al.

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genes and its protein in response to senescence and environmental stresses, including heat stress, which improved the agronomic characters of crop plants.

2.4.4 Stay-Green Gene Photosynthetic responses of annual plants can be improved by extending duration of vegetative growth and delaying leaf senescence (Thomas and Howarth 2000). Stay-green (Sgr) proteins are responsible for the green-flesh and retention of chlorophyll during senescence (Park et al. 2007; Barry et al. 2008). The trait stay-green is divided into five types such as type A, B, C, D, and E on the basis of its chlorophyll retention during leaf senescence (Thomas and Howarth 2000). Overexpression of Sgr gene reduces the loss of chlorophyll and delays early senescence of developing leaves (Park et al. 2007). Sgr synthesis has been reported in many plants such as sorghum (Tao et al. 2000), maize (Rajcan and Tollenaar 1999), rice (Cha et al. 2002; Park et al. 2007), durum wheat (Spano et al. 2003), tomato (Akhtar et al. 1999; Barry et al. 2008), pea (Sato et al. 2007) A. thaliana (Oh et al. 2000; Ren et al. 2007), oat (Helsel and Frey 1978), and Festuca pratensis (Armstead et al. 2006). A stay-green protein potentially downregulates the chlorophyll degradation at transcriptional level and delays senescence (Nam 1997; Park et al. 2007). Delaying leaf senescence resulted in about 11% increase in carbon fixation in Lolium temulentum (Thomas and Howarth 2000). Tollenaar and Daynard (1978) demonstrated that some maize varieties such as L087602 shows stay-green phenotype, which increases the water, carbohydrates, and protein contents in the husks, cobs, and seeds. Nguyen (1999) demonstrated that stay-green genes delay leaf senescence in sorghum and reduce lodging in heat-stressed and low moisture areas. In fact stay-green is used as a selection criterion in warm areas (Acevedo et al. 1991; Kohli et al. 1991). For instance, most lines of wheat are sensitive to heat stress while some lines are heat tolerant due to stay-green character (Rehman et al. 2009).

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3

High Temperature Stress: Management

Despite the fact that heat stress affects all the aspects of growth and development in plants, its effects may be mitigated by adopting various approaches. Some of the pragmatic strategies in this regard are detailed below.

3.1

Exploitation of Genetic Variability

As mentioned above, heat tolerance is a multigenic trait, which offers the opportunity of improving plants against heat stress. Temperate genotypes show less dry matter production and reduced yield due to high temperature stress as compared to tropical ones (Giaveno and Ferrero 2003). Attempts have been made to find the genetic differences in plants based on morphological and physiological criteria (Wahid et al. 2007; Khan et al. 2008). For instance, high temperature stress during grain filling can reduce setting and filling of seed by accelerating senescence thereby reducing crop yields (Harding et al. 1990; Siddique et al. 1999). This is because, resources required are utilized by plants for heat stress tolerance and limited amount is available for reproductive growth (Hall 1992). Search for genotypic variation in heat resistance on the basis of leaf electrolyte leakage is important for improving heat tolerance (Li et al. 1991). It is known that membrane stability is positively associated with crop yield under heat stress (Rahman et al. 2004). Such association was important for survival of wheat when exposed to high temperature at anthesis stage (Saadalla et al. 1990).

3.2

Conventional Breeding and Molecular Strategies

Conventional and modern breeding methods have been practiced for improving plant stress tolerance for the last many decades. For conventional

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breeding, major challenges include germplasm mass screening, selection criteria, and identification of heat tolerant materials (Wahid et al. 2007). Among the several screening and selection methods, heat tolerance index (HTI) based on recovery of plants from heat stress has been proposed in case of sorghum (Young et al. 2001). In tomato, for example, under stress, there was a positive relationship between fruit set and yield. In this regard, many studies suggested that heat stress is not the only reason for impaired seed setting, nonetheless heat stress driven reduction in pollen tube growth and fertilization are rather more important (Wahid et al. 2007). Present-day molecular and biotechnological tools have contributed enormously to understand the complexity and cellular pathways of stress responses and signal transduction mechanisms under abiotic stress responses (Sreenivasulu et al. 2006). Molecular studies involving cDNA arrays have revealed several genes, which are upregulated by several biotic and abiotic stresses (Kawasaki et al. 2001; Provart et al. 2003; Nogueira et al. 2003). Many of these genes are involved in signaling pathways via encoding proteins particularly mitogen-activated protein kinase (MAPK), histidine kinase, Ca2+-dependent protein kinase (CDPK), SOS3 Ca2+ sensor family and numerous transcription factors. Marker-assisted selection (MAS) and genetic transformation are two main approaches to improve heat stress tolerance in plants, which proved worthwhile in improving our understanding of stress tolerance mechanisms at molecular level (Foolad 2005). The use of MAS approach requires identifying genetic markers associated with genes or QTLs contributing to whole or individual stress tolerance. A number of research efforts have identified genetic markers related to different environmental stresses at various growth stages of plants to facilitate understanding of genetic relationships for stresses tolerance (Foolad 2005). However, limited studies have identified genetic markers related to high temperature tolerance in plant. Available studies show that in a number of Arabidopsis mutants, four QTLs were found to be involved in acquiring thermotolerance in heat-sensitive mutants (Hong

and Vierling 2000). Use of restriction fragment length polymorphism (RFLP) mapped 11 QTLs for pollen germination and pollen tube growth in heat-stressed maize plants (Frova and Sari-Gorla 1994; Wahid et al. 2007).

3.3

Seed Treatments and Plant Acclimation

In addition to the above, seed treatment and planting materials and foliar spray of with various organic and inorganic agents has proven their worth in enhancing heat tolerance in a number of plant species. For instance, presowing GB treatment in barley (Wahid and Shabbir 2005), H2O2 treatment in maize (Wahid et al. 2008), and presprouting soaking of sugarcane bud chips with GB and proline (Rasheed 2009) have been implicated with great success in improving high temperature tolerance at germination and subsequent growth stages (Fig. 6.2). Epibrassinolide, a subclass of brassinostreoids, treatment modulated the translational machinery, resulting in higher HSPs synthesis and rapid resumption of protein synthesis during and after the application of high temperature stress (Dhaubhadel et al. 1999; Kagale et al. 2007). More so, precondition of tomato plants (Morales et al. 2003) and acclimation of turfgrass (Xu et al. 2006) to heat stress resulted in better growth of plants in heat-stressed environments.

4

Conclusion and Future Perspective

Responses of plants to heat stress may be symptomatic to quantitative. Despite the fact that heat stress responses are well evident at all growth stages, reproductive growth stages are more prone to heat episodes. Other heat stress effects may entail structural changes in tissues and cell organelles, disturbance of leaf water relations, and decline in the rate of photosynthesis, production of ROS and lipid peroxidation, changes in enzymatic and nonenzymatic antioxidants, and synthesis of secondary metabolites are also important.

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The adaptive mechanisms of plants in response to heat stress include the induction of signaling cascades resulting in expression of specific gene. Synthesis of HSPs is a universal heat stress response in plants. Recently, evidence is pouring in on the synthesis and accumulation of some other stress-related proteins. All such proteins function as molecular chaperones and maintain three-dimensional structure of membrane proteins for sustained cell metabolism and plant survival under heat stress. Use of classical and modern breeding protocols, hunting genetic diversity for high temperature tolerance, use of presowing seed treatments and planting materials, and preconditioning/hardening of plants for high temperature tolerance has been beneficial. Although a considerable progress has been achieved in understanding the heat responses of plants, yet there is need for further understanding the biochemical and molecular basis of heat tolerance for improvement of yield benefits from hot environments. Recent molecular biology and gene transfer protocols can play important roles in this regard. Since benefits of seed treatments have been accrued at advanced growth stages (Wahid and Shabbir 2005), the basis of such changes needs to be explored.

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Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants Dana Zoldan, Reza Shekaste Band, Charles L. Guy, and Ron Porat

Abstract

Many plants of tropical and subtropical origin are severely damaged when exposed to chilling temperatures between 2 and 15°C. In contrast, the cruciferous plant Arabidopsis thaliana is chilling tolerant and, therefore provides an alternative model plant system for the identification of chilling tolerance traits. In this chapter, we describe physiological, biochemical, and molecular responses of Arabidopsis class 1 chilling-sensitive (chs) mutants to low temperatures. These mutants, including chs1, chs2 and chs3, are extremely chilling-sensitive and wilt and turn yellow in just a few days after transfer to low temperatures of 4–13°C. Overall, following exposure to chilling, class 1 chs mutants suffer from: (1) loss of chlorophyll and decrease in photosynthetic efficacy resulting in lack of starch accumulation, (2) damage to cellular membranes resulting in increased electrolyte leakage, and (3) accumulation of the reactive oxygen species (ROS) hydrogen peroxide (H2O2). At the molecular level, transcriptome analysis studies following exposure to 10°C for 48 h using the Affymetrix ATH1 genome array reveal remarkable changes in expression patterns of between 1,500 and 3,000 genes, which are significantly differentially expressed (p £ 0.05 and up- or down-regulated by a factor of at least 4) in chs1, chs2, and chs3 mutants compared to wild-type (WT) plants. The main functional categories of up-regulated genes by chilling include “stress,” “protein,” and “signaling,” whereas the main categories down-regulated by chilling were “photosynthesis,” “tetrapyrrole synthesis,” “carbohydrate metabolism,” “cell wall,” and “lipid metabolism”. Overall, these and other studies using Arabidopsis chilling-sensitive mutants allow the recognition of major genetic traits crucial for plant survival under chilling conditions. R. Porat () • D. Zoldan Department of Postharvest Sciences of Fresh Produce, ARO, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel e-mail: [emailprotected] R.S. Band • C.L. Guy Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611, USA P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_7, © Springer Science+Business Media, LLC 2012

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Keywords

Arabidopsis • Low temperature stress • ROS • Transcriptome • Physiological response • Biochemical response • Gene mapping

1

Introduction

Low temperature is an important environmental factor that greatly influences the growth, development, survival, and geographical distribution of plants (Levitt 1980). Whereas most plant species from temperate regions can acclimatize to cold and can survive exposures to deep freezing temperatures, plants of tropical and subtropical origin are severely injured when exposed to low nonfreezing temperatures between 0 and 15°C (Lyons 1973; Lynch 1990; Wang 1990). Symptoms of chilling injury often include cessation of growth, wilting, chlorosis, necrosis, and eventually plant death (Lyons 1973; Graham and Patterson 1982; Maruyama et al. 1990; Allen and Ort 2001). In addition to its adverse effects on plant growth and development, chilling sensitivity also imposes major limitations on the postharvest storage and handling of many fruits and vegetables, because it necessitates storage at relatively high temperatures that do little to delay deterioration and spoilage (Paull 1990). In contrast to our knowledge of plant responses to other abiotic stresses, such as freezing, drought, salinity, and heat, little is known regarding the molecular basis in regulating chilling tolerance or about the signal transduction networks involved in its acquisition. In previous reviews, the occurrence of chilling damage was attributed mainly to the general disruption or dysfunction of cellular metabolic processes (Lyons 1973; Graham and Patterson 1982; Markhart 1986). In this respect, it has been suggested that an important primary event in the occurrence of chilling injury is an alteration in the physical state of the cellular membranes, which leads to dysfunctional selective permeability and increases in solute leakage from the cells (Lyons 1973; Markhart 1986; Nishida and Murata 1996). Another factor that affects susceptibility to chilling is the status of

the cellular antioxidant defensive system required to avoid accumulation of toxic reactive oxygen species (ROS). Indeed, overexpression of antioxidant defensive genes, such as ascorbate peroxidase, superoxide dismutase, and catalase (CAT) enhanced chilling tolerance (Van Breusegem et al. 1999; Payton et al. 2001), whereas repression of catalase gene expression reduced chilling tolerance (Kerdnaimongkol and Woodson 1999). Finally, several reports have suggested that various stress genes, usually related to other types of stress responses, may also contribute to the acquisition of chilling tolerance. For instance, it has been suggested that heat shock proteins (HSPs) (Sabehat et al. 1998) and dehydrin genes (Ismail et al. 1999) may also be factors in chilling tolerance in plants. In this chapter, we will summarize data obtained from studies on Arabidopsis chillingsensitive (chs) mutants, and will suggest how the adoption of Arabidopsis as a plant model system could improve our basic understanding regarding the molecular and biochemical nature of chilling tolerance. Overall, Arabidopsis has many advantages as a plant model system and its chilling tolerance makes it an excellent study subject for the identification of major genetic traits important for low temperature survival of plants.

2

Using Arabidopsis as a Genetic Resource for Identification of Chilling Tolerance Traits

It is believed that chilling-sensitive species evolved in warm tropical regions where there was no selection pressure favoring growth at low temperatures. In contrary, the dispersal of plants to cooler climates necessitated the acquisition of chilling tolerance and other low temperature

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants

tolerance traits. Indeed, trees and shrubs in temperate regions are able to grow at low chilling temperatures, and become dormant during the coldest portion of the winter when temperatures are below 0°C (Levitt 1980). Many herbaceous annual species such as Arabidopsis normally overwinter in their vegetative state, survive both chilling and freezing conditions, and switch to a reproductive stage during the spring, when temperatures rise. Thus, over-wintering herbaceous annual species can be very resistant to chilling, and continue their growth and development, albeit more slowly, at low temperatures. Indeed, it has been shown that Arabidopsis can complete its entire life cycle and produce fertile seeds when sown and grown under continuous chilling temperatures of 4–6°C (Hasdai et al. 2006). Therefore, naturally chilling-resistant plants, such as Arabidopsis, may provide valuable genetic resources for the identification of chilling tolerance traits which, in the future, may be incorporated into horticultural important chilling-sensitive crops (Tokuhisa and Browse 1999; Porat and Guy 2007). Arabidopsis mutants and forward genetic approaches have become useful tools to study the molecular and physiological responses and traits of plants to low temperature stress. Because Arabidopsis is freezing tolerant and can modulate its freezing tolerance by cold acclimation, and is easily genetically modified, creating and characterization of mutants deficient in freezing tolerance has been a productive strategy to uncover major determinants of signaling pathways and regulators of gene expression at low temperatures. Forward genetic analysis has identified a number of transcription factors like HOS9, HOS10 and other regulators of freezing tolerance like ESKIMO1 (ESK1). HOS9 and HOS10 encode a homeodomain and MYB transcription factors, respectively, (Zhu et al. 2007). Loss-offunction mutations in HOS9 and HOS10 cause significant decreases in basal and acquired freezing tolerance. Mutations in ESK1 (Xin and Browse 1998) result in constitutive freezing tolerance. Also, mutations in a transcriptional adaptor protein ADA2b causes constitutive freezing tolerance (Vlachonasios et al. 2003). Another

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example of forward genetics includes the chy1–10 mutant that is freezing-sensitive after cold acclimation. The chy1 mutant accumulates ROS. Mapbased cloning of CHY1 revealed that the mutant encodes a peroxisomal b-hydroxyisobutyryl– CoA hydrolase needed for valine catabolism and fatty acid b-oxidation, and suggests a role in cold stress signaling, and freezing tolerance for peroxisomal metabolism (Dong et al. 2009). Arabidopsis mutants have also helped to bring light to the relationships of lipid metabolism and low-temperature exposure particularly as the isolation of mutants with altered lipid compositions has assisted in biochemical and molecular approaches to understanding lipid metabolism and membrane function (Wallis and Browse 2002). Further, the availability of a variety of plant lines with specific changes in membrane lipids has afforded valuable resources to study the structural and adaptive roles of lipids. Presently, there are at least five types of Arabidopsis fatty acid metabolism mutants that grow well at 22°C, but are injured at low temperatures (2–6°C). The mutant lines include fab1 (Wu et al. 1997), fad2 (Miquel et al. 1993), fad5, fad6 (Hugly and Somerville 1992), and the fad3-2 fad7-2 fad8 triple mutant (Routaboul et al. 2000). Some examples that could be equated to chilling injury include the fab1 mutant of Arabidopsis thaliana that was derived from the Columbia ecotype following mutagenesis with ethane methyl sulfonate (EMS). The mutant contains increased levels of saturated fatty acids, particularly increased proportions of 16:0 in all of the major membrane lipids of the leaf tissue (Lightner et al. 1994). When subjected to 2°C, after 14 days, major changes in chloroplast ultrastructure were observed (Wu et al. 1997). The mutant chloroplasts were irregular in shape with poorly defined and broken envelopes. By 21 days at a low temperature, chloroplasts had largely disappeared from the mutant, but some chloroplast remnants remained visible in most mesophyll cells. The loss of chloroplast ultrastructure correlated with a major loss of photosynthetic function as indicated by Chlorophyll a fluorescence data. Nevertheless, the fab1 plants were able to largely recover upon return to 22°C. Given that

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fab1 plants are unable to maintain photosynthetic function at 2°C and will die after 5–7 weeks, the sensitivity of fab1 plants to 2°C may result from increased levels of phosphatidylglycerol (PG) having high-melting-point molecular species (containing only 16:0, 18:0, and 16:1, D3-trans fatty acids) (Kim et al. 2010). A fab1 suppressor line, S7, had reduced levels of 16:3 fatty acid in leaf galactolipids compared to WT and was identified by map-based cloning as a hypomorphic allele of lysophosphatidic acid acyltransferase1 (lpat1), lpat1-3. The lpat1-3 mutation was found to strongly affect fatty acid composition of PG, with the proportion of highmelting-point molecular species in PG being reduced from 48.2% in fab1 to 10.7% in fab1 lpat1-3 (S7), a value that was close to the 7.6% found in wild type. The ability to modulate membrane fatty acid unsaturation has long been thought to be critical to membrane function and cell viability for poikilothermic organisms like plants. The Arabidopsis fad3-2, fad7-2, fad8 triple fatty acid desaturase mutant was found to be only subtly impacted upon short-term exposure to low temperatures, with small decreases in photosynthetic quantum yield, FII, were observed in the mutant (Routaboul et al. 2000). However, long-term exposure to 4°C resulted in lower fluorescence parameters, chlorophyll content, photosynthetic processes, and thylakoid membranes in the triplemutant. When taken together, the examples outlined above demonstrate the value in forward genetic approaches to understanding plant lowtemperature responses. Overall, in contrast to nearly all chillingsensitive species, with Arabidopsis it is relatively easy to screen large mutagenized populations for individuals sensitive to chilling phenotype resulting from a loss-of-function mutation in an important gene. Furthermore, there are Arabidopsis populations with insertional mutations, in which the compromised genetic locus can be readily obtained, and the mutated gene can be isolated and sequenced. Additional follow-up complementation studies can confirm the linkage of a mutant gene with the ability of the plant to tolerate chilling.

3

Arabidopsis Chilling-Sensitive Mutants

In order to study the molecular basis of chilling tolerance in Arabidopsis, EMS-mutagenized M2 populations of Arabidopsis were grown at 22°C for 2 weeks after which they were transferred to 10 or 15°C and were screened for the appearance of chilling-sensitive mutants (Schneider et al. 1995a). Of about 20,000 M2 plants examined, 21 mutants were identified that appeared normal at 22°C, but developed chlorosis or necrosis when shifted to lower temperatures (Schneider et al. 1995a). The chilling-sensitive mutants were categorized into four different phenotypic classes according to the severity of their chilling damage symptoms: class 1 mutants (chs1–3) turned yellow, wilted and died, in class 2 mutants (chs4) only the mature leaves became necrotic, and class 3 mutants (chs5–6) developed yellow chlorotic patches, but continued to grow and develop at low temperatures; and in class 4 (chs7–15) only part of the leaf near the rosette turned yellow (Schneider et al. 1995a). Crosses among mutants in different phenotypic classes showed that those in the first three classes were found only in a small number of loci (Schneider et al. 1995a). Detailed characterization of the chs1 mutant revealed that it was sensitive to temperatures below 18°C, and that exposure to low temperatures resulted in defects in chloroplast maintenance and integrity, including disruption of chloroplast protein accumulation and altered steryl-ester metabolism (Hugly et al. 1990; Patterson et al. 1993; Schneider et al. 1995b). Furthermore, it was found that only the leaf tissues of chs1 plants were injured at low temperatures whereas germination, root and callus growth were unaffected by chilling. All these findings suggest that the function of the chs1 gene product may be required to maintain chloroplast function at low temperatures. Overall, the chs1 mutant was found to be extremely sensitive to low temperatures, and after 3 days at 13°C, as the plants become irreversibly injured and could not be rescued upon returning them to normal temperatures (Schneider et al. 1995b). Interestingly, it was also

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants

noted that the chs1 mutants were much more sensitive in terms of leaf yellowing and time till the initiation of wilting to exposure to 15°C than to 5°C (Schneider et al. 1995b). Transcriptome profiling studies among approximately 8,000 Arabidopsis genes that compared gene expression patterns in wild-type (WT) plants with those in 12 chilling-sensitive mutants showed that the expression of more than 1,000 genes in normal plants was unaffected by chilling at 13°C but was affected by at least twofold in class 1 chs mutants (Provart et al. 2003; Zhu and Provart 2003). In the light of these microarray expression data, it was suggested that the normal functions of the mutated chs1, chs2, and chs3 genes might be to prevent widespread chilling damage effects on transcriptional regulation (Provart et al. 2003). It was further observed that the profiles of gene expression of the various class 1 chs mutants (chs1, chs2, and chs3) at 13°C were very similar to each other, which supports the idea that the products of these genes might perform related biological functions. In the future, identification of the gene products of class 1 chs mutants by means of mapping and chromosome walking technologies will certainly provide important insights into the molecular basis of at least some major factors that are crucial for plant survival at chilling temperatures. A recent report on the chs3 mutant has demonstrated the expected arrested growth and chlorosis phenotype when grown at 16°C or when shifted from 22 to 4°C. chs3 plants exhibited chlorotic and spontaneous lesion phenotypes when grown at 16°C as older leaves turned yellow and died, and emerging leaves became watersoaked (Yang et al. 2010). Evidence presented indicated that chs3 plants exhibit an activated defense response at 16°C, which was suppressed to WT levels at 22°C. Map-based cloning of chs3 gene revealed an unusual disease resistance protein belonging to the TIR-NB-LRR class and also having a zinc-binding LIM domain at the carboxyl terminus. The mutation of a G-to-A substitution at the ninth intron–exon junction appears to lead to abnormal splicing and the formation of a truncated protein. Thus, chs3 seems to be a gain-of-function mutation as the mutation led to

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the constitutive activation of the TIR-NB-LRR domain. An activated defense response was supported by hydrogen peroxide (H2O2) analyses with 3,3¢-diaminobenzidine (DAB) staining. Strong staining was observed in chs3 plants grown at 16°C, but not in WT plants under the same conditions indicating H2O2 levels were high in the mutant. The chs3 growth and defense phenotypes could be suppressed by eds1, sgt1b and rar1, and partially suppressed by pad4 and nahG, but not by npr1 and ndr1. These findings reveal an unexpected linkage between defense responses and cold stress, and points to a mutual interaction between cold signaling and defense responses (Yang et al. 2010). Beside chs3, the only other gene among the 21 chs mutants identified by Schneider et al. (1995a) that has been cloned so far is chs5; it belongs to class 3 of chs mutants, which become chlorotic at low temperatures but otherwise continue to grow and develop normally. Genetic and sequence analysis demonstrated that the chs5 mutation occurred in the coding region of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), an enzyme belonging to the nonmevalonate pathway localized in the chloroplast (Araki et al. 2000). DXS functions in the synthesis of isoprenoid compounds like carotenoids, xanthophylls, sterols, and isopentynyl chains of cytokinins and chlorophylls. Once again, as in the case of the chs1 mutation, it seems that among the diverse components of cellular machinery that chloroplast is especially vulnerable to low chilling temperatures. In another independent study, Tokuhisa et al. (1997) identified additional Arabidopsis EMS and T-DNA insertion chilling-sensitive mutants that were indistinguishable from WT plants when grown at 22°C, but exhibited visible chilling symptoms after 42 days of growth at 5°C. Under these conditions, the chilling symptoms that were identified included chlorosis, reduced and impaired growth (small stature, reduced leaf growth, high anthocyanin content, and distorted leaf morphology), necrosis, and death. Thus, two independent populations of chilling-sensitive mutants have been identified so far in Arabidopsis: class 1 chs mutants described by Somerville and coworkers (Schneider et al. 1995a) which showed

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apparent chilling damage symptoms after a short exposure (3–7 days) to mild temperatures (10–15°C), and the mutants described by Browse and coworkers (Tokuhisa et al. 1997) which showed apparent chilling symptoms only after a much longer period of 6 weeks at a very low temperature of 5°C (Schneider et al. 1995a; Tokuhisa et al. 1997). The first type of mutants, especially class 1 chs mutants, most likely encode proteins crucial for basic aspects of cell survival at chilling temperatures, whereas the second type of mutants probably involves governing particular mechanisms of adaptation to low temperatures of older cells and organs or are characteristic of older cells. One of the latter T-DNA-tagged mutants, paleface1 (pfc1), that becomes chlorotic during growth at 5°C, was cloned and encodes a specific 16 S rRNA methylase, which is required for maintenance of a particular step in pre-RNA processing in the chloroplasts, and which is apparently sensitive to low temperatures (Tokuhisa et al. 1998). Further cloning and isolation of additional Arabidopsis chilling-sensitive genes will greatly improve our understanding regarding the molecular basis of chilling tolerance (Tokuhisa et al. 1998; Araki et al. 2000). In the following sections, we will analyze in more details the physiological, biochemical and molecular responses of class 1 chilling-sensitive mutants, including chs1, chs2, and chs3, to low temperatures, and based on that will aim to identify crucial traits required for survival of Arabidopsis plants under chilling conditions.

4

Physiological and Biochemical Responses of chs1, chs2, and chs3 Mutants to Chilling

Class 1 chilling-sensitive mutants include chs1, chs2, and chs3. In addition to previous observations regarding the responsiveness of chs1 plants to chilling (Hugly et al. 1990; Patterson et al. 1993; Schneider et al. 1995b), we hereby show that upon transfer of 2-week-old plants from a normal temperature of 22°C to a moderate chill-

ing temperature of 10°C all class 1 chs mutants, including chs1, chs2, and chs3, turned yellow, wilted and eventually died (Fig. 7.1). The yellowing and growth arrest phenotypes become visible in 7 days after transfer to chilling temperatures, and these phenotypes become more severe as the time of exposure to chilling increased up to 3 weeks (Fig. 7.1). Furthermore, we found that in addition to what has been reported previously for chs1 (Hugly et al. 1990), chs3 mutants are also extremely sensitive to low temperatures, as they showed a severe dwarfism phenotype when grown at a moderate chilling temperature of 18°C (Fig. 7.2). Overall, all class 1 chilling-sensitive mutants are very sensitive to chilling, and turn yellow and wilt in just a few days after exposure to low temperatures. To further evaluate the effects of chilling on chs1, chs2, and chs3 plants, we examined their chlorophyll content, photosynthesis efficacy and electrolyte leakage rates at various periods after exposure to 10°C. It can be seen that the earliest event that was observed within 4 days of exposure to chilling was a sharp decline in chlorophyll content (Fig. 7.3a), and this was followed by a gradual decrease in photosynthetic efficacy (determined by measuring chlorophyll fluorescence; Fv/Fm ratio) which continued up to 21 days of exposure to 10°C (Fig. 7.3b). An increase in electrolyte leakage rates was evident after 7 days at 10°C and gradually became more severe as the plants were kept for longer periods at 10°C (Fig. 7.3c). In accordance with the observed decreases in chlorophyll contents and photosynthetic efficacy, we found that chs1, chs2, and chs3 plants were unable and did not accumulate starch in their leaves as observed in WT plants (Fig. 7.4). Following exposure to low temperatures, WT plants continued to produce assimilates by photosynthesis, but since growth was slowed down they accumulated starch. In contrast, the chs1, chs2, and chs3 mutants were defected in their photosynthetic machinery (as observed by the decrease in chlorophyll content and photosynthesis efficacy) and, therefore, did not produce sugars and did not accumulate starch following exposure to low temperatures (Fig. 7.4).

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Fig. 7.1 Phenotypes of wild-type and chs1, chs2, and chs3 mutants following transfer to chilling conditions. Plants were grown for 2 weeks at 22°C and afterwards transferred to a chilling temperature of 10°C

Fig. 7.2 Effects of a moderate growth temperature of 18°C on the phenotypes of wild-type and chs1, chs2, and chs3 mutants. Plants were grown from sowing at 22 or 18°C, and photographs were taken after 3 weeks of growth at each temperature

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Fig. 7.3 Effects of chilling on chlorophyll content, photosynthesis efficacy, and electrolyte leakage rates of wildtype and chs1, chs2, and chs3 mutants. Plants were grown for 2 weeks at 22°C and afterwards transferred to a chilling

temperature of 10°C. (a) Chlorophyll content, (b) photosynthesis efficacy, and (c), electrolyte leakage. Data are means ± S.E. of five replications

Another response of chs1, chs2, and chs3 mutants to chilling was enhanced accumulation of ROS in general, and H2O2 particularly, as observed by DAB staining experiments (Fig. 7.5). It can be seen that WT plants remained clear and did not accumulate H2O2 following exposure to chilling, whereas leaves of chs1,

chs2, and chs3 mutants were stained by brown spots indicating presence of H2O2 (Fig. 7.5). The accumulation of H2O2 was somewhat more pronounced in chs2 and chs3 plants as compared with chs1, and after exposure to the lower temperature of 4°C as compared with 13°C (Fig. 7.5).

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants

Fig. 7.4 Effects of chilling on starch accumulation in wild-type and chs1, chs2, and chs3 mutants. Plants were grown for 2 weeks at 22°C and afterwards transferred to

5

Mapping of the chs1, chs2, and chs3 Genes

Identification of the gene products of class 1 chs mutants by means of mapping and chromosome walking technologies will provide important insights into the molecular basis of chilling tolerance traits crucial for plant survival at low temperatures. To facilitate the mapping of chs1, chs2, and chs3 genes, we crossed the mutants present in Colombia (Col) background with Lansdberg

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chilling temperatures of 13 and 4°C. Starch accumulation was indicated by iodine staining after 7 days at chilling conditions

erecta (Ler) WT plants, and scanned populations of F2 plants for chilling sensitivity phenotypes upon transfer to low temperatures. Afterwards, we extracted DNA from chilling-resistant plants and searched for possible linkage of PCR markers with the chilling tolerance phenotype. Accordingly, based on polymorphism between Col and Ler ecotypes, we mapped the chs1, chs2, and chs3 genes to the following chromosomal locations (Fig. 7.6): (1) chs1 – was mapped to chromosome 1 in the region between 5.9 and 7.0 Mb.

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Fig. 7.5 Effects of chilling on H2O2 accumulation in wild-type and chs1, chs2, and chs3 mutants. Plants were grown for 2 weeks at 22°C and afterwards transferred to

(2) chs2 – was mapped to chromosome 4 in the region between 9.02 and 9.95 Mb. (3) chs3 – was mapped to chromosome 5 in the region between 5.41 and 7.37 Mb. This initial mapping of the chs1, chs2, and chs3 genes to a final resolution of approximately 1 Mb will facilitate in the future the final cloning and isolation of these important genes that are crucial for survival of Arabidopsis plant at low chilling temperatures. As indicated in Sect. 3, the chs3 gene was recently mapped to the top of chromosome 5 and its sequence was identified (Yang et al. 2010).

chilling temperatures of 13 and 4°C. Accumulation of hydrogen peroxide was indicated by DAB staining after 7 days at chilling conditions

6

Effects of Chilling on the Transcriptome of chs1, chs2, and chs3 Mutants

To identify transcripts that exhibited significant changes in their abundance after exposure to chilling, we performed pair-wise comparisons, and selected transcripts that had ANOVA values of p £ 0.05 and that were induced or repressed by a factor of at least 4 after exposure of chs1, chs2, and chs3 mutants to 10°C for 48 h, as compared with their corresponding expression levels in WT

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants

Fig. 7.6 Chromosomal locations of the chs1, chs2, and chs3 loci. The chs1 gene was mapped to chromosome 1 in the region between 5.9 and 7.0 Mb, chs2 was mapped to

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chromosome 4 in the region between 9.02 and 9.95 Mb, and chs3 was mapped to chromosome 5 in the region between 5.41 and 7.37 Mb

Table 7.1 Effects of chilling (48 h at 10°C) on the transcriptome of chs1, chs2, and chs3 mutants

Pair-wise comparison chs1/wild type chs2/wild type chs3/wild type

Probe sets differentially expressed at p £ 0.05 and induced or repressed by a factor of at least 4 Up-regulated Down-regulated Total 913 591 1,504 1,523 1,717 3,240 1,293 1,426 2,719

Data include numbers of probe sets on the Affymetrix ATH1 Genome Array differentially expressed at p £ 0.05 and induced or repressed by a factor of at least 4

plants. Doing so, led to the identification of 1,504, 3,240, and 2,719 probe sets whose expression significantly changed after exposure to chilling in chs1, chs2, and chs3 mutants, respectively (Table 7.1). To define the degree of similarity and differences between gene expression patterns in the various class 1 chs mutants in response to chilling, we conducted a Venn diagram comparison (Fig. 7.7). It was found that the expression of a large group of 1,426 probe sets was similarly affected by chilling in all class 1 chs mutants, and that of 1,207 additionally genes were similarly

affected in both chs2 and chs3 mutants (Fig. 7.7). Thus, chs1, chs2, and chs3 mutants endure similar molecular responses following exposure to chilling stress. In order to assign the chilling-induced differentially expressed genes in chs1, chs2, and chs3 mutants into corresponding molecular functions, we performed functional categorization analysis using the MapMan software (Thimm et al. 2004). Doing so revealed that the main functional categories up-regulated by chilling in the mutants were “stress,” “protein,” and “signaling,” whereas the main categories down-regulated by chilling

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genes included mainly suppression of transcripts involved in fatty acid synthesis and desaturation; the latter is known to be a crucial factor required for adaptation to chilling (Murata et al. 1992; Nishida and Murata 1996). The overall meanings of these findings are that under chilling conditions class 1 chs mutants are defective in normal gene expression related to the photosynthesis machinery and carbohydrate metabolism, and cell wall and lipid metabolism. In addition, the chs mutants were in severe stress as indicated by massive up-regulation of stress genes, and suffered from imbalanced protein metabolism (suppression of protein synthesis and induction of protein degradation).

7 Fig. 7.7 Venn diagram illustrating the overlapping and differences in gene expression patterns among the various chs1, chs2, and chs3 chilling-responsive regulons. The numbers on the diagram indicate the amount of overlapped probe sets

were “photosynthesis” and “tetrapyrrole synthesis,” “major carbohydrate metabolism,” “cell wall,” and “lipid metabolism” (Table 7.2). A more or less similar response consisting differential expression of stress, photosynthesis, and protein, carbohydrate and lipid metabolism genes, following exposure to chilling were reported also in several other chilling-sensitive commodities, such as rice, sunflower and potato as well as chilling-tolerant poplar trees (Yan et al. 2006; Fernandez et al. 2008; Oufir et al. 2008; Maestrini et al. 2009). The up-regulated category of “protein” included massive up-regulation of genes involved in protein degradation, and particularly transcripts encoding members of RING finger and F-BOX proteins belonging to the ubiquitin protein degradation pathway. In addition, the “protein” category further included massive down-regulation of genes involved in protein synthesis. The up-regulated category of “signaling” included mainly up-regulation of receptor kinase genes and calcium signaling genes, as previously reported (Bhattacharjee 2009). The observed down-regulation in “lipid metabolism”

Conclusion and Future Perspective

From the current evaluations of the physiological, biochemical and molecular responses of class 1 chs mutants to chilling, we have arrived at the following main conclusions: (1) Under chilling conditions, class 1 chs mutants are defective in normal gene expression related to photosynthesis, chlorophyll synthesis and major carbohydrate metabolism (Table 7.2). Furthermore, these molecular observations were confirmed by biochemical measurements indicating a rapid loss of chlorophyll content and decrease in photosynthetic efficacy (Fig. 7.3), leaf yellowing (Fig. 7.1), and inability to accumulate starch as observed in WT plants (Fig. 7.4). (2) Under chilling conditions, class 1 chs mutants are defective in normal gene expression related to lipid metabolism including downregulation of fatty acid synthesis and desaturation (Table 7.2). Furthermore, these molecular observations were supported by conductivity measurements indicating a continuous increase in electrolyte leakage rates upon transfer of the chs mutants to chilling, thus indicating accumulated damage to cellular membranes (Fig. 7.3). (3) Under chilling conditions, class 1 chs mutants activated a battery of stress-related genes,

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants

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Table 7.2 Functional categorization of chs1, chs2, and chs3 regulon genes Functional categorization Photosynthesis Major CHO metabolism Minor CHO metabolism Glycolysis Fermentation Gluconeogenesis OPP TCA/transformation Electron transport/ATP Cell wall Lipid metabolism N-metabolism Amino acid metabolism S-assimilation Metal handling Secondary metabolism Hormone metabolism Tetrapyrrole synthesis Stress Redox regulation Polyamine metabolism Nucleotide metabolism Misc RNA and transcription DNA Protein Signaling Cell “Micro RNA” Development Transport Not assigned

chs1 regulon Up Down 1 16 2 12 9 3 2 3 0 0 1 0 0 1 0 0 4 2 15 35 9 31 1 0 10 9 0 3 4 3 19 17 27 25 0 11 73 15 12 7 0 1 6 2 59 48 64 48 4 6 115 32 103 21 18 7 0 0 11 10 43 32 296 223

chs2 regulon Up 2 4 15 4 2 2 2 0 5 27 24 3 31 0 6 33 47 0 99 21 1 9 91 120 9 211 141 28 0 19 68 496

Down 48 26 16 5 1 0 3 5 3 83 57 3 36 4 8 46 46 21 47 17 1 19 113 133 18 140 72 44 0 23 61 579

chs3 regulon Up 2 2 12 4 1 1 2 0 4 22 18 2 19 0 5 39 41 0 81 19 1 7 83 105 7 177 120 27 0 15 54 433

Down 38 21 12 3 0 0 2 5 3 77 49 3 24 4 7 27 42 17 44 12 1 11 101 113 13 105 66 35 0 18 56 495

Functional categorization was performed according to MapMan software (http://gabi.rzpd.de/projects/MapMan/). The functional groups of “Photosynthesis,” “Major CHO metabolism,” “Cell wall,” “Lipid metabolism,” and “Tetrapyrrole synthesis” down-regulated in chs mutants are marked by light shading, while the groups of “Stress,” “Protein,” and “Signaling” up-regulated in chs mutants are marked by dark shading

indicating that the plants were under severe stress conditions (Table 7.2). This observation was further supported by our findings regarding enhanced accumulation of ROS, as observed by DAB staining for detection of H2O2 levels (Fig. 7.5). (4) Following exposure to chilling, class 1 chs mutants suffered from imbalanced protein metabolism, demonstrated by suppression of

transcripts involved in protein synthesis and massive induction of transcripts belonging to the ubiquitin protein degradation pathway. These processes obviously lead to progressive destruction of normal cellular activity. Overall, based on our studies with Arabidopsis chilling-sensitive mutants, we conclude that several biochemical and molecular traits are apparently crucial for plant survival under chilling

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temperatures; these include maintenance of photosynthetic activity and carbohydrate metabolism, maintenance of normal lipid metabolism, maintenance of stress tolerance and capability to detoxify accumulation of ROS, and maintenance of normal cellular function including normal protein turnover and cellular signaling processes. Acknowledgements This chapter is a contribution from the Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel, no. 597/10.

References Allen DJ, Ort DR (2001) Impacts of chilling temperatures on photosynthesis in warm climate plants. Trends Plant Sci 6:36–42 Araki N, Kusumi K, Masamoto K, Niwa Y, Iba K (2000) Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-D-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis. Physiol Plant 108:19–24 Bhattacharjee S (2009) Involvement of calcium and calmodulin in oxidative and temperature stress of Amaranthus lividus L. during early germination. J Environ Biol 30:557–562 Dong CH, Zolman BK, Bartel B, Lee BH, Stevenson B, Agarwal M, Zhu JK (2009) Disruption of Arabidopsis CHY1 reveals an important role of metabolic status in plant cold stress signaling. Mol Plant 2:59–72 Fernandez P, Rienzo D, Fernandez L, Hopp HE, Paniego N, Heinz RA (2008) Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis. BMC Plant Biol 8:11 Graham D, Patterson BD (1982) Responses of plants to low nonfreezing temperatures: proteins, metabolism, and acclimation. Annu Rev Plant Physiol 33:47–72 Hasdai M, Weiss B, Levi A, Samach A, Porat R (2006) Differential responses of Arabidopsis ecotypes to cold, chilling and freezing temperatures. Ann Appl Biol 148:113–120 Hugly S, Somerville C (1992) A role for membrane lipid polyunsaturation in chloroplast biogenesis at low temperature. Plant Physiol 99:197–202 Hugly S, McCourt P, Browse J, Patterson GW, Somersville C (1990) A chilling sensitive mutant of Arabidopsis with altered steryl-ester metabolism. Plant Physiol 93:1053–1062 Ismail AM, Hall AE, Close TJ (1999) Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence. Proc Natl Acad Sci USA 96:13566–13570 Kerdnaimongkol K, Woodson WR (1999) Inhibition of catalase by antisense RNA increases susceptibility to

D. Zoldan et al. oxidative stress and chilling injury in tomato plants. J Amer Soc Hort Sci 124:330–336 Kim HU, Vijayan P, Carlsson AS, Barkan L, Browse J (2010) A Mutation in the LPAT1 gene suppresses the sensitivity of fab1 plants to low temperature. Plant Physiol 153:1135–1143 Levitt J (1980) Responses of Plants to Environmental Stresses. Academic, New York, NY Lightner J, Wu J, Browse J (1994) A mutant of Arabidopsis with increased levels of stearic acid. Plant Physiol 106:1443–1451 Lynch DV (1990) Chilling injury in plants: The relevance of membrane lipids. In: Katterman F (ed) Environmental Injury to Plants. Academic, San Diego, CA, pp 17–34 Lyons JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24:445–466 Maestrini P, Cavallini A, Rizzo M, Giordoni T, Bernardi R, Durante M, Natalim L (2009) Isolation and expression of low temperature-induced genes in white poplar (Poplus alba). J Plant Physiol 166:1544–1556 Markhart AH (1986) Chilling injury: a review of possible causes. HortScience 21:1329–1333 Maruyama S, Yatomi M, Nakamura Y (1990) Response of rice leaves to low temperature. I. Changes in basic biochemical parameters. Plant Cell Physiol 31:303–309 Miquel M, James D, Dooner H, Browse J (1993) Arabidopsis requires polyunsaturated lipids for low-temperature survival. Proc Natl Acad Sci USA 90:6208–6212 Murata N, Ishizaki-Nishizawa O, Higashi S, Hayashi H, Tasaka Y, Nishida I (1992) Genetically engineered alteration in the chilling sensitivity of plants. Nature 356:710–713 Nishida I, Murata N (1996) Chilling sensitivity in plants and cyanobacteria: the crucial roles of membrane lipids. Annu Rev Plant Physiol Plant Mol Biol 47:541–568 Oufir M, Legay S, Nicot N, Van Moer K, Hoffmann L, Renaut J, Hausman JF, Evers D (2008) Gene expression in potato during cold exposure: changes in carbohydrate and polyamine metabolisms. Plant Sci 175:839–852 Patterson GW, Hugly S, Harrison D (1993) Sterols and phytyl esters of Arabidopsis thaliana under normal and chilling temperatures. Phytochemistry 33:1381–1383 Paull RE (1990) Chilling injury of crops of tropical and subtropical origin. In: Wang CY (ed) Chilling Injury of Horticultural Crops. CRC Press, Boca Raton, FL, pp 17–36 Payton P, Webb R, Kornyeyev D, Allen R, Holiday S (2001) Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activity. J Exp Bot 52:2345–2354 Porat R, Guy CL (2007) Arabidopsis as a model system to study chilling tolerance mechanisms in plants. Plant Stress 1:85–92 Provart NJ, Gil P, Chen W, Han B, Chang HS, Wang X, Zhu T (2003) Gene expression phenotypes of Arabidopsis associated with sensitivity to low temperatures. Plant Physiol 132:893–906

7 Understanding Chilling Tolerance Traits Using Arabidopsis Chilling-Sensitive Mutants Routaboul JM, Fischer SF, Browse J (2000) Trienoic Fatty Acids Are Required to Maintain Chloroplast Function at Low Temperatures. Plant Physiol 124:1697–1705 Sabehat A, Lurie S, Weiss D (1998) Expression of small heat shock proteins at low temperature: A possible role in protecting against chilling injuries. Plant Physiol 117:651–658 Schneider JC, Hugly S, Somerville CR (1995a) Chillingsensitive mutants of Arabidopsis. Plant Mol Biol Rep 13:11–17 Schneider JC, Nielsen E, Somerville CR (1995b) A chillingsensitive mutant of Arabidopsis is deficient in chloroplast protein accumulation at low temperature. Plant Cell Environ 18:23–32 Thimm O, Bläsing O, Gibon Y, Nagel A, Mayer S, Krüger P, Selbig J, Müller LA, Rhee SY, Stitt M (2004) MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 37:914–939 Tokuhisa J, Browse J (1999) Genetic engineering of plant chilling tolerance. Genet Eng 21:79–93 Tokuhisa JG, Feldmann KA, LaBrie ST, Browse J (1997) Mutational analysis of chilling tolerance in plants. Plant Cell Environ 20:1391–1400 Tokuhisa JG, Vijayan P, Feldmann KA, Browse J (1998) Chloroplast development at low temperatures requires a hom*olog of DIM1, a yeast gene encoding the 18 S rRNA dimethylase. Plant Cell 10:699–711 Van Breusegem F, Slooten L, Stassart J, Botterman J, Moens T, Van Montagu M, Inzé D (1999) Effects of overproduction of tobacco MnSOD in maize chloroplasts on foliar tolerance to cold and oxidative stress. J Exp Bot 50:71–78

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Vlachonasios KE, Thomashow MF, Triezenberg SJ (2003) Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression. Plant Cell 15:626–38 Wallis JG, Browse J (2002) Mutants of Arabidopsis reveal many roles for membrane lipids. Prog Lipid Res 41:254–278 Wang CY (1990) Chilling Injury of Horticultural Crops. CRC Press, Baca-Raton, FL Wu J, Lightner J, Warwick N, Browse J (1997) Lowtemperature damage and subsequent recovery of fab1 mutant Arabidopsis exposed to 2 degrees C. Plant Physiol 113:347–356 Xin Z, Browse J (1998) Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc Natl Acad Sci USA 95:7799–7804 Yan SP, Zhang QY, Tang ZC, Su WA, Sun WN (2006) Comparative proteomic analysis provides new insights into chilling stress responses in rice. Mol Cell Proteom 5:484–496 Yang H, Shi Y, Liu J, Guo L, Zhang X, Yang S (2010) A mutant CHS3 protein with TIR-NB-LRR-LIM domains modulates growth, cell death and freezing tolerance in a temperature-dependent manner in Arabidopsis. Plant J 63:283–296 Zhu T, Provart NJ (2003) Transcriptional responses to low temperature and their regulation in Arabidopsis. Can J Bot 81:1168–1174 Zhu J, Dong CH, Zhu JK (2007) Interplay between coldresponsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr Opin Plant Biol 10:290–295

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8

Root Form and Function in Plant as an Adaptation to Changing Climate Maria Rosa Abenavoli, Maria Rosaria Panuccio, and Agostino Sorgonà

Abstract

Climate variables including temperature, atmosphere CO2, and precipitation are expected to change during this century. As consequence, in the short- and long-term, the increase of soil temperature, salinity, drought, and waterlogging stresses could be the more exceeding problems for agricultural productivity and the functioning of the natural ecosystems. Root system represents the first and more sensitive target of the climate change, being seriously damaged in its form and function, and consequently strongly contributes to limit plant growth, development and crop productivity. This review focuses on changes of root morphology, architecture, distribution and dynamics, and on essential root physiological processes, such as water and nutrient uptake in response to soil warming, salinity, drought, and waterlogging. The literature appear sometimes to be controversial due to the complexity of root system characterized by different root types, genetically, developmentally, and functionally distinct, and by diverse root morphological parameters such as total root length, biomass, specific root length (SRL), and tissue density and fineness differently involved on root stress responses. For example, the change on total root length and dry weight, the lateral root formation, the depth of rooting and the root dynamics represent the preferential strategy for plant species in waterlimited environments. Whereas, the development of aerenchyma, tissue containing enlarged gas spaces with a low-resistance pathway to oxygen, often accompanied by the aerotropic and extensive lateral roots formation, the herringbone-type root architecture, the emergence of adventitious roots and the presence of anatomical barriers are expressed in flooded root. Salinity reduces plant growth and yield by two mechanisms, osmotic stress and ion cytotoxicity. It is difficult to separate the osmotic effect A. Sorgonà () • M.R. Abenavoli • M.R. Panuccio Dipartimento di Biotecnologie per il Monitoraggio Agro-Alimentare ed Ambientale, Università Mediterranea di Reggio Calabria, Contrada Melissari – Lotto D, 89124 Reggio Calabria, Italy e-mail: [emailprotected] P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_8, © Springer Science+Business Media, LLC 2012

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from specific ion effects that overlap during the development of salinity stress, thereby some uncertainty exists regarding the relative importance of both mechanisms. The responses of root cells are finalized to maintain their own correct functionality, despite the condition of elevated Na+ concentration. The genetic diversity of the root system in the plant response to climate change was also reported. Behind the root “form” changes, many metabolic and physiological pathways are involved in the plant adaptation to climate change. The maintenance of lower respiration rate, carbohydrate metabolism and cell expansion and elongation, often mediated by hormones, are expressed in roots grown in dry soil. At molecular level, the deposition of proline, metabolite responsible of the total osmotic adjustments, the higher expansin and xyloglucan endotransglycolase/hydrolase (XTH) activities, enzymes of the cell wall extension, represent the physiological mechanisms implemented by plants for improving their drought tolerance. In waterlogging soils and also in presence of salinity, the adaptation of physiological mechanisms are addressed to improve the cellular energy status and reduce the accumulation of toxic end products that acidify the cytosol or damage membrane integrity. Remarks on proteomic and molecular aspects which represent a future approach to individuate the plant strategies for their adaptation to the climate change are also included. Keywords

Root system • Drought • Precipitation • Salinity • Temperature • Waterlogging

1

Introduction

Climate change variables such as temperature (warming), precipitation (drought and flooding), and atmospheric CO2 concentrations (CO2 fertilization) have greater impact on agricultural productivity, ecosystem structure and function. Plant has a strong photohydraulic system involved in water and nutrient uptake, a sink for the photoassimilates in which the root system plays a key role in determining plant responses to climate change. Further, the root system, by its respiration, turnover, exudation processes, and interactions with the soil biota, plays a critical role in controlling the soil C storage and cycling and ultimately in the feedbacks of terrestrial C cycling to climate change. Hence, the root system could be considered as sink and source of carbon dioxide, the main driver of climate change, and a better understanding

of its responses could provide useful information in the plant adaptation to the future atmospheric composition. In the present chapter, we will mainly focus on how direct (temperature, drought, flooding) and indirect (salinity) components of climate changes influence the root form and function in higher plants. Let us consider the root form as a “photographical description” of the root system at the tri-, bi-, and one-dimensional levels, as determined by biometric parameters. The root form includes the root morphology, architecture, distribution, and dynamics. Root morphology means “superficial features of the whole and single root axis” (Lynch and Nielsen 1996) such as the length, mass, surface area, volume, and diameter. Further morphological parameters derived from the formers and having a functional significance (Ryser 1998) are the root length ratio (RLR) (root length per unit of the plant’s dry mass), root mass

8 Root Form and Function in Plant as an Adaptation to Changing Climate

ratio (RMR) (root mass per unit of the plant’s dry mass), specific root length (SRL) (root length per unit of root dry weight), root fineness (RF) (root length per unit root volume) and root tissue density (RTD) (root dry mass per unit root volume). The root architecture is defined as the spatial configuration of the root system (Lynch and Nielsen 1996) and is generally estimated in terms of topology (Robinson et al. 2003). Root topology, which refers to the distribution of the branches within the system, can lie within two extreme types: the “herringbone” type in which branching is confined to the main axis and the “dichotomous” type exhibiting a more random branching (Fitter and Stickland 1991). The root distribution, which refers to the deployment of the root axis in terms of biomass or length along the soil profile is described by root mass density (RMD) (root mass per unit soil volume) and root length density (RLD) (root length per unit soil volume). Finally, the root dynamics includes the root production, mortality, and turnover (ratio of root number present at a time point to the number of roots produced up to that time) and life span. The root system as defined by Robinson et al. (1991) “…is the result of an evolution strategy to solve the problems of soil resources acquisition….” Hence, the main function of the roots is the capture of belowground resources, such as water and nutrients, from that “…heterogeneous and porous system …” (Robinson et al. 1991) which is the soil environment. The climate change has impact on the plant C allocation and respiration, water and nutrients uptake which are important physiological processes in terms of C (Hinsinger 1998, 2001; Hinsinger et al. 2003). Finally, we will discuss the impact of climate changes on the root’s “secondary functions” such as storage of carbon and nutrients and supply of energy to belowground food web and microorganisms (Hinsinger et al. 2005, 2006).

2

Roots and High Temperature

The Fourth Assessment Report (AR4) of the Inter governmental Panel on Climate Change (IPCC) of United Nations predicted an approximately 1.8–4°C increase in global mean air temperature

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during this century (IPCC 2007). Soil temperatures are also expected to increase reflecting the future atmospheric temperature trend (Pollack et al. 1998). The plant root system will be affected by soil temperatures which will have a significant impact on its form and function and therefore on plant development and productivity. Lots of study have been done on the effects of temperature on the root system (Cooper 1973; Voorhes et al. 1981; Kaspar and Bland 1992; McMichael and Burke 2002), relatively few information have focused on integrated root development, growth, metabolic responses to soil warming and how roots preserve its form and function under warming soil conditions is not completely clarified. Considerable evidence indicates that the root growth increased in response to increased soil temperature up to an optimum threshold, typical for each species and depending partly on their native temperature regime (McMichael and Burke 1998), beyond which root growth decreased. Faster elongation rates of whole root system were observed in the temperature range of 5–23°C for Eucalyptus species (Misra 1999), 10–30°C for sunflower (Seiler 1998), 10–15°C for winter wheat (Gavito et al. 2001) and bog and fen plant communities (Weltzin et al. 2000). Supraoptimal soil temperatures, on the other hand, reduced the root growth in many species such as Agrostis stolonifera (>35°C, Huang et al. 1998), Lactuca sativa (>35°C, Qin et al. 2007; He et al. 2009) and wheat (>38°C, Tahir et al. 2008). McMichael and Burke (2002) grouped the different taxa in relation to optimum of temperature for the root growth, pointing out the influence (incidence) of diverse genetic background on temperature-dependent root growth pattern among the plant species due to their different acclimation strategies. For example, a diverse response and adaptation, in terms of both root length and mass, was evident among genetically diverse sunflower (Seiler 1998) wheat genotypes (Tahir et al. 2008) and in two Agrostis species; where the root system of A. scabra was more thermotolerant growing up to 45°C (Terceck et al. 2003) than that of A. stolonifera which grew upto 23°C only (Pote et al. 2006). These observations suggested that genetic diversity in root growth contributes to the survival of

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plant species and to improve their productivity under high soil temperature conditions and deserves further studies. To better understand the temperature-induced root responses between and within plant species is needed to consider that the root systems comprise of different root types which are distinct genetically, developmentally, and functionally and differently respond to soil environmental stresses (Waisel and Eshel 2002). Several examples can be mentioned in this regard: the first root axes of pearl millet showed a higher elongation rate in response to the increase of temperature (from 20 to 32°C) with respect to the second one (Gregory 1986), the primary root of sunflower was less inhibited at temperature above 35°C than lateral roots (Seiler 1998), the fine roots were more sensitive to soil warming (Pregitzer et al. 2000), the SRL (root length/root mass) and specific root area (root area/root mass) increased in warmer soil in the root finest fraction (40°C) strongly affected the bacterial infection and N2 fixation, at different degree among the host plant species (Zaharan 1999). The total N2 fixation of Trifolium repens was enhanced by an increase of the temperature in the 7–13°C range while it was not influenced by temperatures above 13°C (MacDuff and Dhanoa 1990). Warmer soil also affected other main symbiotic relationship of the higher plants, that is, the plant host-mycorrhizal fungi interactions. Increasing the soil temperature, root length colonization (LRC) was improved (Heinemeyer and Fitter 2004) and an increase in development of arbuscular mycorrhizal hyphae was observed which consequently helped in higher P uptake by roots of pea plants (Gavito et al. 2003). However, Olsrud et al. (2004) pointed out that the positive relationship between the mycorrhizal development and warmer soil was an indirect effect due to an increased C allocation toward the roots in response of the concomitant low soil moisture content rather than a direct temperature effect on the root system (Olsrud et al. 2004).

3

Roots and Altered Precipitation

The climate change models of the Intergovernmental Panel on Climate Change predicted that, as a consequence of temperature increases, the precipitation pattern will vary (IPCC 2007). At high latitudes, it should increase in winter and decrease in summer, and in various regions of Central and South Europe will also increase the frequency and duration of summer precipitation. Thus, the climate change will be responsible for determining an increased risk of both soil drought and waterlogging. Generally, water is believed to be available for plant uptake at soil water potential greater than

8 Root Form and Function in Plant as an Adaptation to Changing Climate

wilting point, −1.6 MPa, which is considered the limiting value for the growth and development of many mesophytic plants. Suboptimal water availability, that is, drought soil condition is in fact considered a major constraint limiting the crop productivity. Soil drought more severely affect shoots than roots (Spollen et al. 1993) which have the ability to grow under mild stress condition. This differential sensitivity represents an important advantage to plants and allows them a greater exploration of soil for ensuring a supply of water and increasing their probability to survive at dry condition. Drying soils produce a range of effects on the plant root system and varies from species to species. The total root length and dry weight, the lateral root production, the depth of rooting and the root dynamics represent the major traits influenced by water deficit. Several evidences indicated that the primary root length of maize was inhibited at −0.5 MPa water potential after 24 h (Fan and Neumann 2004). The reduction in the primary root elongation of Arabidopsis thaliana was observed at moderate water stress (−0.2 MPa) (van der Weele et al. 2000), similar results were observed in Pinus pinaster whose root system was increased at −0.15 MPa and reduced at −0.66 MPa) (Triboulot et al. 1995). In White oak (less sensitive) whose root elongation rate was reduced in the range of −0.4 and −0.8 MPa and completely ceased at −1.2 MPa soil water potential (Kuhns et al. 1985). The rapid soil drying conditions determined an intense reduction of the elongation rate of the Opuntia ficus-indica roots after 3 days of water deficit while the gradual drought stress caused the root inhibition after 9 days of water stress (Dubrovsky et al. 1998). Additionally, as response to soil drying, lateral roots varied in length in a specie-specific manner. The length of the first-order lateral roots was more reduced in maize than in wheat making this species less sensitive to water deficit (Ito et al. 2006). What physiological mechanisms are involved for maintaining the root elongation at low water potential? Over the last 20 years, Sharp and coworkers (Sharp et al. 2004; Ober and Sharp 2007) pointed out detailed results on the regulation of maize primary root growth under soil

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drought conditions. In particular, they observed that elongation rate of the apical region (7 mm, region 3, respectively). The mechanisms involved in the maintenance of the elongation rate in the region 1 may be due to the following reasons: (1) accumulation of the plant hormone abscissic acid (ABA), (2) the osmotic adjustments, and (3) the cell wall extension properties. Saab et al. (1990) and Sharp et al. (1994) showed that endogenous ABA accumulated in the apical region of waterstressed maize roots causing prevention of excess ethylene production and playing a regulatory role in the ion homeostasis (Ober and Sharp 2003). Accumulation of proline was responsible for as much as 45% of the total osmotic adjustments in the apical root region of maize seedlings under water stress (Voetberg and Sharp 1991). Further, Wu et al. (1996) reported differential responses of cell wall extension properties in maize seedlings. The expansion and xyloglucan endotransglycolase/hydrolase (XTH) activities were higher in the apical region of water-stressed than wellwatered roots. It is interesting to note that ABA accumulation was involved in both processes increasing the proline transport and the XTH activity in the apical region of water-stressed roots underlying its regulative role in drought stress (Ober and Sharp 1994; Wu et al. 2001). In addition, Fan and Neumann (2004) found that the water stress induced an acidification in the apical region of maize roots which maintained the growth in presence of drought stress. Consistent with these results, transcriptomic and proteomic analyses of root growth of maize seedlings in responses to soil drought revealed that the water stress induced gene expression and the cell wall protein abundance were largely region specific. The apical region exhibited a greater gene and protein numbers involved for the root adaptation to drought (Yamaguchi and Sharp 2010). Under water stress conditions, beyond the root length, other root morphological parameters were modified such as root diameter or SRL. Water-stressed roots became thinner than that of

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well-watered plant such as, A. thaliana (van der Weele et al. 2000), rice (Trillana et al. 2001), maize ((Liang et al. 1997; Hund et al. 2009) and rootstocks of Vitis vinifera (Baurle et al. 2008). Root thinning as adaptive traits in dry environments could imply: (1) a lower construction cost for unit of root length (Sharp et al. 1988; Ho et al. 2005), (2) a faster elongation rate of root axis (Thaler and Pages 1996; van der Weele et al. 2000), (3) a greater root proliferation in moisturedisturbed soils (Eissenstat 1991), and (4) a limitation on radial expansion to conserve water (van der Weele et al. 2000). However, since the thicker roots were positively correlated with an increase of water transport (Passioura 1988; Doussan et al. 1998) and a greater RLD in deep soil layers ( Azhiri-Sigari et al. 2000; Kato et al. 2006). The reduction of the root diameter as plant adaptive traits in drying soils was still questioned. Generally, the soil dried gradually from the upper layers producing a sharply vertical soilmoisture gradient. Padilla and Pugnaire (2007) observed that in a semi-natural field during the summer, the soil moisture was 20% at 5 and below 30 cm of soil depths, respectively, and this difference disappeared in the spring season. This vertical soil-moisture gradient in the rainfed or non-irrigated conditions was also observed in field experiments by Songsri et al. (2008), Bucci et al. (2009), and Cheng et al. (2009). As the soil environment became drier, root system changed its distribution becoming deeper since deep-rooted plants had an improved ability to extract water from well-watered deep soil layers compared to shallow-rooted. This shift on root distribution in response to water stress has been confirmed by Schenk and Jackson (2005) who collected data of >1,300 records of root distribution of individual plants from deserts, scrublands, grassland, and savannas along the soil depths, showed that the “absolute” rooting depth was more strongly correlated with the mean annual precipitation in all plant growth except shrubs and trees. However, Bucci et al. (2009) observed that shallow-rooted shrub species of Patagonia steppe were correlated with leaf negative water potential than deeply rooted ones. Padilla and Pugnaire (2007) observed that species

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characterized by deep roots (Salsola oppositifolia and Retama sphaerocarpa) had consistent access to deep water content and pointed out a successful survival compared with shallow-rooted plants (Ephedra fragilis, Olea europaea, and Pinus halepensis) which died as drought progressed. Further, root depth was found to be the preferential strategy for plant species in dry ecosystems (Canadell et al. 1996), long dry periods (Paz 2003), dry sandy soils (Yamada et al. 2005), and seedling of dry forest (Markesteijn and Poorter 2009). The root depth as drought-adaptive trait was exploited in crop breeding programs for improving the yield in water-limited environments. Two drought-tolerant maize hybrids exhibited around three times more axile roots in the deeper soil layers compared drought-sensitive ones (Wan et al. 2000). Drought-tolerant genotypes of sorghum were characterized by deeper roots (Ludlow et al. 1990) and the ability to produce roots in deeper soil layers could markedly improve the drought tolerance of wheat cultivars (Manschadi et al. 2006). Change in root distribution under water stress was reported among genotypes in cowpea (Matsui and Singh 2003), white clover (Annicchiarico and Piano 2004), and chickpea (Kashiwagi et al. 2006). Beside the deep rooting, the higher RLD at lower soil depths has been identified as a drought adaptative trait that permits to stabilize the pod yield and the harvest index in drought-avoiding peanut genotypes (Songsri et al. 2008) and the ability to produce roots in deeper soil layers could markedly improve the drought tolerance of wheat cultivars (Manschadi et al. 2006). However, the direct relationship between the deeply rooted system and increased water absorption from deeper soil layers has not been clearly demonstrated and it was generally based on indirect evidences with the above-ground biomass and/or yield parameters. This consideration pointed out the following question: did root form changes root functional modification? Hund et al. (2009) demonstrated that the root system of CML444, drought-tolerant maize germoplasm, compared with that of SC-Malawy, moderate drought-tolerant, exhibited a deeper roots accompanied by higher ability to absorb water from deep layers. Further, the

8 Root Form and Function in Plant as an Adaptation to Changing Climate

deep-rooting is an important root architecture strategy for efficient water uptake in dry environments. Indeed, Manschadi et al. (2008) observed that the narrower angle of the seminal roots causing a deeply rooted architecture was a trait to exploit in breeding for improved wheat cultivars for water-stressed environments. Further, the investment in terms of carbon allocation toward specific root types, such as tap root in bean, determining a deeply rooted architecture improved the water acquisition efficiency (Ho et al. 2005). In the agro-ecosystems, plants usually faced with several different environmental stresses acting in combination or in sequence and then it must optimize their resource allocation for the construction and maintenance of root systems. For example, the reduction of the soil water status caused the copresence of both water stress and mechanical impedance to root growth (Whitmore and Whalley 2009). Ho et al. (2005) demonstrated that the “dimorphic” root system exhibited the best performance in environments characterized by multiple stresses. The bean genotype BAT477, exhibiting root architecture with both shallow and deep root localization, was well adapted in the presence of suboptimal water and phosphorus availability. Besides the root morphological and architectural traits, the regulation of root water flow represented a physiological adaptive mechanism to soil water limitation. Root hydraulic conductance (Kr) was generally reduced when soil dried (North and Nobel 1991, 1992; Nardini et al. 2002). However, it has been observed that, when seedlings were exposed to moderate water stress, drought-sensitive plants increased the Kr (Nardini et al. 1998) and drought-tolerant plants decreased it (Lo Gullo et al. 1998). The capacity and the time-course necessary to recover Kr was a key factor of plant adaptation to seasonality in water availability under water stress, especially in Mediterranean species. The timing of recovery seemed to be correlated with the recovery of growth of root tip pre-existing and the new lateral root formation (Lo Gullo et al. 1998; Dubrovsky et al. 1998). Other than apoplastic pathway, the root hydraulic conductance was also related with water movement occurred along the cell-to-cell path by the aquaporins, water channel proteins

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(Javot and Maurel 2002; Tyerman et al. 2002; Maurel 2007). Several studies suggested that aquaporin-mediated transport was important in the regulation of root water flow under drought stress (Siemens and Zwiazek 2004), although their actual function was still unclear. Indeed roots of sunflower exposed to water limitation exhibited an up- and downregulation of different aquaporin genes (Sarda et al. 1999) and transgenic plants of Arabidopsis and tobacco, that constitutively over-expressed PIP1b, PIP1;4 or PIP2;5, had adverse effects on plant growth under drought stress (Aharon et al. 2003; Jang et al. 2007). The drought stress altered the root functionality such as the nutrient uptake. Substantially, drought reduced the uptake of phosphorous (P) in barley (Shone and Flood 1983) and rye grass (Jupp and Newman 1987) and nitrogen (N) in maize (Buljovicic and Engels 2001), Pseudoregneria spicata (BassiriRad and Caldwell 1992a), and Artemisia tridentata (BassiriRad and Caldwell 1992b). Conversely, N uptake was not affected by drought stress in Agropyron desertorum (BassiriRad and Caldwell 1992a) while P uptake was increased in Artemisia tridentata (Matzner and Richards 1996). These controversial results could be due to the wide influence of drought stress on all other factors/components involved in actual rate of nutrient uptake from soil, such as (1) soil processes that provide the nutrient availability at root surface; (2) anatomical (endo- and exodermis), morphological (length and surface area), and architectural (shallow and deep rooting) root characteristics; and (3) physiological (nutrient transporters) root characteristics.

4

Root and Excess Water

Excess water in soil determines the full filling of the pore space restricting the diffusion of oxygen by 104-fold than in air (Drew and Armstrong 2002) and causing a stress, named waterlogging, with dramatic impact on plant growth and productivity. Climate change provisions provide that, as a consequence of anomalous weather patterns, waterlogging could be a more exceeding problem for many plant communities.

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Under oxygen absence (anoxic soil) or under severe hypoxic conditions, the cytochrome oxidase activity of the plants became oxygen limited with a consequent reduction of ATP and pH of the cytoplasm, carbohydrate starvation, and accumulation of the toxic products due to a switch to a fermentative pathway (Drew 1997; Geigenberger 2003; Bailey-Serres and Chang 2005). Within short-time (few minutes or hours), these toxic effects caused severe damages to the plant growth and ultimately leads to death of many plant species. However, the supply of small amount of oxygen (hypoxic soil) stimulated several acclimative mechanisms which allowed the plants to survive to the transient waterlogging. In waterlogging soils, root system represented the first and more sensitive target of plants which could be seriously damaged in its form and function. Significant inhibition of the root growth, exposed to waterlogging stress were observed in Arabidopsis (van Dongen et al. 2009), Trifolium glomeratum (Gibberd et al. 1999), wheat (Malik et al. 2002), maize (Wei and Li 2000; Qiu et al. 2007), woody species (Poot and Lambers 2003; Nicoll and Ray 1996; Nicoll and Coutts 1998; Coutts and Philipson 1978). At the same time, the root system was also able to engender several adaptative responses to waterlogging that could enhance the plant survival in flooded soils. The root adaptation mechanisms were addressed to improve the cellular energy status and reduce the accumulation of toxic end products that acidify the cytosol or damage membrane integrity. To provide sufficient oxygen for maintaining the root respiration and, consequently, the ATP production it was essential to improve the root energy status and, ultimately, the root growth in anaerobic and chemically reduced soils. Aerenchyma, a plant tissue containing enlarged gas spaces, is an important trait for the root growth and function which provides a low-resistance pathway to obtain the oxygen from the atmosphere to the flooded below-ground organ. The development of aerenchyma has been associated with the tolerance to waterlogging in many plant species (Colmer et al. 1998). Evans (2003) distinguished two types of aerenchyma: (1) the schizogeneous derived from a differential cell expansion and

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specific pattern of cell separation with subsequent creation of cell spaces and (2) the lysigenous produced by the death and dissolution of the root cortical cells. While the former was common in various wetland species as Rumex (Laan et al. 1989), the lysigenous was typical of many crop species as soybean (Thomas et al. 2005), rice (Kaway et al. 1998), maize (Drew et al. 1979; Gunawardena et al. 2001), wheat (Huang et al. 1994), and pasture species (Gibberd et al. 2001; Ashi-Smiti et al. 2003). However, there was a third type of aerenchyma defined secondary aerenchyma, a white spongy tissue filled with gas spaces, which was found in stem, hypocotyls, tap roots, and root nodules of Glycine max (Shimamura et al. 2003), Lotus uliginosus (James and Sprent 1999), and Sesbania rostrata (Shiba and Daimon 2003). Generally, the aerenchyma was constitutively expressed in rice and wetland species. Recently, Seago et al. (2005) described the pattern of aerenchyma formation in 85 species representing 41 families of wetland plants. On the other hand, the aerenchyma development was also induced by flooding and other stresses, e.g., nutritional and drought in many field crops. For example, soybean, very sensitive species to flooding stress during the vegetative stage developed a secondary aerenchyma in stems, roots, and root nodules within few weeks of stress (Shimamura et al. 2003). Complex physiological and molecular mechanisms were involved in the development of aerenchyma in plants subjected to the flooding stress (Colmer 2003a; Evans 2003). In maize roots, the hypoxia conditions (3–12% oxygen) promoted the ethylene biosynthesis which triggered a signal transduction cascade involving Ca2+ and protein kinases, inducing a programmed cell death in target cells of the root cortex (Drew et al. 2000). The genetic variability of the plant species in the differing tolerance to waterlogging was associated with the aerenchyma formation which determined higher root porosity. Indeed, the superior tolerance to waterlogging of Trifolium tomentosum, T. fragiferum and T. repense than T. subterraneum var. subterraneum and T. glomeratum, more sensitive species were due to the development of aerenchyma in adventitious roots

8 Root Form and Function in Plant as an Adaptation to Changing Climate

(Gibberd et al. 1999). The different ability to form aerenchyma and, hence, to exhibit a greater tolerance to the waterlogging stress were observed among the genotypes of soybean (Bacanamwo and Purcell 1999), maize (Zaidi et al. 2004), wheat (Boru et al. 2003), and woody species (Aguilar et al. 1999). Further, the wetland Rumex species tolerant produced a higher root porosity than sensitive ones (Laan et al. 1989). Beside the aerenchyma, other root mechanisms which improved the presence of oxygen in plant tissues subjected to the flooding stress, such as the aerotropic roots and extensive lateral roots (Gibberd et al. 2001), the herringbone-type root architecture (Bouma et al. 2001), the emergence of adventitious roots (Mergemann and Sauter 2000) and the anatomical barriers such as exoderism (Colmer 2003b) were suggested to be part of the basis of tolerance to waterlogging. The recovery of the root cellular energy status in oxygen deficiency conditions was obtained through different metabolic adaptative mechanisms: the switch to a fermentative pathway (Sachs et al. 1996; Chang et al. 2000), the global depression of ATPconsuming processes (van Dongen et al. 2009), the death of metabolically intensive tissues as root tip (Subbaiah and Sachs 2000; Subbaiah and Sachs 2001) and the induction of anaerobic protein synthesis (Lal et al. 1998; Mujer et al. 1993; Chang et al. 2000). During waterlogging, the roots were more prone to oxidative stress which caused the generation of “reactive oxygen species” (ROS), including superoxide anion radicals (O2−), hydroxyl radicals (OH), hydrogen peroxide (H2O2), alkoxy radicals (RO), and singlet oxygen (O1/2) (MunnéBosch and Peñuelas 2003). The production of ROS led to enhanced peroxidation of membrane lipids and degradation of nucleic acids, and both structural and functional proteins. In this respect, the induction of free radical scavenging enzymes observed in wheat roots subjected to hypoxia determining tolerance to the waterlogging stress (Biemelt et al. 1998). Proteomic and genomic analysis supported the morphological and physiological mechanisms that made up the root acclimative responses to the waterlogging stress. Indeed, the roots stressed by water shortage exhibited a large-scale repro-

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gramming of gene expression and metabolism: (1) up- and downregulation of genes in Arabidopsis that mainly encoded proteins involved in fermentative process and energy-consuming processes (transport, lipid and secondary metabolism), respectively (van Dongen et al. 2009), (2) a quantitative trait locus containing the ethylene response factor-like genes was regulated by submergence in rice (f*ckao et al. 2006), and (3) several quantitative trait loci associated with waterlogging tolerance were detected in maize plants (Qiu et al. 2007).

5

Root and Salinity

Salinity reduces plant growth and yield by two mechanisms, osmotic stress and ion cytotoxicity (Munns and Tester 2008). Munns 2002 proposed a two-phase model of salt injury where growth is initially reduced by osmotic stress and then by Na+ toxicity. According to this biphasic model, growth is first reduced by the decrease in soil osmotic potential (ψo), caused by salt outside the plant rather than within it. The induced osmotic stress is controlled by inhibitory signals from the roots and, genotypes differing in salt resistance, respond identically in this first phase. Ionic stress develops over time and is due to a combination of ion accumulation in the shoot and to an inability to tolerate ions that have accumulated. The adaptation responses to ionic stress by plants are of two distinct types: Na+ exclusion and tissue tolerance, and is the result of different abilities by plants to exclude or to sequester toxic ions into vacuoles. In this second phase of growth reduction, genotypes varying in salt resistance may respond differently. Secondary stresses induced by salinity, such as nutritional imbalances and oxidative stress, are also responsible of reduced plant growth. It is difficult to separate the osmotic effect from specific ion effects that overlap during the development of salinity stress, thereby some uncertainty exists regarding the relative importance of both mechanisms. Rengasamy (2010) conducted a pot experiment on wheat growth where the plants were exposed to NaCl or to a

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Hoagland nutrient solution at different salinity levels. The results evidenced that the osmotic effect is continuous but, at low level of salinity, the ionic effect may be significant in reducing growth and, the application of nutrients (Hoagland solution), alleviates the salinity stress on plants. However, above a threshold value of soil solution salinity, the osmotic effect becomes the dominant mechanism limiting the growth. Although the term salinity implies high concentration of salts in soil NaCl contributes the most part in soil salinity and this explains why all plants have evolved some mechanisms to regulate NaCl accumulation or exclusion. Moreover, the specific-ion toxicity of NaCl is not only the result of an excessive Na uptake, but a combined contribution of both Na+ and Cl− as well. In fact Cl− concentrations may be higher than those of Na+ ions, cations that can be adsorbed by soil particles. Generally, anions like Cl− are repelled from soil surface and retained in soil solution where they can accumulate also at large amount, controlling the overall salt concentration of the soil solution. For most species Na+ appears to reach a toxic concentration before Cl− does; however, for some crops, such as soybean, citrus, and grapevine, Cl− is considered to be the more toxic ion (Storey and Walker 1999).

5.1

Hydroponic vs. Soil Systems

The majority of works regarding salt effects and developing selection criteria for improved salt tolerance in plants has been done using solution culture, assuming that responses in hydroponics mimic those in soil. In a recent study on barley, Tavakkoli et al. (2010) show that the effects of salinity on plants differed between the hydroponic and soil systems. The salt concentration in the rhizosphere may increase, as a result of decreasing water content in the vicinity of the roots, due to the high transpiration demand and low hydraulic conductivity of soil. This does not occur in solution culture where no ion gradients will build up and neither depletion nor salt accumulation in the rhizosphere will occur. In soil sudden changes of salt concentration are unlikely

because of the soil-buffering capacity, associated with the cation exchange soil complex (Vetterlein et al. 2004). Thus, plants in soil have more time to adapt to the increase of salinity than plants in hydroponic system. This is of a particular importance for cellular homeostasis adjustments that require ion uptakes and compatible solute accumulation. A result consistent with the two systems is the more significant negative effect of osmotic stress on plant growth, in comparison with the specific ion effects (Tavakkoli et al. 2010). This agrees with assumptions by Munns (2005) and Rengasamy (2010) which indicated that the biggest reduction in growth is caused by the osmotic stress and a relatively smaller effect is due to the genetic differences in ion exclusion.

5.2

Soil Constraints on Root Growth in Saline Environment

The evaluation of the average soil salinity and water content of a specific soil layer cannot be considered comprehensive to calculate the effective soil solution salinity roots are exposed to. In fact, it does not consider aspects concerning interactions between roots and soil at the root/ soil interface. Driven by transpiration of the shoot, saline soil solution moves from the bulk soil to the root surface where water uptake occurs but most ions are excluded. Consequently, rhizospheric soil can be up to 15 times more saline than the bulk soil and this gradient is also more expressed under conditions of higher ET demand. The osmotic water potentials of the soil solution contacting the root surface are significantly lower than the bulk soil and this gradient initiates a flow of soil solution directed to the root surface (mass flow). Then, the increase in soil salinity, as result of evaporation, occurs at the soil interface, while the site of separation of salts from the soil water, due to root water uptake, takes place at the soil– root interface. In many saline soils a deterioration of the structure leads some physical constraints that in the root zone appear principally in the form of compaction and crusting. Low porosity restricts rates of water and nutrient uptake by roots as well

8 Root Form and Function in Plant as an Adaptation to Changing Climate

as gas exchange, whereas high soil strength directly inhibits root elongation and expansion. Soil oxygen movement to roots is critical to maintain adequate respiration for plant growth. Under anoxic conditions some bacteria shift metabolic pathways so as to utilize alternative terminal electron acceptors and produce some substances, such as hydrogen sulfide, that are toxic for plants. Roots need nutrition, water aeration, and low mechanical strength to grow and function in the soil environment. The study of interactions between root properties (morphology and activity) and soil conditions are relevant to assess the water supply of plants and the salt tolerance of plants. If root growth and physiological processes in the root are affected, adverse leaf water status and top growth can occur through both hydraulic and biochemical signals.

5.3

Na+ Uptake and Accumulation in Roots

In most plants, roots should exclude 98% of the salt in soil solution allowing only 2% to be transported to the shoot. Then roots filter out most of the salt in the soil while taking up water and play a fundamental role in protecting the plants from excessive uptake of salts. Furthermore, roots have a remarkable ability to control their Na+ and Cl− concentration, which is rarely much higher than in external solution. (Munns 2005) Unidirectional influx and efflux provide the two main components of the currently accepted model of Na+ uptake in plants. Na+ ions passively enter the cell, down the ion’s electrochemical potential gradient, and exit the cell via a secondarily active proton-driven sodium with a probable Na+:H+ stoichiometry of 1:1 (SOS1; Shi et al. 2002). This process consumes significant cellular energy. Recently, Malagoli et al. (2008) showed that the energy predicted to drive active Na+ efflux in rice roots was much greater than the measured one. This discrepancy may indicate the involvement of more Na+-specific transport systems and interestingly, a sodium– potassium–chloride transporter has recently

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been discovered in A. thaliana (ColmeneroFlores et al. 2007). Then, it was suggested a possible mechanism in which active Na+ efflux is energized differently from current models, possibly via its coupling to passive fluxes of ions other than protons. The compartmentation in root vacuoles of remaining Na+ is achieved by tonoplast Na+/H+ antiporters. A passive leakage of Na+ back to the cytosol (possibly via tonoplast nonselective cation channels) requires a constant resequestration of Na+ into vacuoles (Apse et al. 1999). This mechanism allows plants to minimize or delay the toxic effects of high concentrations of salts, so genotypes with a poor ability to sequester salts have a greater rate of leaf death. Therefore, an efficient sequestration system may improve tissue tolerance by plants, perhaps by reducing cytosolic Na+ concentrations. As the water moves from the soil across the root cortex ions are transported by this stream toward the stele. Some X-ray microanalysis on roots of wheat plants showed that the root cortex is the main barrier to Na+ transport into the stele, rather than the endodermis (Läuchli et al. 2005) and the highest concentration of Na+ was in the cell layer of pericycle. Similar results show substantial sequestering of large amount of Na+ and Cl− in vacuoles of pericycle cells in grapevine roots, grown at relatively low salinity (25 mM NaCl), suggesting an important role of pericycle in the radial transport of Na+ and resulting xylem loading (Storey et al. 2003).

5.4

How Salinity Is Sensed in Roots

The perception of salinity is achieved by both ionic and osmotic stress signals in plants. The responses of root cells are finalized to maintain their own correct functionality, despite the condition of elevated Na+ concentration. Long distance signals to shoots are activated in the form of hormones or their precursors; in fact the reduction of leaf growth under salinity is independent of carbohydrate supply and water status (Turka and Demiral 2009). ABA plays a central role in root-to-shoot and cellular signaling, but gibberellins

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are also involved. ABA can inhibit leaf elongation by lowering the content of active GA, as observed in barley leaves (Munns et al. 2006). Root growth is usually less affected by salinity than leaf growth. Root elongation rates recover remarkably well after exposure to NaCl or other osmotica and, unlike leaves, the recovery takes place despite turgor. Changes in wall properties must occur, but the mechanism is unknown. With time, reduced initiation of new lateral or seminal roots is evident. Signals within root cells are likely independent from ABA. Plants respond directly and specifically to the addition of Na+ within seconds. Then, a plasma membrane protein must be the sensor, but this is still obscure. The first recorded response in roots is an increase in [Ca+2]cyt from an influx across the plasma membrane and also from the tonoplast. This perturbation in Ca+2 level activates salt stress signaling, sensed by a protein (SOS3) that interacts with a protein kinase, identified as SOS2. The complex SOS3/ SOS2, enabling the phosphorylation, activates the membrane bound Na+/H+ antiporter, SOS1, that is responsible of Na+ efflux. The discovery of the SOS (Salt-Overlay-Sensitive) pathway in Arabidopsis clarified how Na+ (ionic stress) is sensed and the relationship between ion homeostasis and salinity tolerance. However, Arabidopsis is a glycophite species, sensitive to moderate levels of NaCl, and the adaptive responses to Na+ in this plant should be extrapolated with caution. In fact, if Arabidopsis remains a useful model to study and discover plant Na+ transport processes, the identification of signaling pathways in salt tolerant species is more relevant to define adaptive rather than dysfunctional responses to salinity. The relationship between Na+ tolerance and Na+ accumulation is different in Arabidopsis and cereals (Tester and Davenport 2003). More work is necessary for the identification of the different mechanisms that are fundamental to specific aspects of salinity tolerance, and also the evaluation of the time of exposure and the severity of salt treatment are important, because they determine the physiological and molecular changes that are detected.

5.5

Root Form and Function in Saline Environment

Root system is the main interface between plants and their environment, and shows a high degree of plasticity in its development in response to local heterogeneity of the soil. On the level of the individual root and the entire root system, various morphological parameters such as length, section, surface area, root hairs are used as potential indicator of root plasticity. Moreover, responses of biomass allocation patterns and structural traits such as SRL, RTD, and root diameter distribution, are associated with acquisition capacities for below-ground resources and respond to stresses and environmental changes. Therefore, some morphological modifications, at the individual root level can affect the structural and physiological characteristics of the entire root system and this can change water uptake and nutrient supply by plants. Rice is considered as a moderately salt-sensitive crop, although a large variability exists among cultivars, as well as between developmental stages (Bahaji et al. 2002). A delay in the emergence of primary, adventitious, and lateral roots and a subsequent inhibition of root development, in terms of number and length were common responses to osmotic and saline treatments. However, some specific NaCl responses were detected and concerned lateral root development. In particular, lateral roots were thicker, as well as more densely arranged, and more irregular spaced than those of control plants. Furthermore, some bifurcations were occasionally noticed in primary and adventitious roots of NaCl stressed rice seedlings. In rice under salinity silicon can accumulate to high levels and it reduces Na+ loading to xylem in plants. X-ray microanalysis of root transverse sections showed that the greatest silicon deposition was in the endodermis. Silicon deposition restricted the movement of water and ions through the apoplast so the Na+ uptake was reduced by blocking the influx through the apoplastic pathway. It has been reported a positive role for silicon in reduction of salt stress in many crop grasses including wheat, maize, and barley (Munns 2002; Munns et al. 2006; Flowers 2004).

8 Root Form and Function in Plant as an Adaptation to Changing Climate

Wang et al. (2009) showed that high salt exposure suppressed lateral root initiation and organogenesis in Arabidopsis thaliana, resulting in the abortion of lateral root development but, on the other hand, salt stress markedly promoted lateral root elongation. The lateral root shaping is considered a prime example of developmental plasticity because both number and placement of lateral roots are highly responsive to external cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the “decision” to form a lateral root at a particular time and place. Auxin plays a key role in shaping plant architecture and it mediates responses to a broad range of external signals. Histochemical staining, physiological experiments using transport inhibitors and genetic analysis revealed that the quantity of auxin and its patterning in roots were both greatly altered by exposure to high concentrations of salt stress and auxin transport pathway is important for adaptive root system development under salt stress (Malamy 2005). Root hairs can make up 70–80% of the root surface area. They play an important role in nutrient uptake, and root hair number and density generally increase as a consequence of a nutrient stress (Glory and Jones 2000). Wang et al. (2008) showed that in Arabidopsis thaliana root hair number and density decreased significantly under salinity, in a dose-dependent manner, and they reported a physiological mechanism for root hair development in response to salt stress. They hypothesize that salt stress may affect cell-fate specification and the reduction in root hair number is likely caused by a decrease in the epidermal cells differentiating into trichoblasts. The inhibition was sensitive to ions but not to osmotic stress, and was considered an adaptive mechanism to avoid excessive ion uptake, by reducing the absorptive area when ion disequilibrium occurs in roots. Furthermore, because of high sensitivity of root hairs toward salt, they suggest a possible role of root hair alteration as an early indicator of salt stress and plant response. Salinity stress is also responsible for thickening of roots. Some studies were carried out on growth and changes in structure of root cells in

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Kikuyu seedlings grown in Hoagland nutrient solution with different salt concentrations (Panuccio et al. 2002, 2003; Muscolo et al. 2003). The cross sections of the primary structure of Kikuyu grass roots exposed to 50–100 mM NaCl did not show significant changes in the cortex growth and stele development; in contrast, 200 mM NaCl caused a significant reduction in the relative volume of the endodermis around the central cylinder, a thickness of the Casparian band and an increase in the number and diameter of root metaxylem vessels. These anatomical modifications may increase the mechanical resistance and decrease the root permeability to avoid the toxic effects of ions in excess. Lens culinaris has always been considered as a salt-sensitive species, but the microsperma landrace “Ustica” is a genotype that behaves like a salt-tolerant one because of its adaptation to the particular environment of the hom*onymous little island (North of Sicily). Some studies have been conducted to evaluate salt effects and plant responses in Ustica seedlings, grown for 20 days in microcosms, using agrilite as solid substrate and in the presence of different salt concentrations (0, 50, 100, 200 mM NaCl) (unpublished data). Various morphological root parameters such as length (cm), diameter (mm) and surface area (cm2) were tested, by using an image analysis system. The results were also compared with those of a commercial cultivar (Eston), as they are valuable parameters when describing and comparing root systems. In both cultivars, the length of lateral roots was more affected than that of primary roots, but to different extent (Fig. 8.1). The parameter “root length” is considered more important than the “root weight” to indicate the root functionality, because it expresses the potential for solute and water uptake (Ryser 2006). In Eston seedlings, exposed to 100 mM NaCl, no lateral roots were expressed while seedling of Ustica showed an inhibition of the lateral root length even though the number was not significantly influenced. Generally, a water supply reduction in plants brings to a lower lateral root production (Fig. 8.1). The SRL values were higher in Ustica than in Eston seedlings (Table 8.1). SRL is the length-to-mass ratio; it is believed to characterize economic aspect of the

Fig. 8.1 Effects of salinity (0–200 mM NaCl) on primary and lateral root length and on lateral number of a landrace (Ustica) and a commercial cultivar (Eston) of Lens culinaris seedlings Table 8.1 Effects of salinity (0–200 mM NaCl) on specific root length (cm g−1) and root tissue density (g cm−3) of a landrace (Ustica) and a commercial cultivar (Eston) of Lens culinaris seedlings Ustica

Eston

NaCl (mM) 0 50 100 200 0 50 100 200

Specific root length (cm g−1) 8.0 ± 1.9 10.6 ± 2.9 2.8 ± 0.3 4.5 ± 2.0 4.9 ± 0.3 3.5 ± 0.5 1.1 ± 0.1 –

Root tissue density (g cm−3) 48.1 ± 6.1 42.0 ± 9.3 73.1 ± 1.9 82.2 ± 10.7 47.9 ± 1.7 63.3 ± 4.7 106.1 ± 11.2 –

8 Root Form and Function in Plant as an Adaptation to Changing Climate

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Fig. 8.2 Effects of salinity (0–200 mM NaCl) on the frequency (%) of different root diameter classes of primary (a) and lateral roots (b) of Lens culinaris landrace (Ustica)

root system and is frequently used as indicator of root fineness. Then, higher SRL results from longer and thinner roots per unit construction cost (root mass) and this root apparatus is more effective in water and nutrient uptake (Fitter 2002). SRL is a complex parameter that includes variations in root diameter and RTD, which respond to environmental conditions differently (Ryser 2006). In the Ustica variety, a salinity increase leads to an increase in SRL (Table 8.1), due to thicker lateral roots and the root diameter distri-

bution was shifted toward larger diameter classes (Fig. 8.2a, b). Root diameter distribution is usually expressed as the mean diameter but sometimes it does not necessarily characterize a response of root system structure adequately. In fact, fine and coarse roots show different responses, indicating that root diameter classes should be considered as functionally distinct and regarded separately to fully understand stress responses of root systems. It is known that roots with a smaller root diameter can contact a larger soil volume per

M.R. Abenavoli et al.

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unit root surface area; however, the maintenance carbon cost of producing finer roots may be higher as these will have to be replaced more frequently (Fitter 2002). In Ustica plants, coarse roots, for both principal and lateral prevailed under high salinity conditions. This result can be explained by considering that under salinity, the construction costs per root length should be minimized because of the onset of growth-limiting conditions, and the root development resulted further inhibited to counter water stress and ion toxicity due to the salt around the root. Apart from the effects on root biomass production, contrasting root morphological responses of ecotypes to salt treatments might be partially responsible for dissimilar abilities to tolerate salinity. Structural and morphological differences in roots certainly play an essential role for nutrient and water uptake by plants from saline soil and the study of these parameters can help to determine different mechanisms underlying salt toxicity and the way plants can cope with saline conditions. Some modifications of root morphology should not be considered a simple growth stopping, but rather an induced reorientation of growth which is related to stress avoidance. This information could be considered an important tool in studies that involve salt tolerance improvements in plants.

6

Conclusion and Future Perspectives

As detailed above, the root system may have a fundamental role in relieving the disturbances caused by the variables of the climate change on the plant growth, development, and production. However, an exciting challenge will be to understand the following key aspects regarding the impact of the root system on plant adaptation to the climate change: 1. An increase of the knowledge on the root responses to the interactive effects of the climate variables change (high temperature vs. drought and/or salinity and/or drought) that usually occur together 2. An greater understanding, in an integrated view, of physiological processes (development,

growth, metabolic) involved in root responses to the warmer, drought, and salinity environments 3. A study of changes in proteins, metabolites, and other compounds inside the root cells by advanced genomic techniques for better understanding the molecular mechanisms implemented by the plant in response to temperature, water availability, and salinity change 4. An increase of the knowledge on the genetic diversity of the root system in plant response to climate variables change An improved understanding of these aspects together with genomics, proteomics, and transcriptomic approaches are likely to pave the way for engineering roots that can withstand and give satisfactory economic yield under climate change.

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Reactive Oxygen Species and Nitric Oxide in Plants Under Cadmium Stress: From Toxicity to Signaling Luisa M. Sandalio, Maria Rodríguez-Serrano, Dharmendra K. Gupta, Angustias Archilla, Maria C. Romero-Puertas, and Luis A. del Río

Abstract

The toxicity of heavy metals as a result of increasing environmental pollution in living organisms has become a major focus of research in recent decades. Among the heavy metals cadmium is one of the most dangerous heavy metals because of its high mobility in plants. It causes severe disturbances in plant metabolism that affect photosynthesis and water/ nutrient balance, and it also causes oxidative damage. Although there is an enormous literature on the tolerance and accumulation of cadmium in plants, very little research has been performed on the molecular mechanisms and signaling events underlying plant responses to Cd toxicity. The dual role as both oxidative damage inducers and signaling molecules of ROS and NO in heavy metal toxicity has been demonstrated by many workers. In this chapter, we review the contribution of different ROS and NO sources in cells and their role in regulating cellular responses to Cd. Keywords

Cadmium stress • ROS • NO • Photosynthesis • Metal transporters • GSH metabolism • Signaling

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L.M. Sandalio () • M. Rodríguez-Serrano • D.K. Gupta • A. Archilla • M.C. Romero-Puertas • L.A. del Río Department of Biochemistry and Molecular and Cellular Biology of Plants, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas (CSIC), Mail box 419, E-18080 Granada, Spain e-mail: [emailprotected]

Introduction

Heavy metals such as Cd, Hg, Pb, and Al are major environmental pollutants, particularly in industrial areas. The heavy metals are generated as a result of anthropogenic activities such as metal working industries, cement factories, smelting plants, refineries, traffic, and heating systems (Sanitá di Toppi and Gabbrielli 1999). Much of the arable soil around the world has been moderately contaminated by Cd through the

P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_9, © Springer Science+Business Media, LLC 2012

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use of phosphate fertilizers, sludge, and irrigation water (Sanitá di Toppi and Gabbrielli 1999). In polluted soils, Cd is generally present as a free ion or in other soluble forms, and its mobility depends on pH as well as the presence of chelating substances and other cations. Cadmium has a toxic impact on all living organisms by entering the food chain and accumulating by humans and animals (Nordberg 2004). Prolonged exposure to Cd by humans can cause renal dysfunction, lung damage, acute gastrointestinal problems, depression of immune system, increased cancer risk, and anemia (Nordberg 2004). The accumulation of cadmium in plants causes chlorosis, growth reduction, and even cell death. The cellular toxicity of this metal results from its various direct and indirect effects on cell metabolism and can be explained by its chemical characteristics. Cd can bind to SH groups of proteins and enzymes, leading to misfolding, enzyme inhibition, and interferences in redox regulation. Cadmium can also displace other cations from proteins and enzymes, which affects their functioning (Van Assche and Clijsters 1990). Like most heavy metals, cadmium induces oxidative stress by generating reactive oxygen species which causes oxidative damage to biomolecules such as membrane lipids, proteins, nucleic acids, etc. (Sandalio et al. 2009). Cadmium is one of the most dangerous heavy metals in nature, and at low concentrations it adversely affects the plant growth and development. Strong evidence have shown that Cd-induced generation of reactive oxygen species plays an important role in cellular toxicity, and the effects produced are dose- and species-dependent (Benavides et al. 2005; Sandalio et al. 2009). However, ROS are double-faced molecules acting as signal molecules that regulate a large gene network involved in cell response to biotic and abiotic stress. Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signaling role in many developmental and cell response processes (Besson-Bard et al. 2008). This molecule can also interfere with the ROS metabolism. There are many reports that showed the role of NO in the alleviation of the toxicity caused by heavy metals including Cd and As (Xiong et al. 2010). Several defense strategies to

avoid metal toxicity have been developed by plants, which include preventing the entry of metal through exudation of metal-complexing agents (citrates and phytosiderophores) by roots and metal immobilizing pectic sites and histidinyl groups in the cell wall (Sanitá di Toppi and Gabbrielli 1999; Clemens 2006). A second line of defense involves the induction of specific peptides called phytochelatins (PCs) which chelate the metal. PC–Cd complexes are transported into the vacuole to protect cells from toxicity (Cobett 2000). The isolation of an Arabidopsis cad1 mutant, which is defective in PC activity and hypersensitive to Cd, has demonstrated the importance of this mechanism in defending plants against Cd (Howden et al. 1995). Cd and other metals can also be complexed by metallothioneins and nicotianamine (Sharma and Dietz 2006).

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Cadmium Toxicity in Plants

The toxic effects of cadmium on several plant species have been reported by different authors (Sanitá di Toppi and Gabbrielli 1999; Sandalio et al. 2001; Schützendübel et al. 2001; Benavides et al. 2005), although the mechanisms involved in cadmium toxicity are still not fully understood. Cadmium inhibits seed germination, decreases plant growth, induces premature senescence, and can even trigger cell death in cell suspension cultures (Fotjová and Kovařik 2000; McCarthy et al. 2001; Rodríguez-Serrano et al. 2009; De Michele et al. 2009). At cellular levels, Cd produces alterations in membrane functionality by inducing changes in lipid composition and by promoting lipid peroxidation (Ouariti et al. 1997; Hernández and Cooke 1997; Sandalio et al. 2001); it also produces disturbances in photosynthesis by affecting CO2 fixation and by inhibiting PSII photoactivation because of competition with essential Ca2+ sites (Faller et al. 2005; Baryla et al. 2001). Cadmium toxicity is associated with modifications in both the uptake and distribution of macro- and micronutrients (Hernández et al. 1998; Rogers et al. 2000; Sandalio et al. 2001; Tsyganov et al. 2007) and can therefore compete with other cations for

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Reactive Oxygen Species and Nitric Oxide in Plants Under Cadmium Stress...

protein- and transporter-binding sites (Clemens 2006). Cd uptake occurs through plasma membrane transporters similar to those used for other cations such as K+, Ca2+, Mg2+, Fe2+, Mn2+ or Cu2+ (Clemens 2006). Cadmium reduces Ca2+ content, which can then affect the activity of calmodulindependent proteins (Rivetta et al. 1997; Rodríguez-Serrano et al. 2009). Cd tolerance and Ca2+ homeostasis in a Cd-resistant pea mutant (SGECdt) have been observed to be interrelated (Tsyganov et al. 2007), while in radish and Arabidopsis seedlings, calcium has been reported to alleviate Cd toxicity by reducing Cd uptake (Rivetta et al. 1997; Suzuki 2005). The role of oxidative stress in Cd toxicity has been established in different plant species by analyzing oxidative damage to proteins and lipids as well as by studying disturbances in antioxidative defenses caused by this metal (Benavides et al. 2005; Sandalio et al. 2009; Remans et al. 2010). Although Cd is a bivalent cation unable to participate in redox reactions in the cell, most transcriptome studies show upregulation of genes encoding proteins involved in defense against oxidative stress and ROS production (Suzuki et al. 2001; Zhao et al. 2009). These results suggest that oxidative stress is one of the primary effects of Cd exposure. Reactions involving oxygen free radicals are an intrinsic feature of plant senescence and stimulate the process of oxidative deterioration that leads to cell death (del Río et al. 2009). Cd induces senescence and cell death in both cell culture and plant tissues characterized by the induction of the glyoxylate cycle, protein oxidation, and proteolytic activities (McCarthy et al. 2001; Romero-Puertas et al. 2002). Senescence is considered to be a type of plant programmed cell death (PCD), and various studies have indicated that Cd induces PCD in cell cultures (Fotjová and Kovařik 2000; De Michele et al. 2009). There are evidence to support the possibility of dose dependence and intensity of the onset of the senescence process and the final cell death event (De Michele et al. 2009). Condensation of chromatin, fragmentation of DNA, as visualized by TUNEL assay, and induction of SAG12 expression are some of the symptoms of PCD and have been observed in

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Arabidopsis and tobacco cell cultures exposed to Cd (Fotjová and Kovařik 2000; De Michele et al. 2009). Cadmium-dependent senescence and PCD are regulated by ROS and NO, although the mechanisms involved are not fully understood (Yakimova et al. 2006; De Michele et al. 2009; Rodríguez-Serrano et al. 2009). Lipid signaling and Ca2+ also play an important role in Cd-induced cell death (Yakimova et al. 2006).

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Sources of ROS in Plants Exposed to Cadmium

The reactive oxygen species are mainly singlet oxygen (1O2), superoxide radical (O2·−), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) which are by-products of normal aerobic metabolism such as respiration and photosynthesis. Their steady-state levels are determined by the interplay of different ROS-producing and ROSscavenging mechanisms. This balance is maintained by enzymes such as superoxide dismutase (SOD), which remove O2− radicals, and catalase (CAT), peroxidase (POX), and peroxiredoxin, which decompose H2O2 and use metabolites such as glutathione (GSH) and ascorbate (ASC), to control ROS accumulation in different subcellular compartments. An excess of ROS is dangerous mainly because of reactions with lipids, proteins, and nucleic acids, giving rise to lipid peroxidation, membrane leakage, enzyme inactivation and DNA breaks or mutations, which can cause severe damage to cell viability. Subtle control of ROS production enables these species to act as signaling molecules which are involved in the regulation of processes such as mitosis, tropism, cell death and cell response to biotic and abiotic stresses. Compared with other ROS, H2O2 is a relatively long-lived molecule that is able to diffuse across cell membranes and acts as a signaling molecule during growth and development (Van Breusegem and Dat 2006). However, although we have a clear understanding of the toxic effects of ROS induced by metals as well as detoxification mechanisms, information on their role in regulation and signal transduction under metal stress remains quite limited.

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The electron transfer chains associated with chloroplasts and mitochondria are the main sources of ROS generation. However, this view has changed and the oxidative metabolism of peroxisomes is now seen as a very important source of ROS under different stress conditions (del Río et al. 2006, 2009). In peroxisomes purified from pea leaves, a Cd-dependent increase in the H2O2 concentration was observed, mainly as a result of the activation of glycolate oxidase, a key enzyme in the photorespiration cycle (Romero-Puertas et al. 1999). In pea leaves, it has been demonstrated through the use of a cytochemical approach that Cd-dependent H2O2 production occurs in peroxisomes, in the outer mitochondrial membrane, and mainly in the plasma membrane, where the NADPH oxidase (NOX) is the main source of ROS (Romero-Puertas et al. 2004). In peroxisomes, H2O2 was located in close contact with other organelles, which suggests possible cross-talk with other cell compartments (RomeroPuertas et al. 2004). In mitochondria, the Cd-dependent H2O2 produced could be because of increased O2− production at the complex III site of the electron transport chain, as reported in animals treated with Cd (Wang et al. 2004) and also suggested for soybean roots (Heyno et al. 2008). H2O2 was also observed in the tonoplast from bundle sheet cells and plasma membrane from epidermal and transfer cells (RomeroPuertas et al. 2004). Cd-dependent superoxide radical accumulation was demonstrated in the tonoplast from bundle sheet cells and plasma membrane from mesophyll cells, although the source has not been identified (Romero-Puertas et al. 2004). Accumulation of both H2O2 and O2− was also observed in vascular tissues from Cd-treated pea plants using confocal laser microscopy, electron microscopy, and cytochemistry (Romero-Puertas et al. 2004; Rodríguez-Serrano et al. 2006, 2009). This ROS accumulation is associated with lignifications processes which are highly active in vascular tissue under physiological conditions and are also induced in response to metal toxicity (Schützendübel et al. 2001; Rodríguez-Serrano et al. 2009). Results using different inhibitors and modulators of signal transduction demonstrated that the earliest

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control point in ROS production induced by Cd is at the level of protein phosphorylation/dephosphorylation. A comparative transcriptomic study using different metals and sodium chloride in Arabidopsis thaliana showed that Cd specifically induced genes coding kinases (Zhao et al. 2009), which demonstrates the importance of these processes in regulating cell response to Cd. Calcium ions are also important in the regulation of ROS production induced by Cd, and cGMP is also involved in this process, probably as result of a transient increase in Ca2+ concentration (RomeroPuertas et al. 2004). NOXs are located in the plasma membrane and catalyze the production of O2−, which can be converted into H2O2, spontaneously or in the reaction catalyzed by SOD. Ten genes encoding NOXs in Arabidopsis have been described and are termed respiratory burst oxidase hom*ologs A–J (rbohA–J) given their hom*ology with the catalytic subunit gp91 phox (Nox2) of the NOX complex of mammalian phagocytes (Torres and Dangl 2005). The role of NOX as the main source of ROS under Cd stress has also been demonstrated in tobacco cell cultures (Olmos et al. 2003; Garnier et al. 2006; Horemans et al. 2007) and alfalfa roots (Ortega-Villasante et al. 2005). In Arabidopsis plants, the analysis of transcript levels of different NOXs shows a transient increase in the expression of rbohF in response to Cd, while the expression of rbohC and rbohD remained unchanged (Horemans et al. 2007). However, the contribution of NOXs to cadmiuminduced ROS production is a subject of debate (Heyno et al. 2008). In tobacco cell cultures, Cd induced cell death, which was preceded by three successive waves of ROS production. The first wave was because of an NOX followed by an accumulation of O2− and fatty acid hydroperoxides (Garnier et al. 2006). Before the first oxidative burst induced by Cd, a rapid and transient induction of cytosolic Ca2+ concentration takes place, which requires protein phosphorylation and IP3mediated release of calcium from internal stores (Garnier et al. 2006). Downstream, protein phosphorylation, calmodulin, and Ca2+ may directly regulate NtrbohD activity (Garnier et al. 2006). The disturbances caused by Cd in the mitochondrial

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Fig. 9.1 Sources of reactive oxygen species activated in response to cadmium. Cd-dependent ROS production takes place in different compartments: plasma membraneassociated NADPH oxidase, electron transport chain from

mitochondria and peroxisomes (mainly glycolate oxidase). Overaccumulation of H2O2 produces redox imbalance and oxidative damage, but also can regulate gene expression in order to improve plant survival

electron transport chain stimulate a second wave of ROS production possibly because of an increase in the semi-ubiquinone radical concentration (Garnier et al. 2006). A third wave of ROS coincides with cell death and involved membrane peroxidation as a result of increase in ROS production caused by mitochondria (Garnier et al. 2006). Remans et al. (2010) have recently demonstrated a Cd-dependent induction of NOX and differential regulation of gene expression by Cd and Cu in Arabidopsis plants and have suggested a link between NOX and lipoxygenase gene expression. A diagram showing the different subcellular locations of ROS production is provided in Fig. 9.1. The inhibition of antioxidative enzymes may also lead to a cadmium-mediated increase in the level of cellular ROS (Sandalio et al. 2001, 2009; Romero-Puertas et al. 2002; Schützendübel and Polle 2002; Benavides et al. 2005). One of the consequences of plant cell exposure to cadmium is the rapid consumption of GSH for sequestration of the metal and synthesis of PCs. This limits the GSH level required to maintain the redox

balance of the cell which then increases ROS accumulation (Romero-Puertas et al. 2007a, b).

4

NO Production in Plants Exposed to Cadmium

Nitric oxide is a simple gaseous signaling molecule which, in many plant tissues, regulates a wide range of physiological and biochemical processes as well as plant responses to biotic and abiotic stresses (del Río 2011; Delledonne 2005; Siddiqui et al. 2010). An increasing number of studies have reported the role played by NO in plant response to heavy metals including cadmium, although the source of NO and its role in metal toxicity and plant responses are not yet clearly established (Xiong et al. 2010). NO can be generated enzymatically by nitrate reductase and nitric oxide synthase (NOS)-like activities and can also be produced nonenzymatically by reduction of apoplastic nitrite under acid conditions and by reduction of nitrite to NO in the mitochondria (Neill et al. 2008; del Río 2011).

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There is still some uncertainty concerning NOS in plants. Although there is strong evidence to show the presence of L-arginine-dependent NOS activity in plants (Barroso et al. 1999; del Río 2011), the only NOS from the plant kingdom to be fully characterized so far is the enzyme from the Ostreococcus tauri green alga (Foresi et al. 2010). NOS activity has been shown to be present in peroxisomes from pea leaves (Barroso et al. 1999). This enzyme uses L-arginine as substrate and requires NADPH, Ca2+/calmodulin, BH4, FAD, and FMN, although its gene has not yet been characterized (Corpas et al. 2004; del Río 2011). The generation of NO in peroxisomes by this NOS activity has also been reported by Corpas et al. (2004). In addition, chloroplasts have recently been identified as a source of NO via arginine and nitrite, although the enzyme involved has not been characterized yet (Jasid et al. 2006; del Río 2011). A nitriteNO oxidoreductase enzyme (Ni-NOR) associated with root plasma membrane may also contribute to NO production (Stöhr and Stremlau 2006). However, there are other potential enzymatic sources of NO in plants (del Río et al. 2004; del Río 2011) such as xanthine oxidase which can produce NO under hypoxic conditions (Millar et al. 1998; Harrison 2002). Regardless of the source of NO involved, the mechanisms determining the effects of NO are far from being fully understood, while a number of downstream signaling pathways involving Ca2+, cyclic GMP, and cyclic ADP-Rib have been described (Besson-Bard et al. 2008). NO is able to react with oxygen radicals such as O2−, generating peroxynitrite (ONOO−), and also to control ROS levels in cells and vice versa (Delledonne et al. 2001). NO can also react with GSH to produce S-nitrosoglutathione (GSNO), which is regarded as a long-distance-signaling molecule and a natural reservoir of NO (del Río 2011). NO directly or indirectly can regulate gene expression and protein functions. It therefore reacts very rapidly with heme groups and thiols, thus regulating enzymatic activities (Moreau et al. 2010). The protein S-nitrosylation of cystein residues has been demonstrated to be very important in regulating the enzymatic

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activity of certain proteins (Lindermayr et al. 2006; Lindermayr and Durner 2009; RomeroPuertas et al. 2007b, 2008). Some studies of NO production during the exposure of plants to heavy metals have reached controversial conclusions. The cell cultures of soybean (Kopyra et al. 2006) and Arabidopsis (De Michele et al. 2009) exposed to Cd showed an increase in NO and was dependent on NOS-like activity (De Michele et al. 2009), whereas, in pea leaves and roots, prolonged exposure to 50 mM Cd reduced NO accumulation (Rodríguez-Serrano et al. 2006, 2009). Bartha et al. (2005) reported increased NO in the roots of Brassica juncea and Pisum sativum exposed to 100 mM Cd, Cu, and Zn. Besson-Bard et al. (2009) have shown that NO production in Cd-treated roots is related to Cd-induced Fe deficiency. The discrepancies observed in these results could be because of differences in Cd exposure duration, with NO increasing after a short period of Cd treatment and decreasing after a prolonged treatment (Fig 9.2). The metal concentrations, plant ages, and plant tissues used could also explain these discrepancies (Rodríguez-Serrano et al. 2009). Exogenously supplied NO has been demonstrated to alleviate heavy metal toxicity (Kopyra and Gwóždž 2003; Hsu and Kao 2004; Yu et al. 2005; Wang and Yang 2005; Laspina et al. 2005; Xiong et al. 2010) possibly because of its ability to act as an ROS-scavenging antioxidant such as SOD and CAT (Wang and Yang 2005; RodríguezSerrano et al. 2006; Singh et al. 2008; Siddiqui et al. 2010). Exogenous NO application also affects root cell walls and helps in metal accumulation. NO cause increases in cytosolic Ca2+ concentrations by regulating Ca2+ channels and transporters, which may be involved in the signaling cascade that regulates gene expression under stress conditions (Besson-Bard et al. 2008). Recently, cross-talk between Cd, Ca2+, ROS, and NO has been detected in pea leaves (Rodríguez-Serrano et al. 2009). The supply of exogenous Ca2+ to pea plants exposed to Cd reduced Cd-dependent O2− accumulation and restored NO accumulation to the level observed in control plants (Rodríguez-Serrano et al. 2009).

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cadmium can be categorized into different protein groups in terms of photosynthetic processes, signal transduction, and transcriptional regulation, cellular defenses, ROS detoxification and repair, hydric balance, metal transport, cell wall metabolism, sulfate and GSH metabolism and protein degradation.

5.1

Fig. 9.2 Cadmium induces differential response in plants depending on the period of treatment. A short period of treatment produces oxidative and NO burst, which induces gene expression to prevent oxidative damages caused by the metal. Long-term treatment produces overaccumulation of ROS and a reduction of NO giving rise to severe damages. Gene regulation in long-term treatment is focused on repairing oxidative damages and cell death. PCs phytochelatins, HSPs heat shock proteins

5

Plant Responses to Cadmium

Information on molecular mechanisms and signaling events underlying plant transcriptional responses to Cd is rather limited as compared to research in the field of cadmium toxicity. The mechanism by which Cd modulates the levels of expression of most genes is not clearly understood, while our knowledge of global changes in the expression of Cd-responsive genes is also limited. A number of studies have been carried out involving both small-scale experiments and whole-genome approaches. Their findings suggest that gene expression is time- rather than dose-regulated in response to Cd and is differentially regulated in roots and leaves (Herbette et al. 2006; Ogawa et al. 2009). Genes regulated by

Photosynthesis Regulation

The decrease in chlorophyll content has been considered to be one of the early symptoms of cadmium toxicity. The inhibition of chlorophyll biosynthesis has been suggested to be a primary event in Cd toxicity (Baryla et al. 2001). A substantial number of genes involved in photosynthesis were downregulated in the leaves of Arabidopsis plants grown with 5–50 mM Cd. For example, the genes involved in the photochemical process of photosynthesis, such as the chlorophyll synthesis pathway, glutamyl tRNA reductase, hydroxymethylbilane synthase, and Mg chelatase, some proteins of PSI and PSII, electron transporters, enzymes involved in Calvin cycle and Rubisco are downregulated (Herbette et al. 2006). Genes encoding enzymes in the pentose phosphate pathway were also downregulated by Cd (Herbette et al. 2006). These results have been corroborated by proteomic approaches (Álvarez et al. 2009) and correlate with the reduction observed in the photosynthesis net rates for different plant species (Sandalio et al. 2001; Faller et al. 2005). Downregulation of photosynthesis-related genes is a primary response under different stress conditions probably to avoid oxidative damage (Mittler 2002).

5.2

Signal Transduction and Transcriptional Regulation

Numerous genes involved in signal transduction were regulated in response to Cd in different plant species showing that signal transduction pathways are rapidly activated by the presence of Cd (Suzuki et al. 2001; Herbette et al. 2006; Ogawa et al. 2009). These include genes encoding

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mitogen-activated protein kinases (MAPKs), calmodulins and calcium-dependent protein kinases (CDPKs) (Suzuki et al. 2001; Herbette et al. 2006; Ogawa et al. 2009), which suggests that Cd interferes with the Ca2+ signaling pathway, as demonstrated by Rodríguez-Serrano et al. (2009). MAPKs and CDPKs are involved in biotic and abiotic stress responses and participate in cross-talk with ROS production activities (Kobayashi et al. 2007). Transcription factors belonging to different families, such as WRKY, bZip, MYB, DREB, NAC, and AP2, are induced by Cd in different plant species (Herbette et al. 2006; Weber et al. 2006; Ogawa et al. 2009). The inductions by Cd of transcripts for bZIP, MYB, and zinc finger transcriptional factors have also been demonstrated in the root of the metal accumulator B. juncea (Fusco et al. 2005). Genes involved in hormone signaling, mainly ABA and ethylene and jasmonic acid, have also been shown to be regulated in response to Cd (Herbette et al. 2006; Minglin et al. 2005).

5.3

Cellular Detoxification and Repair

Several genes associated with cellular detoxification and repair have been shown to be induced by treatment with cadmium. Chitinases and heat shock proteins (HSPs) are induced in response to heavy-metal stress and are regarded as a second line of defense under these stress conditions (Metwally et al. 2003; Békésiová et al. 2008; Rodríguez-Serrano et al. 2009; Zhao et al. 2009). Transgenic plants expressing fungal chitinases actually showed enhanced tolerance to metals (Dana et al. 2006), while chitinase isoforms are differentially modified by certain metals (Békésiová et al. 2008). Chitinases are regulated by ROS and are possibly part of the general defense response program of cells under heavymetal stress (Békésiová et al. 2008; RodríguezSerrano et al. 2009). Other pathogenesis-related proteins (PRPs) are upregulated by Cd (Fusco et al. 2005; Rodríguez-Serrano et al. 2009). ROSdependent up-regulation of PRP4A has been demonstrated in pea plants exposed to Cd, whose

transcripts were specifically accumulated in palisade mesophyll cells, as evidenced by in situ hybridization (Rodríguez-Serrano et al. 2009). These results point to an overlap in the regulatory mechanisms underlying these processes, with ROS production being a common event in these situations. HSPs are upregulated by heat stress and can act as molecular chaperones favoring the transport of proteins to organelles and preventing protein aggregation (Ma et al. 2006). Induction of HSPs by Cd has been observed in different plant species (Sanitá di Toppi and Gabbrielli 1999; Rodríguez-Serrano et al. 2009) and is regulated by H2O2 overproduction (Rodríguez-Serrano et al. 2009). The transcription factors involved in HSP expression can act as H2O2 sensors (Miller and Mittler 2006). In B. juncea, Cd upregulates a DNAJ HSP (BjCdR57), a chaperone involved in protein protection against stress, which confirms that protein denaturation is one of the effects of Cd toxicity (Suzuki et al. 2001; Fusco et al. 2005). GSH S-transferases catalyze the conjugation of xenobiotics with GSH and participate in the removal of ROS and are upregulated in response to Cd (Suzuki et al. 2002; Fusco et al. 2005; Ogawa et al. 2009). Antioxidative defenses such as glutaredoxin, thioredoxin, GSH reductase, monodehydroascorbate reductase, SOD, CAT, and POXs are upregulated by Cd in order to deal with oxidative damage caused by this metal (Lemaire et al. 1999; Herbette et al. 2006; Romero-Puertas et al. 2007a, b; Smeets et al. 2005; Ogawa et al. 2009). Enzymes involved in vitamin E biosynthesis are upregulated in response to Cu and Cd in Arabidopsis plants, while vitamin E-deficient mutants (vte1) showed enhanced oxidative stress and sensitivity to both metals, suggesting that Vitamin E also contributes to defense against heavy metals (Collin et al. 2008). The regulation of these antioxidative enzymes is mainly dependent on H2O2 (RomeroPuertas et al. 2007a, b; Rodríguez-Serrano et al. 2009), although GSH metabolism also plays an important role in controlling the gene regulation of antioxidants in response to Cd stress (Cuypers et al. 2011). The activity of glucose-6-P dehydrogenase (G6PDH), malic enzyme (ME), and

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NADP isocitrate dehydrogenase (NADP-ICDH) is stimulated by Ni, Zn, and Cd (Van Assche and Clijsters 1990; León et al. 2002), while, in pepper cultivars with different levels of sensitivity to Cd, tolerance to this heavy metal was more dependent on the availability of NADPH than on its antioxidant capacity (León et al. 2002).

5.4

Metal Transporters

Some of the genes regulated by Cd, such as AtPcr1 (Song et al. 2004) and those belonging to the ABC, MATE, cation diffusion facilitator (CDF), heavy metal P-type ATPase (HMA) and ZIP families, are involved in Cd transport (Ogawa et al. 2009). Fe and Zn transporters are also often involved in Cd transport because of their low substrate specificity. The iron transporters ZIP, AtIRT1, OsIRT1111, and OsIRT2 as well as the Zn transporter OsZIP1 have been shown to transport Cd. The HMA family is also involved in Cd detoxification in addition to CDF transporters and natural resistance-associated macrophage protein (NRAMP) family transporters (Ogawa et al. 2009). Pleiotropic drug resistance (PDR) family proteins are involved in Cd tolerance via export out of the cytoplasm (Kim et al. 2007). AtPDR8 is a cadmium extrusion pump, while AtOSA1 could be involved in the signal transduction pathway in response to oxidative stress (Kim et al. 2007; Jasinski et al. 2008). Cd-binding proteins such as Cdl19 could be involved in maintaining heavy-metal homeostasis and/or detoxification (Suzuki et al. 2002).

5.5

Cell Wall Metabolism

The cell wall is one of the first structures to be directly exposed to Cd and has the ability to bind metals, which is regarded as a mechanism of metal tolerance. Most of the heavy metals associated with the cell wall are linked to polygalacturonic acids, whose metal ion affinities vary depending on the metal in question. The plant cell wall is mainly composed of cellulose and matrix polysaccharides, which are divided into

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pectins and hemicelluloses, both of which are rich in polygalacturonic acids (Xiong et al. 2010). Cellulose is a key component in plant cell walls, and it has been reported that NO affects the cellulose content of tomato roots in a dose-dependent manner. Low concentrations of sodium nitroprusside (SNP) increase cellulose content in roots, while higher concentrations have the opposite effect (Correa-Aragunde et al. 2008). Exogenous NO increases Cd tolerance in rice plants by increasing pectin and hemicelluloses content in the root cell wall and by decreasing Cd accumulation in the soluble fraction of cells in rice leaves (Xiong et al. 2009). H2O2 may also trigger secondary defenses, causing cell wall rigidification and lignifications in Cd-exposed cells (Schützendübel and Polle 2002). The transcript levels of genes involved in cell wall metabolism are modulated in response to Cd. The proteins involved in lignification and extension were therefore upregulated (Fusco et al. 2005; Herbette et al. 2006), whereas expansins and pectin esterases were downregulated (Herbette et al. 2006).

5.6

Sulfate and GSH Metabolism

One of the best described mechanisms induced under heavy-metal toxicity is the chelation of the metal by PCs and GSH. PCs have the general formula (gGlu-Cys)n-Gly (with n = 2–11) and are synthesized enzymatically through the transpeptidation of gGlu-Cys moieties of GSH onto another GSH molecule by the phytochelatin synthase (PCS) enzyme, which is known to be activated posttranslationally by a range of heavy metal metalloids (Grill et al. 2006). Chelation of metals by PCs and the compartmentalization of PC–metal complexes in vacuoles (Clemens 2006; Grill et al. 2006) are generally considered as firstline defense mechanisms. The rate-limiting step for PCs and GSH biosynthesis is the availability of reduced sulfur to the roots. Various genes involved in the sulfate metabolism are induced in response to Cd, which include sulfate transporters from roots (Sultr1; 1; Sultr1; 2), enzymes involved in sulfate reduction

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to sulfide (ATP sulfurylase), and those involved in PC biosynthesis (PC synthases) (Herbette et al. 2006; Ramos et al. 2007). One of the steps in PC biosynthesis is the synthesis of cysteine catalyzed by O-acetylserine(thiol)lyase (OASTL) which is upregulated by Cd (Fusco et al. 2005). Arabidopsis plants over expressing OASTL were highly Cd resistant, which suggests that cysteine pool required for GSH biosynthesis is one of the principal factors affecting Cd tolerance (DomínguezSolis et al. 2001). A deficiency in the major OASTL isoform in the cytosol from Arabidopsis plants, OAS-A1, causes aH2O2 homeostasis imbalance (López-Martín et al. 2008).

5.7

Hydric Balance

The plant–water balance is also disturbed by Cd, and the stomatal opening is inhibited (Poschenrieder et al. 1989; Sandalio et al. 2001; Perfus-Barbeoch et al. 2002). Sequence analysis of Cd-responsive genes in the metal accumulator B. juncea revealed the induction of genes encoding aquaporins, which facilitates the movement of water through cellular membranes. In addition, other drought and ABA-responsive genes, such as BjCdR39 (the aldehyde dehydrogenase) and BjCdR55, (RNA-binding protein), are also upregulated by Cd, which confirms the existence of cross-talk between Cd-induced and water stressinduced signaling using ABA as a signal transducer. Stomatal closure, a symptom of water stress mediated by ABA, is one of the principal responses of higher plants to Cd (Sanitá di Toppi and Gabbrielli 1999).

5.8

Protein Degradation

Oxidative damage to proteins has been observed in different plant species exposed to Cd and is regarded as an oxidative stress marker (Sandalio et al. 2001; Pena et al. 2007; Djebali et al. 2008; Paradiso et al. 2008). Some of the proteins undergoing oxidative modification have been identified in pea leaves and include CAT, GR, Rubisco, and Mn-SOD (Romero-Puertas et al. 2004). Increased

proteolytic activity in leaves following Cd treatment and more efficient degradation of the oxidized proteins have been observed (McCarthy et al. 2001; Romero-Puertas et al. 2004). Similar results have been reported by Pena et al. (2006) in Helianthus annus and by Djebali et al. (2008) in Solanum lycopersicum. A proteomic study of A. thaliana cells has also reported an increase in several proteases after Cd treatment (Sarry et al. 2006). Cd treatment has also been shown to increase polyubiquitinated protein accumulation (Pena et al. 2007). The proteasome–ubiquitin system is the major proteolytic pathway in eukaryotes and is also involved in the depredation of oxidized proteins (Pena et al. 2007). The plant proteasome was upregulated at transcriptional and translational levels under oxidative conditions caused by cadmium stress (Pena et al. 2006, 2007; Djebali et al. 2008; Polge et al. 2009). Using in vivo experiments with A. thaliana mutants, it has been demonstrated that 20S proteasomes are preferentially involved in the degradation of oxidized proteins (Kurepa et al. 2008). The remobilization of oxidized proteins may be a protective mechanism under stress conditions to prevent further damage to other macromolecules and to facilitate the recycling of amino acids for protein biosynthesis.

6

Signal Transduction Under Cadmium Stress

The response to heavy metals depends on a complex signal transduction pathway within the cell which begins with the sensing of heavy metal and converges in transcription regulation of metal-responsive genes (Sing et al. 2002), although much remains to be learned about the molecular components of metal-induced signal transduction. Various transcription factors (TFs) involved in the regulation of cell response to metal stress have recently been identified (Sect. 21.5.2). The modulation of different groups of TFs highlights the complex response of plants to Cd (DalCorso et al. 2008). ROS and NO are important players in the regulation of plant response from signal perception to the intracellular

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transduction cascade, triggering the activation of genes involved in the induction of different metabolic pathways to deal with Cd toxicity. Hydrogen peroxide governs the transduction of cellular response in different abiotic stresses including those caused by heavy metals. The transduction of H2O2 signals into biologically relevant information is coordinated by a complex network of sensors and receptors, such as MAPKs, and transcription factors and is thought to be evolutionarily conserved (Vandenbroucke et al. 2008), although there are around 400 H2O2-responsive protein families in A. thaliana and may vary depending on the plant species in question (Vandenbroucke et al. 2008). There are several elements in the signal transduction pathway of ROS-sensitive plants which include the MAPK, MAPKK, MAPKKK, AtMPK3/6, AtANP1, NtNPK1, Ntp46MAPK, and calmodulin (Mittler 2002; Vanderauwera et al. 2009). The increase in H2O2 levels induced by Cd can be perceived by oxidative protein modifications. The protein thiol groups tyrosine, tryptophan, and histidine can be oxidised by H2O2 and O2−. The redox changes in the Cys residues of transcription factors directly regulate nuclear gene expression. However, transcriptional modifications may also require additional upstream sensing and transduction of ROS and ROSderived signals, being involved MAPKs and several protein phosphatases (Vanderauwera et al. 2009). Salicylic and jasmonic acid as well as ethylene can also participate in signal transduction under Cd stress (Rodríguez-Serrano et al. 2009; Ogawa et al. 2009). Salicylic acid (SA) acts as an important signaling element in plants and has been observed to alleviate Cd-induced growth inhibition and oxidative damage (Metwally et al. 2003). Although this mechanism is not fully understood, it has been suggested that SA may induce H2O2 signals involved in Cd tolerance, such as repair processes, Cd binding and compartmentation (Metwally et al. 2003). The Cd-induced ethylene biosynthesis has been reported to occur in various plant species (Sanitá di Toppi and Gabbrielli 1999; Rodríguez-Serrano et al. 2006), although the molecular relationships

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between ethylene biosynthesis and Cd stress have yet to be clearly determined. Transcriptomic studies of Arabidopsis plants have detected Cd-dependent up-regulation of ACC oxidase and ACC synthase as well as the ethylene responsive factors ERF2 and ERF5 (Herbette et al. 2006). JA content increases in response to heavy metals in various plant species (Wang and Wu 2005; Rodríguez-Serrano et al. 2006, 2009). JA regulates genes involved in GSH and PCS in Arabidopsis plants under Cd treatment (Xiang and Oliver 1998). In different plant species, Ca2+, calmodulin, CDPK and an MAPK act as signaling molecules which regulates cell response to cadmium stress (Romero-Puertas et al. 2004, 2007a, b; Herbette et al. 2006; Yeh et al. 2007; Rodríguez-Serrano et al. 2009). Several studies have provided genetic evidence for the importance of NO in gene regulation. Two studies involving large-scale transcriptional analysis of A. thaliana have revealed NO-dependent regulation of genes involved in signal transduction, disease resistance, stress response, photosynthesis, and basic metabolism (Grün et al. 2006); however, the intracellular signaling pathway involved has not yet been defined. Most of the information available relates to plant defense and wounding and suggests that NO and salicylic and jasmonic acids are interrelated (Grün et al. 2006). The activity of different nuclear regulatory proteins is dramatically affected by NO. Modification by S-nitrosylation can regulate the activity and function of some regulatory proteins and transcription factors. Although no plant transcription factor has been observed to be regulated by this process, some regulatory proteins could be S-nitrosylated (Grün et al. 2006). NO can regulate cell signaling by controlling Ca2+ homeostasis. Most Ca2+ channels are regulated by NO either directly through S-nitrosylation or indirectly through cyclic ADP-ribose (cADPR) involving GMP (Courtois et al. 2008). NO-dependent activation of protein kinases, MAPK, and CDPK has been reported in various plant species. The activation of these kinases by NO is thought to be involved in defense responses and/or cell death (Courtois et al. 2008).

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A hypothetical model depicting some of the players involved in ROS, NO perception, and signal transduction pathways in response to cadmium is shown in Fig. 9.3. Cadmium promotes an increase of ROS production in different cell compartments and their accumulation could give rise to oxidative damages affecting to lipids and proteins. However, ROS can also trigger defense cellular responses indirectly acting as signaling molecules, by promoting changes in Ca2+ concentration, through GMP or by altering the redox status of several proteins, and further activating the MAPK cascade. But ROS can also directly regulate nuclear gene expression by affecting the redox state of transcription factor Cys residues.

In this scenario, NO plays an important role in the regulation of cell responses to this metal, but further works are needed to better understand the coordinated role of ROS and NO in both toxicity and regulation of cell response to cadmium. The role of some hormones, such as JA and ET, in the cell response to Cd is also an interesting point which deserves more research in order to understand the cross-talk between hormone balance/ ROS and NO in the regulation of plant defense against heavy metals. In response to Cd the upregulation of some defense genes takes place, although some of them are also induced during pathogen attack, which suggests an overlap in the regulatory mechanisms governing these processes. However, the role of genes such as HSPs or chitinases in the mechanisms of tolerance to Cd has not been explored in depth so far, and they could be important in the development of

Fig. 9.3 Hypothetical model showing signal transduction of cell response to cadmium toxicity. Cd-dependent changes in H2O2 and NO levels can be perceived by changes in Ca2+ concentration, and oxidation or S-nitrosylation of

proteins or transcription factors. The transcriptional response can also require more upstream transduction involving mitogen-activated protein kinases (MAPKs) and hormones such as jasmonic acid (JA) and ethylene (ET)

7

Conclusion and Future Perspectives

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Reactive Oxygen Species and Nitric Oxide in Plants Under Cadmium Stress...

new strategies for phytoremediation. Further studies will be necessary to understand the role of the post-translational modification of proteins in the perception of metal toxicity and also in the transduction and regulation of cell response to Cd. An integrated study of all the players mentioned in this chapter at biochemical, molecular, and cellular levels is needed in order to understand the complex network involved in perception, transduction, and development of cell responses to cope with adverse conditions caused by heavy metals. This could allow the development of new and more efficient strategies for phytoremediation. Acknowledgments This work was supported by ERDFcofinanced grants from the Ministry of Education and Science (Grant BIO2008-040067) and Junta de Andalucía (Project P06-CVI-01820), Spain

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Reactive Nitrogen Inflows and Nitrogen Use Efficiency in Agriculture: An Environment Perspective

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Khalid Rehman Hakeem, Ruby Chandna, Altaf Ahmad, and Muhammad Iqbal

Abstract

Increased use of nitrogenous (N) fertilizer has significantly altered the global N-cycle and produced nitrogenous gases of environmental consequence. While nitrous oxide (N2O) emissions contribute to global greenhouse gas accumulation and the stratospheric ozone depletion, degradation of groundwater quality by N use in agriculture is fundamentally a nitrate leaching problem. Despite these evident negative environmental impacts, consumption of N fertilizer cannot be reduced in view of the food security for teeming population in the developing countries. Various strategies, from agronomic to genetic engineering, have been tried to tackle this problem. Split application of N, use of slow-release fertilizers, nitrification inhibitors, and the use of organic manures are some agronomic techniques adopted. One of the important goals to reduce N-fertilizer application can be effectively achieved by choosing N-efficient (i.e., which can grow under low N conditions), ensuring their optimum uptake of applied N by application of adequate amounts of fertilizer nutrients in a balanced manner and knowing the molecular mechanisms for their uptake as well as assimilatory pathways. Newer approaches like quantitative trait locus and proteomics could also help us in understanding these processes fully, hence could contribute greatly in enhancing nitrogen use efficiency and reduction of N pollution in the environment. Keywords

Reactive nitrogen • NUE • QTL • Proteomics • N pollution

K.R. Hakeem () • R. Chandna • A. Ahmad • M. Iqbal Molecular Ecology Laboratory, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi 110062, India e-mail: [emailprotected] P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_10, © Springer Science+Business Media, LLC 2012

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1

Introduction

Nitrogen (N) represents one of the most important nutrients found in terrestrial ecosystems. It is an important constituent of a number of complex organic molecules viz., proteins, nucleic acids, etc. Atmosphere is the main reservoir of nitrogen (N2), which stores around one million times more N than contained in all the organisms. Oceans and organic matter in soil are the other major store houses of nitrogen. N is often considered as an important limiting nutrient for plant growth and development, despite its remarkable abundance in the atmosphere. This is the reason for the past half a century, supply of nitrogen through fertilizers has been an influential application for increasing the growth and yield of cultivated plants such as cereals. To meet the increasing demand for food, farmers apply more fertilizers in their bid to increase the agricultural productivity. Fertilizer nitrogen has provided food security particularly to developing nations including India, as the cereal production has kept pace with its ever-increasing population. Today, India occupies the third rank in the world in fertilizer N consumption and second in fertilizer N production (FAI 2008). The consumption of fertilizer nitrogen in India increased from a mere 55,000 metric tons in 1950–1951 to over 14.2 million tons in 2007–2008 and is still increasing (FAI 2008). With the current rate of N fertilization, the requirement of nitrogen will be 22–25 million tons/year in 2020 (FAI 2008). However, it is remarkable that utilization of applied fertilizer nitrogen in field by most cereal crops does not exceed 50% and around 70% of the total nitrogenous fertilizer is applied for rice and wheat cultivation (Abrol et al. 1999). Therefore, with the increase in agricultural food production worldwide in last 50 years, the N fertilization of crop plants has increased more than 20-fold (Shrawat and Good 2008). However, the use of this fertilizer is generally inefficient, as lesser amount of applied N (around 30–40%) is actually utilized by cereal crops, and the major part (60–70%) is lost from the plant–soil system which has caused severe impacts on the ecosystems of the

non-agricultural neighboring bacteria, animals, and plants. As a result of leaching, the unused N fertilizer causes impacts like eutrophication of freshwater (London 2005) and marine ecosystems (Beman et al. 2005). In addition, gaseous augmentation of N oxides reacting and affecting with stratospheric ozone and the volatilization of toxic ammonia into the atmosphere (Stulen et al. 1998) has also been linked to unused N fertilizers. The toxic effects of nitrate are due to its endogenous conversion to nitrite and this ion has been implicated in the occurrence of methaemoglobinemia, gastric cancer, and many other diseases (Anjana et al. 2007). Presently the human population is more than 6.5 million, which is expected to increase around 10 billion by 2025 (Hirel et al. 2007), therefore, the major challenge will be to reach a highly productive agriculture without degrading the quality of our environment. Efficient farming techniques and choosing plant varieties/genotypes that have better nitrogen use efficiency (NUE) could be the tools to tackle this problem. The development of such varieties/genotypes, through conventional plant breeding techniques or by using recombinant DNA technology, will be more proficient with a better understanding the physiological, genetic, and molecular bases of NUE among cereal crops. Therefore, there is an urgent need of a “second green revolution” that does not rely on exhaustive use of inorganic fertilizers rather would aim at improving crop yields in soils by developing varieties with better adaptation to low-fertility soils (Yan et al. 2006). In the present chapter, we have discussed the inflow and effects of reactive N in the environment and then summarized the strategies adopted to develop the crop varieties/genotypes with high NUE.

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Reactive Nitrogen Inflow

Reactive nitrogen (Nr) is usually referred to all the nitrogen species that are biologically active, photochemically reactive, and radiatively important N compounds in the atmosphere and biosphere of the earth (Galloway 1995). Thus, Nr includes

10 Reactive Nitrogen Inflows and Nitrogen Use Efficiency in Agriculture¼

reduced inorganic forms of N (NH3, NH4+), oxidized inorganic forms (NOx, HNO2, N2O, NO3−), and organic compounds (urea, amines, proteins, nucleic acids). There are numerous sources in environment that contribute to Nr and total nitrate content of natural waters, e.g., atmosphere, geological features, anthropogenic sources, atmospheric nitrogen fixation, and soil nitrogen. However, detailed hydro geological investigations conducted have indicated a heterogeneous pattern of nitrate distribution. Soils with low water-holding capacity (sandy soil) and high permeability, movement of pollutants like chloride and nitrate is much quicker than in clayey soil. This is probably the main cause for high nitrates in areas with sandy soil. Vegetables account for more than 70% of the nitrates ingested in the human diet. The remainder of nitrate in a typical diet comes from drinking water (21%), meat and meat products (6%) (Prasad 1999). The form of added N plays a role in regulating N losses and influencing NUE. Among these forms, NO3 is the most susceptible to leaching, NH4 the least, and urea moderately susceptible. Ammonia and urea are more susceptible to volatilization loss of N than fertilizers containing NO3. Urea is the most widely used N fertilizer in India. The studies showed the importance of selecting ammonium-based N fertilizer early in the season to reduce N leaching due the mobility of urea and nitrate source in irrigated rice and wheat systems (Prasad and Prasad 1996). Nitrate containing fertilizers when applied to rice proved less efficient because nitrate is prone to be lost via denitrification and leaching under submerged soil conditions in normal and alkali soils (Prasad 1998). In saline soils, however, it is beneficial to use NO3 containing N fertilizers as it compensates the adverse effects of Cl− and SO42− on absorption of NO3 by plants (Choudhary et al. 2003). Nitrogen losses from soil–plant system. Once inorganic N has appeared in the soil, it can be absorbed by the roots of higher plants or still metabolized by other microorganism during nitrification. This process is carried out by a specialized series of actions in which a few species of microorganisms oxidize NH4+ to NO2 or NO2− to NO3−. Ammonium ion reacts with excess hydroxyls in

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soil solutions, which leads to N losses to the atmosphere by NH3 volatilization (Wood et al. 2000). This represents an important source of N loss in agricultural soils under favorable conditions. Due to extensive use of N fertilizers and nitrogenous wastes, the amount of N available to plants significantly exceeds the N returned to the atmosphere by gaseous losses of N through volatilization and denitrification (Martre et al. 2003). Minimizing drying of surface soil and providing additional source of urease enzyme can minimize NH3 volatilization. A portion of this excess N is leached out in the soil profile as NO3− or carried in runoff waters. These are conductive conditions for N losses in agricultural soils, thus reducing the NUE (Delgado et al. 2001). With transport of N in water ways and neighboring ground-water systems, the N concentration could exceed the levels acceptable for human consumption. Nitrate in soil profile may be leached into groundwater when percolating water moves below the rooting depths of crop and provides leaching potential. Paramasivam et al. (2002) have reported a potential leaching of NO3− in arid regions and sandy soils. Losses of N by leaching are affected by local differences in rainfall, water-holding capacity of soil, soil-drainage properties, and rates of mineralization of soil organic N (Delgado et al. 1999). Processes such as adsorption, fixation, immobilization, and microbial assimilation of added NH4-N in soils are of great importance as they affect NUE and have the corresponding environmental repercussions (Kissel et al. 2004). In many field situations, more than 60% of applied N is lost due in part to the lack of synchrony of plant N demand with N supply. The remainder of the N is left in the soil, or is lost to other parts of the environment through leaching, runoff, erosion, NH3 volatilization, and denitrification. The cereal NUEs are 42% in developed and 29% in developing countries (Raun and Johnson 1999). Many 15N studies have reported N fertilizer losses in cereal production from 20 to 50% with higher values in rice than in wheat (Ladha 2005). Prasad (1998) reported that apparent recovery of N applied to wheat varies from 40 to 91%. It has been estimated that rice and wheat N recovery efficiency ranging from 30 to 40% are occurring

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Fig. 10.1 Diagrammatic representation of N inflow and N loss in ecosystem

in irrigated conditions. An N recovery efficiency exceeding 40% is expected to occur in response to improved N management practices. In a rice– wheat cropping system of Punjab, recovery of 15 N by the first wheat crop was 30–41%, the soil at wheat harvest retained 19–26%, and the succeeding rice recovered 5.2% of the 120 kg N ha−1 applied (Singh and Singh 2001). Total losses of applied N (not recovered from soil–plant system) were about 42% in rice and 33% in wheat grown on a typical sandy loam soil in north-west India. The main causes of for low N recovery are usually attributed to (1) ammonia volatilization, (2) denitrification, (3) leaching, and (4) runoff and erosion (Fig.10.1). Loss of N via NH3 volatilization can be substantial from surface-applied urea in both rice and wheat, which can exceed 40%, and generally greater with increasing soil pH, temperature, electrical conductivity, and surface residue (Singh et al. 2003; Choudhary et al. 2003). Water management in rice and wheat fields influences the extent of N losses due to nitrification–denitrification and NH3 volatilization. Available research results from ideal rice soils suggest that NH3 volatilization rather than denitrification is an more important gaseous loss mechanism for fertilizer N applied to continuously flooded, puddled rice soils of the tropics. The picture is quite opposite in highly permeable porous soils under rice. There exist two mechanisms in such soils due to which losses due to denitrification assume more importance than NH3

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volatilization losses. Firstly, in porous soils under rice it is difficult to maintain continuous flooding. Rather there occur very frequent alternate aerobic– anaerobic cycles, which lead to very fast formation of nitrate under aerobic conditions and their subsequent denitrification under anaerobic conditions that develop due to application of irrigation (Singh and Singh 2001). Secondly, due to high permeability of coarse textured porous soils, urea as such is rapidly transported to subsoil where even after it is hydrolyzed to NH4, it is not prone to losses via NH3 volatilization (Sangwan et al. 2004a). Sangwan et al. (2004a, b) have shown that NH3 volatilization losses from urea increases with the increase in soil salinity, sodicity, and the rate of N applied. The losses of fertilizer N as NH3 in rice decreased with increasing floodwater depth and depth of placement (Singh et al. 1995a), and with the application of organic manures (Sihag and Singh 1997). Alkalinity, pH, and NH3 concentration in flood water control the extent of NH3 loss from flooded soils (Singh et al. 2003). Sarkar et al. (1991) reported a loss of 15–20% of applied N when urea was broadcast in a wheat field. Prasad (1999) reported a marked reduction in the loss of applied N when the urea was deep placed as compared with surface broadcast on a moist soil. They have reported 13.5% N losses as ammonia after 1 week of urea application under submerged conditions. The high pH or alkalinity resulted in high losses of ammonia by volatilization, which can be nearly 60% of applied N at field capacity. Submergence decreases pH as well as losses to 35% of applied N. The reclamation of sodic soils using gypsum has been found to decrease N losses through ammonia volatilization (Choudhary et al. 2003). The timing of fertilization and irrigation could further influence the losses of urea applied to porous soils. If applied on the wet soil surface following irrigation, as much as 42% of the applied 15N was lost, most likely due to volatilization (Sangwan et al. 2004a). Singh et al. (1995b) showed that application of urea before irrigation increased the NUE by 20% as compared to its surface application after irrigation or broadcast application and surface mixing of urea at field capacity in a clay loam soil.

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In nonideal porous soils under rice, there exist every possibility that applied urea N is preferentially lost via denitrification rather than NH3 volatilization. Direct measurement of denitrification losses made by Aulakh et al. (2001) showed that denitrification is a significant N loss process under wetland rice amounting to 33% of the applied N. In excessive N fertilizer application (i.e., at rates in excess of that needed for maximum yield in cereal crops), NO3 leaching can be significant, particularly from the coarse-textured soils. Residual N is then available in soil profile for potential leaching. High levels of NO3-N in the region’s groundwater have been reported by Singh et al. (1995b). There is not much information available on leaching losses of N. In a pot culture study, the leaching loss was 11.5% of the applied urea N and was reduced to 8.7% when urea was coated with neem cake (Prasad and Prasad 1996). In a field study at Pantnagar on a silty clay loam soil, 12% of the applied N was lost by leaching and these losses were reduced to 8% when urea was blended with neem cake (Singh et al. 1995b).

3

Nitrogen Removal by Crops

From the human nutrition point of view, rice and wheat are the most important cereals and their production in north-west India in rice–wheat cropping system, which covers about 10 million hectares, is the backbone of the India’s food security (Prasad 2005). Rice–wheat cropping system produces 5–14 ton/ha/year grain and this depends heavily on nitrogen fertilization which ranges from 100 to 150 kg N/ha/crop or even more, especially in rice. From the animal nutrition point of view, maize, sorghum, and pearl millet stovers which contain 27–51% of nitrogen harvested by the crop in stover are more important both for milch as well as draught cattle. On the contrary, rice and wheat straw is low in nitrogen content and is a poor protein source. Nevertheless, they meet majority calorie requirements of the cattle. Also most sorghum and pearl millet is grown in rainfed areas where nitrogen application rates are low and even response to N application is low. Nitrogen removal per metric ton as

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well as its percentage in grain in pulses depends very much upon the plant stature and its vegetative growth. For example, Prasad (2004) reported a removal of 50.6 kg/ton in chickpea and 92.1 kg/ton in pigeon pea; these are the two major pulse crops in India. Most of this nitrogen is obtained by N fixation by Rhizobia as very little fertilizer N is applied to pulses. Again depending upon the plant stature and vegetative growth, 63.3% of total N removed by chickpea was contained in its grain, while the values for pigeon pea, a tall and heavily fertilized plant, was 31.6%. The protein-rich pulse foliage is widely used for enriching rice or wheat straw fed to cattle. Before the mechanization of Indian agriculture which is even now limited mostly to north-western India, draught animals were the major source of farm power and the Indian agriculture provided a characteristic “humans–animals–crops” ecosystem where man survived on the grains and the animals on the straw/stover. Taking an average N contribution by grain legumes at 30 kg N/ha, about 0.66 million metric tons of N is annually added to soil on 22 million hectares occupied by them. Another 0.34 million metric tons N may be added by leguminous trees and plants in forests and grasslands, and by leguminous oilseed crops such as groundnut. Thus the N contribution of legumes in Indian soils can be roughly estimated at least at 1 million metric tons, it is likely to be much more. In addition, some N is added by rains and use of N-fixing biofertilizer such as Azotobacter, Azospirillum, Acetobacter, Bluegreen algae, and Azolla.

4

Concept of NUE

NUE at the plant level is its ability to utilize the available nitrogen (N) resources to optimize its productivity (Raghuram et al. 2006). As a concept, NUE includes N uptake, utilization, or acquisition efficiency, expressed as a ratio of the total plant N, grain N, biomass yield, grain yield (output) and total N, soil N, or N-fertilizer applied (input) (Pathak et al. 2008). NUE is quantified based on apparent nitrogen recovery using physiological and agronomic parameters. Agronomic

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efficiency is an integrative index of total economic outputs relative to the available soil N (native and applied). Apparent nitrogen recovery is related to the efficiency of N uptake; physiological NUE deals with N utilization to produce grain or total plant dry matter. NUE in the context of photosynthesis is called as photosynthetic nitrogen use efficiency (PNUE), which is determined by the rate of carbon assimilation per unit leaf nitrogen (Kumar et al. 2002). The most suitable way to estimate NUE depends on the crop, its harvest product, and the processes involved in it.

5

Strategies for Minimizing N Pollution in Agriculture

Various strategies were adopted to minimize the N loss from the agricultural fields. Split application of N, use of slow-release fertilizers, nitrification inhibitors (NIs), and the use of organic manures are some agronomic techniques used. Bulk of the fertilizer nitrogen in India is broadcast on surface and both surface runoff (on sloppy lands) and ammonia volatilization lead to N losses. This can be easily overcome by deep placement of N a few centimeters below soil surface. For example, Sarkar (2005) showed that in wheat surface broadcast application of urea as band or top dressing caused 15–20% loss of N due to agriculture volatilization. Surface broadcast application followed by its mixing with top soil reduced the volatilization loss to 10%, while side band placement of urea reduced it further to only 5%. Thus the farmers need to be told about the advantage of incorporation in surface soil or if possible its placement using a ferti-drill or a pore in upland crops. Split application is a wellestablished technique for increasing NUE. In wheat and maize, studies with 15N showed that application of 40 kg N/ha as basal followed by 60 kg N/ha at crown root initiation (CRI) gave significantly higher yield than all basal application and other split application combinations (Sachdev et al. 2000; Narang et al. 2000). Havangi and Hegde (1983) showed in pearl millet also two or three split applications were found to be better than a single application. In rice, two split

applications are recommended for short and medium duration varieties, while three split applications are recommended for long duration varieties (Prasad 1999). Another way is NIs, these are a group of chemicals that are toxic to Nitrosomonas sp. and Nitrosomonas sp. involved in the conversion of NH4 to NO2− as well as to Nitrobacter sp. involved in the conversion of NO2 to NO3 and therefore, inhibits nitrification, which reduces losses due to leaching and denitrification. The most widely tested NIs are 2-chloro-6-trichloromethyl pyridine (N-serve), 2 amino-4-chloro-6 methyl pyrimidine (AM), dicyandiamide (DCD), and sulfathiazole (ST) (Prasad and Power 1995). Research on the use of NIs for reducing N losses and increasing NUE from the soil was initiated in India by Prasad (1999) at the Indian Agricultural Research Institute (IARI), New Delhi, with a field experiment on rice. Treatment of ammonium sulfate with N-serve significantly increased rice yield and nitrogen uptake by the rice crop. Prasad (2005) showed from a laboratory experiment that N losses due to denitrification could be considerably reduced by treating ammonium sulfate with NIs N-serve and AM. Prasad and Prasad (1996) showed through field experiments that treatment of urea with NIs, N-serve, and AM significantly increased rice yield and N uptake. Das et al. (2004) showed the effect of N-serve and AM on nitrification under field capacity moisture (upland) and water-logged (low-land paddy) conditions at New Delhi. Both the NIs were effective in retarding nitrification. The nitrification rate (nitrates expressed as percentage of total mineral N) after 40 days of incubation was 78% with N-serve at 2 ppm and 76% with AM at 10 ppm (mg/kg) as against 100% with untreated urea. Slow-release N fertilizers (SRFs) were developed with an aim to slowdown the dissolution of applied N so that most of it is taken up by crop plants rather than be subjected to N-loss mechanisms. There are two kinds of SRFs, namely, coated fertilizers and inherently slow dissolution rate materials. The examples of coated SRFs are sulfur-coated urea (SCU) (developed by TVA, USA), lac-coated urea (developed by Indian Lac Research Institute), polymer-coated urea, and to some extent neem cake-coated urea. The other

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kind of slow-release fertilizers are generally urea–aldehyde condensates, e.g., urea-form (urea and formaldehyde products developed in USA), isobutylidene diurea (IBDU, urea and isobutyraldehyde product developed in Japan and USA), and CD-urea (urea and crotonaldehyde product developed in Germany) (Prasad 2005). After 20 days of incubation under field capacity conditions, the mineral N (NH4+ NO3−) in soil was 67, 43, 31, and 27 ppm (mg/kg soil) with urea, oxamide, IBDU, and SCU, respectively. As would be expected under submerged conditions, NO3-N was not detected and the NH4+-N content in soil after 20 days of incubation was 67, 61, 46, and 15 ppm with urea, oxamide, IBDU, and SCU, respectively. Thus, of the three SRFs oxamide released, the N the fastest and SCU the slowest.

6

Physiological and Molecular Aspects for Improving NUE

NUE at the plant level is its ability to utilize the available nitrogen (N) resources to optimize its productivity. In terms of agriculture, it is the optimal utilization of nitrogenous manures or fertilizers for plant growth, yield, and protein content, as atmospheric nitrogen gas is not utilized by higher plants, except symbiotic legumes. The inherent efficiency of the plant to utilize available N for higher productivity needs to be tackled biologically (Abrol et al. 1999; Abdin et al. 2005). This includes uptake, assimilation, and redistribution of nitrogen within the cell and balance storage and current use at the cellular and whole plant level. Moreover, since N demand and its actual availability tend to vary in time, space, and environmental conditions, the regulation of plant nitrogen metabolism must be responsive to nutritional, metabolic, and environmental cues.

6.1 Regulation of Nitrate Uptake Plants have evolved an active, regulated, and multiphasic transport system making their NO3− uptake scheme efficient enough to transport

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sufficient NO3− to satisfy total nitrogen demand of the plant in face of varying external NO3− concentrations. Plants can also take up other forms of nitrogen, such as amino acids and ammonium ions. Root NH4+ uptake is carried out by both high-affinity and low-affinity NH4+ transporters that are encoded by a multigene family (Glass et al. 2002). However, nitrate is the most abundant form of nitrogen available to the plant roots in aerated soils. Nitrate influx is an active process driven by the H+ gradient and can work against an electrochemical potential gradient (Vidmar et al. 2000). The uptake involves high- and low-affinity transport systems, also known as HATS and LATS, respectively (Forde 2000). One of the high-affinity systems is strongly induced in presence of NO3− and is known as inducible highaffinity transport system (or iHATS), while the second high-affinity system (the cHATS) and LATS are constitutively expressed (Aslam et al. 1993; Glass and Siddiqi 1995; Forde 2002). The Km values of iHATS, cHATS, and LATS for nitrate are in the ranges of 13–79 mM, 6–20 mM, and >1 mM, respectively. The iHATS is a multicomponent system encoded partly by genes of the NRT2 family or nitrate–nitrite porter family of transporters. Recently, two dual affinity transporters have been identified in Arabidopsis, AtKUP1, and AtNRT1.1, of which the latter is induced as HATS by phosphorylation at threonine residue 101. This family of transporters is recognized as being exceptional in both the variety of different substrates which its members can mobilize (oligopeptides, amino acids, NO3−, chlorate) and in the ability of individual transporters to handle substrates of very different sizes and charges. Nitrate acts as a regulator for its own uptake, a specific property which is not seen in other ion transport systems such as phosphate, sulfate, etc. On exposure of the cells to external NO3−, the uptake capacity increases after a lag period of 0.5–1.5 h and reaches a new steady state after 4–6 h. Use of RNA and protein synthesis inhibitors provided early evidence that induction of the iHATS involves gene expression and the synthesis of new transporter protein (Aslam et al. 1993). The evidence that the inducer of iHATS is indeed

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nitrate ion and not its downstream metabolite came from NR-deficient mutants of Arabidopsis and N. plumbaginifolia (Krapp et al. 1998; Lejay et al. 1999). Studies in the last decade have shown that enhancing the uptake of N by overexpressing transporters may not necessarily improve NUE. For example, transgenic overexpression of a CHL1 cDNA (representing the constitutive HATS) driven by the cauliflower mosaic virus 35S promoter in a chl1 mutant, recovered the phenotype for the constitutive phase but not for the induced phase (Liu et al. 2003). Similarly, the NO3− contents in transgenic tobacco plants overexpressing the NpNRT2.1 gene (encoding HATS), were remarkably similar to their wildtype levels, despite an increase in the NO3− influx. These findings indicate that genetic manipulation of nitrate uptake may not necessarily lead to associated improvement in nitrate retention, utilization, or NUE, though it remains to be seen whether different plants respond differently to the overexpression of different transporters (Pathak et al. 2008). Light as an important abiotic factor is known to enhance NO3− uptake in a number of plant species and diurnal changes in nitrate uptake have been observed (Anjana et al. 2007). These changes seem to be linked to the imbalance between nitrate uptake and reduction due to the light regime and as well as to the rate of photosynthesis in shoots. Reduced nitrate uptake during darkness could be reversed by exogenous supply of sugars (Raghuram and Sopory 1995). Recent evidence on the upregulation of AtNRT1.1 gene expression by auxin (Li et al. 2007) suggests that nitrate transporters may also be regulated by hormones.

6.2

Physiology of Nitrate Reduction in Crops

A portion of the nitrate taken up is utilized/ stored in the root cells, while the rest is transported to other parts of the plant. Due to the abundant availability of photosynthetic reductants, leaf mesophyll cells are the main sites of nitrate reduction. This is initiated by the NAD/ NADP-dependent NR enzyme, which converts

nitrate to nitrite by catalytic reaction in the cytosol. Nitrite is transported into the chloroplast, where it is further reduced into ammonium ion by a ferredoxin-dependent NiR. Being the first, irreversible, and often rate-determining step of the N-assimilatory pathway, nitrate reduction has been a favorite step for physiological and biochemical approaches to optimize fertilizer N use.

6.3

Developing Plants with Transport Gene Systems Using Genetic Engineering Tools

Plants receive N from the soil in the form of nitrate or ammonia, however, some may utilize amino acid as an important sources of N. Specific transporters located in the root cell membrane are responsible for uptake of N from the soil. Subsequent to its uptake, NO3− is assimilated via a series of enzymatic steps. Nitrate reductase being the first enzyme in nitrate assimilatory pathway and thus an important gene for manipulation. NR activity in leaf blades, express either as seasonal average or converted into seasonal input of reduced N, has been related to total reduced N, grain N, and grain yield of cereals. The pattern of nitrate assimilation from different plant parts, viz. the main shoot of wheat, developing ear of wheat plants grown at different soil N levels, and in the leaf blades at different stages of growth has revealed a direct positive correlation between increasing NR activity and increasing rates of nitrogenous fertilization. Most plant tissues have the capacity to assimilate nitrate, though their NR activity varies widely. Several endogenous as well as exogenous factors have been found to influence the expression of NR genes at both translational as well as transcriptional levels. Andrews et al. (2004) reported that overexpression of either the NR or the NiR gene often affects N uptake by increasing mRNA levels in the plants. However, this does not seem to increase the growth or yield of plants, irrespective of N source. It is believed to be due, in part, to the complex regulation of both NR and the

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pathway as a whole. Transcriptional regulation of NR has only minor influence on the levels of free amino acids, ammonium, and nitrate, whereas posttranslational regulation of NR strongly affects these compounds (Lea et al. 2006). The light/dark conditions affect NR activity; heterotrophic nitrate assimilation in darkness is closely linked to the oxidative pentose phosphate pathway and the supply of glucose-6-phosphate. Under photoautotrophic conditions, glucose-6phosphate dehydrogenase is inhibited by reduction with thioredoxin in light, thus replacing the heterotrophic dark nitrate assimilatory pathway with regulatory reactions functioning in light. These studies as well as bioenergetic calculations have indicated that both yield and N harvest or protein can be increased to some extent with adequate nitrogen supply by altered management practices, thus improving the fertilizer NUE. Genotypic differences in the NR levels also provide insight in the relation of varietal differences in N assimilation. The genotypic differences in NR expression have been reported in corn, wheat, sorghum, and barley. In sorghum, a positive relationship between decline in the height of the plant and enhancement of NR activity was observed, though no such relationship was evident in tall and dwarf cultivars of wheat, T. aestivum. Wheat genotypes revealed over twofold variability in NR activity, which supports genetic findings that the enzyme level is highly heritable, its differences are reflected in N harvest and that hybrids could be bred with predictable NR levels by selecting parents appropriately. In the high NR genotypes, higher levels of NR activity were found under low N levels, often with significantly higher N concentration in the grains. They also have sustained activity at later stages of growth, such as flag leaf emergence and anthesis. The reasons for these genetic differences are not fully understood, except that the regulation operated at the level of gene expression and that low levels of NADH might limit NR activity in low NR genotypes. Similarly, overexpressing NiR genes in Arabidopsis and tobacco resulted in increased NiR transcript levels but decreased enzyme activity levels, which were attributed to posttranslational modifications.

6.4

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Glutamine Synthetase and Glutamate Synthase (GOGAT) Gene Systems

Glutamine synthetase (GS) catalyzes the critical incorporation of inorganic ammonium into glutamine. In higher plants, it is represented by two groups of protein – the cystolic and plastidic forms (Miffin and Habash 2002). Cystosolic GS (GS1) is known to be encoded by a complex multigene family, whereas plastidic GS (GS2) is encoded by a single gene. Glutamate synthase (Glutamine (amide): 2-oxoglutarate aminotransferase, GOGAT) catalyses the reductive transfer of the amide group of glutamine (produced by GS) to 2-oxoglutarate (a-keto glutarate) to form two glutamate molecules (Lea and Ireland 1999). GS/GOGAT pathway is of crucial importance since the glutamine and glutamate produced are donors of amino groups for the biosynthesis of major N-containing compounds, including amino acids, nucleotides, chlorophylls, polyamines, and alkaloids (Lea and Ireland 1999; Hirel and Lea 2001). A direct correlation was reported between an enhanced GS activity in transgenic plants in some cases, which is depicted by an increase in biomass or yield by transforming novel GS1 construct. Similarly, Kozaki and Takeba (1996) constructed transgenic tobacco plants enriched or reduced in plastidic glutamine synthetase (GS2, a key enzyme in photorespiration). Ectopic expression of GS1 has been shown to alter plant growth (Fuentes et al. 2001; Oliveira et al. 2002) and the overexpression of GS1 in transgenic plants could cause the enhancement of photosynthetic rates, higher rates of photorespiration and enhanced resistance to water stress (Fuentes et al. 2001). The overexpression of soybean cytosolic GS1 in the shoots of Lotus corniculatus was reported to accelerate plant development, leading to early senescence and premature flowering, particularly when plants were grown under conditions of high ammonium (Vincentz et al. 1993). Man et al. (2005) provided additional empirical evidence for enhanced nitrogen-assimilation efficiency in GS1 transgenic lines. However, differences in the degree of ectopic GS1 expression have been reported (Fuentes et al. 2001) and attributed to

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positional effects, effectiveness of chimeric constructs, or differences in growth conditions. This may be due to lack of correlation between the enhanced expression of GS1 and concomitant growth (Vincentz et al. 1993; Ortega et al. 2001). A significant increase in leaf area, plant area, plant height, and dry weight has been recorded in poplar trees transformed with conifer gs1a gene. Striking differences were observed at low nitrate concentration. Furthermore, higher rates at 15N incorporation into the transgenic plants demonstrate that the transformed plants have increased NUE (Man et al. 2005). Transgenic overexpression and antisense technology have been employed recently to modulate the expression of NADH-GOGAT in alfalfa and rice plants (Yamaya et al. 2002). The studies on transgenic rice plants expressing antisense RNA for either GS1 or NADH-GOGAT point towards the possible involvement of GS1 in the export of N via phloem in senescing leaves. On the other hand, in case of developing leaf blades and spikelets, NADH-GOGAT was implicated in the utilization of glutamine transported from senescing organs (Yamaya 2003). While these genes appear to be good candidates for improving NUE in the short run, the degree of improvement may vary with the crop and cropping conditions. Therefore, the utility of transgenic overexpression of N-assimilatory genes for major improvements of NUE remains uncertain, though the possibility that different crops respond differently cannot be ruled out yet.

6.5

Other Gene Systems Regulating N Metabolism and Their Manipulation

Enzymes like asparagine synthetase (AS), that catalyzes the formation of asparagine (Asn) and glutamate from glutamine (Gln) and aspartate. In higher plants, AS is encoded by a small gene family (Lam et al. 1998). Together with GS, AS is believed to play a crucial role in primary N metabolism. The observation made by Carvalho et al. (2003) that the levels of AS transcripts and polypeptides in the transgenic nodules of

Medicago truncatula increase when GS is reduced suggests that AS can compensate for the reduced GS ammonium assimilatory activity. However, it was also demonstrated that GS activity is essential for maintaining the higher level of AS. Thus, GS is required to synthesize enough Gln to support Asp biosynthesis via NADH-GOGAT and AspAT (Carvalho et al. 2003). A reduction in GS activity in transgenic Lotus japonicas is also correlated with an increase in Asn content (Harrison et al. 2007), supporting the hypothesis that when GS becomes limiting, AS may be important in controlling the flux of reduced N into plants. With the aim of increasing Asn production in plants and to study the role of AS, several researches have attempted to clone AS genes and to examine the corresponding gene expression in plants. Lam et al. (2009) showed overexpression of the ASN1 gene in Arabidopsis and demonstrated that the transgenic plants have enhanced soluble seed protein content, enhanced total protein content, and better growth on N-limiting medium. Arabidopsis plants overexpressing the ASN2 gene accumulate less endogenous ammonium than wild-type plants when grown on medium containing 50-mM ammonium. This study indicates that signaling processes may provide an attractive route for metabolic engineering. In comparison to GS/GOGAT enzymes, the physiological role of glutamate dehydrogenase (GDH) has been less clear (Dubois et al. 2003). In an attempt to investigate the role of GDH by expressing a bacterial gdhA gene from E. coli in tobacco, Ameziane et al. (2000) found that biomass production is consistently increased in gdhA transgenics, regardless of whether they are grown under controlled conditions or in the field.

7

Signaling and Regulation of Nitrogen Metabolism

It is a well-known concept in signal transduction that whenever multiple genes are subject to transcriptional regulation by a common signal, it is mediated through a regulatory sequence that exists in all the genes that respond to the signal.

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These signature sequences, commonly known as response elements, are identified by mutations that abolish their function, and their conserved nature as revealed by hom*ology comparisons. Early experiments in transgenic Nicotiana plants using GUS gene fused to NR and NiR promoter sequences clearly demonstrated for the first time that nitrate induction of gene expression requires some sequence(s) associated with the NR and NiR promoters (Raghuram et al. 2006). Subsequent studies in transgenic tobacco incorporating the 5¢ flanking regions of the nitrate reductase genes NR1 and NR2 (designated NP1 and NP2), in case of Arabidopsis thaliana, demonstrated that 238 and 330 bp of NP1and NP2, respectively, are sufficient for nitrate-dependent transcription (Lin and Demain 2006). These nitrate-responsive elements (NREs) are composed of several copies of a core A[G/C]TCA sequence motif preceded by an ~7-bp AT-rich sequence present in the 5¢ flanking regions of nitrate reductase (NR1 and NR2) genes. This particular sequence motif was also found to be very well conserved in the 5¢ flanking regions of NR and NiR genes from eight other plants. Sarkar (2003) compared the flanking sequences of all available plant nitrate-responsive genes and found that the NRE core sequence (A[C/G] TCA) was present in multiple copies on both strands in all the known nitrate-responsive genes in many dicots, monocots, and cyanobacteria. Though most of the NREs examined contained both the core sequence and a proceeding AT-rich sequence, there were some cases which had GC-rich regions or did not reveal any AT/GC bias. A more detailed bioinformatic analysis of the entire Arabidopsis genome in our lab revealed that the proposed NREs are randomly distributed, with no difference between nitrate-responsive genes and the presumably nonresponsive genes and intergenic regions in the rest of the genome (Raghuram et al. 2006). These findings raise doubts on the validity of the proposed NRE as comprising of (A[C/G] TCA) elements preceded by AT-rich sequence. Further work in this area will need a combination of bioinformatic and experimental approaches to redefine the NREs that mediate the expression of all

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nitrate-responsive genes in all plants. The discovery of NREs is important, as it provides an end point for nitrate signal transduction.

8

QTL Approach to NUE

NUE in plants is a complex quantitative trait that depends on a number of internal and external factors in addition to soil nitrogen availability, such as photosynthetic carbon fixation to provide precursors required for amino acid biosynthesis or respiration to provide energy. Although this trait is controlled by a large number of loci acting individually or together, depending on nutritional, environmental, and plant developmental conditions, it is possible to find enough phenotypic and genotypic variability to partially understand the genetic basis of NUE and thus identify some of the key components of yield for markerassisted breeding. Thus the development of molecular markers has facilitated the evaluation of the inheritance of NUE using specific quantitative trait loci (QTLs) that could be identified. In maize, Hirel et al. (2001) and Masclaux et al. (2001) analyzed recombinant inbred lines for physiological traits such as nitrate content, NR and GS activities. When the variation in these traits and yield components were compared, it was found that there was a positive correlation between nitrate content, GS activity, and yield. When the loci that govern quantitative traits were determined on the map of the maize genome, the positions of QTLs for yield components and the locations of the genes for cytosolic GS (GS1) coincided. In maize, studies on different genotypes or populations of recombinant inbred lines based on NUE components, chromosomal regions, and putative candidate genes have hinted at some factors that might control yield and its components directly or indirectly, when the amount of N fertilizers provided to the plant is varied (Hirel et al. 2007). Similar results were obtained in rice by Obara et al. (2001), confirming the earlier indications that the GS1 enzymatic activity in the leaf cytosol is one of the major steps controlling organic matter reallocation from source to sink organs

K.R. Hakeem et al.

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during senescence and for grain-filling in cereals. Previous studies have already demonstrated that when GS1 is over expressed in Lotus, nitrogen remobilization was prematurely induced leading to early senescence of the plant (Vincentz et al. 1993). In rice (Yamaya et al. 2002) and wheat (Habash et al. 2001), preliminary investigations with enhanced or decreased GS1 activity indicated that grain yield and grain nitrogen content were modified. In other species such as tobacco (Migge et al. 2000) or poplar (Gallardo et al. 1999), overexpression of GS2 or GS1 significantly increased plant biomass production at early stages of plant development. With these experiments, two out of seven QTLs for GS1 protein content were detected in different regions from other physiological and biological traits. In maize, QTLs for the activities of acid-soluble invertase and sucrosephosphate-synthase were detected in the regions where each structural gene was mapped (Ishimaru et al. 2001). Thus, quantitative studies of genetic variability for NUE using molecular markers and combining agronomic and physiological studies will be increasingly used in the future to identify new genes or loci involved in the regulation of these metabolic pathways and their interconnection with carbon assimilation and recycling and to select genotypes that assimilate or remobilize nitrogen more efficiently.

9

Proteomics Approach to NUE

The ability of crop plants to cope up with the variety of environmental stresses depends upon a number of changes in their proteins, which may be up- and downregulated as a result of altered gene expression. Under a stressful condition, the modifications in the expression levels of these proteins could provide us valuable information about the nature of stress factor as well as the physiological and molecular state of a biological system. Hence, provides us some clues to understand the nature of defensive mechanism and adaptability, besides stress monitoring in these biological systems.

Proteomic-based technologies have been recently applied for the systematic analysis of the induced gene products in a number of plant species subjugated to a wide range of abiotic and biotic challenges. Proteome analysis is becoming a powerful tool in the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up new perspectives to analyze the complex functions of model plants and crop species at different levels. Improvements in proteomic technology regarding protein separation and detection, as well as mass spectrometry-based protein identification, have an increasing impact on the study of plant responses to salinity stress (Parker et al. 2006; Qureshi et al. 2007; Caruso et al. 2008). Proteomics has provided valuable information in various fields of plant biology. Construction of several plant protein databases is in progress for Arabidopsis, rice, maize, and some trees, where different genetic, cellular, and physiological information is available, such as expression in various organs or tissues, response to treatments, cellular localization, and genetic bases (Thiellement et al. 1999). Recent advances in MS techniques will facilitate protein identification so that in the future this will not be a limiting factor in the interpretation of variations detected on 2D gels. By providing information on affected and unaffected proteins, large-scale protein identification will simplify determining the consequences of mutations, plant transformation, or natural polymorphism for plant metabolism, as well as interpreting the effects of protein changes on development, or in response to biotic and abiotic stress. Studies in Saccharomyces cerevisiae, for which hundreds of proteins have been identified, show the power of the proteomic approach in the study of the regulation of metabolic pathways. Schiltz et al. (2005) studied that during seed filling, the accumulation of proteins in the seeds relies on the nitrogen supply from the mother plant, and a proteomic approach was used to study the mobilization of proteins from the leaves to the filling seeds in pea. Two contrasting N-responsive wheat varieties have differential expressions of root as well as leaf

10 Reactive Nitrogen Inflows and Nitrogen Use Efficiency in Agriculture¼

proteins when grown under controlled conditions at different N levels (Bahrman et al. 2004, 2005). These proteins were grouped into two categories, one involved in carbon metabolism and the other associated with other pathways and functions like thiol-specific antioxidant proteins, etc. This study revealed that levels of gene expressions are modified with the varying levels of nitrate supply, even if only a few polypeptides appear, disappear, or change. Sarry et al. (2006) have demonstrated the protein level changes associated with nitrogen and sulfur metabolism, and their interaction. With the help of high throughput proteomic tools, they were able to detect various enzymes including ATP sulfurylase, sulfite reductase, cysteine synthase, S adenosylmethionine synthase, glutamine synthase, aspartate aminotransferase, GDH, etc., involved directly or indirectly in S and N metabolism. Recently a study for the detection of low nitrogen-responsive proteins in cultivated rice species was done by Kim et al. (2009). Studies at constructing 2-D gel reference map for use in comparative proteomics among cultivars for N-responsive proteins might provide an insight for precise identification of potential molecular protein markers to assist the breeders for screening N-efficient genotypes and help in understanding how crop adapts to low N availability. Correlations between the level of expression and NUE might bring information on the possible role of the genes involved in nitrogen metabolism.

10

Conclusion and Future Perspectives

Present review provides an overview of plant nutriomics, which is still at a conceptual stage. Although considerable efforts are in progress with the aim at enhancing plant nutrient efficiency through molecular and genetic approaches. We have focused here largely on nitrogen with which we have been working on along molecular biology lines. Crop response to N and NUE is very low in developing countries including India. Use of NIs and slow-release nitrogen fertilizers and efficient crop and fertilizer management can significantly increase NUE. It is clearly evident

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that optimizing the plants, NUE goes beyond the primary process of uptake and reduction of nitrate, involving quality of events, including metabolite partitioning, secondary remobilization, C–N interactions, as well as signaling pathways and regulatory controls outside the metabolic cascades. Despite the various attempts to manipulate each of the above steps in some plant or the other, we are far from finding a universal switch that controls NUE in all plants. However, transgenic studies, QTL, and proteomics approaches seem to increasingly suggest that the enzymes of secondary ammonia remobilization are better targets for manipulation, followed by regulatory processes that control N–C flux, rather than the individual genes/enzymes of primary nitrate assimilation. There is an urgent need of large-scale, co-ordinated research on plant nutriomics, involving sincere efforts from both national and international researchers to develop the nutrient-efficient, high-yielding, and stresstolerant genotypes/varieties that will contribute to both environmental safety as well as food security worldwide.

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Arbuscular Mycorrhizal Symbiosis and Other Plant– Soil Interactions in Relation to Environmental Stress

11

Patrick Audet

Abstract

In this chapter, focused on the arbuscular mycorrhizal (AM) fungi and their mostly mutualistic association with the vast majority of herbaceous plant species, we examine the cellular, molecular, and physiological mechanisms by which the mycorrhizal symbiosis can enhance plant stress tolerance in relation to a number of abiotic environmental stressors, such as macro- and micronutrient deficiency, drought, and metal toxicity. Overall, the primary mechanisms of interaction discussed here include: (1) the enhanced uptake of macro- and micronutrients and water; and (2) the stabilization of the soil architecture via mycorrhizal-enhanced soil aggregation and metal biosorption processes. A key facet of this analysis involves the identification of direct vs. indirect benefits of interactions, and their distinctive impacts toward plant development as well as the proximal growth environment. Accordingly, due to the significant and widespread effects of these direct and indirect processes toward plant physiological and soil ecological function, it is suggested that the mycorrhizal symbiosis should constitute an extrinsic stress tolerance strategy that could complement the inherent resistance mechanisms of plants when subjected to an array of potential stressors, and also buffer the growth environment. For this reason, it is recommended that future studies take into account such multitrophic interactions (e.g., above- and belowground relationships) to better depict physiological and ecological phenomena in relation to environmental stress. Keywords

Mutualism • Macro- and micronutrients • Drought • Soil stabilization

P. Audet () Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Brisbane, QLD 4072, Australia e-mail: [emailprotected] P. Ahmad and M.N.V. Prasad (eds.), Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change, DOI 10.1007/978-1-4614-0815-4_11, © Springer Science+Business Media, LLC 2012

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P. Audet

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1

Introduction

1.1

Intrinsic vs. Extrinsic Stress Tolerance

In the field of plant physiology, environmental stress (or strain) refers to a state or event causing a deviation in plant growth relative to its optimal

development (Larcher 1987, 2003). As shown in other chapters of this volume, potential environmental stress factors are many (e.g., radiation, temperature, water, nutrient availability) and often grouped according to their abiotic or biotic origins (Fig. 11.1). From this definition, such stressors can be either localized (root vs. shoot predation) or systemic (heat shock, frost), and typically incur a complex cascade of physiological effects ranging

Environmental Stress Factors

Abiotic Radiation Deficiency Excess UV Temperature Heat Cold Frost Water Dry air Dry soil Flooding Gases Oxygen deficiency Volcanic gases Minerals Deficiency Excess (Toxicity) Imbalances Salinity Acidity Alkalinity

Biotic Plants Crowding Allelopathy Parasitic plants Microorganisms Viruses Bacteria Fungi Predation disturbances Grazing Trampling Anthrophogenic activity Industrial pollution Agrochemicals Soil compaction Fire Ionization radiation Electromagnetic fields

Mechanical disturbances Wind Sulifluction Burial Snow cover Ice sheets

Fig. 11.1 Abiotic and biotic environmental stress factors (adapted from Marschner 1995)

11

AM Symbiosis and Other Plant–Soil Interactions in Relation…

from temporary (reversible) to permanent (irreversible) adaptive responses depending on the relative intensity of the given stress factor. In order to increase their survivorship and reproductive success, plants have developed a remarkable array of stress tolerance (endurance), resistance (acclimation), and (or) avoidance (prevention) mechanisms to circumvent a number of environmental challenges and avoid any permanent associatedstress injuries. Further to these more conventional descriptors of adaptive stress tolerance (e.g., tolerance, resistance, and avoidance), two alternative descriptors have been proposed relating to plant investment (or resource and energy allocation) in either intrinsic (e.g., metabolic) or extrinsic (e.g., symbiotic) tolerance strategies1 (Audet and Charest 2007b, 2008). Here, intrinsic stress tolerance refers to plant investment in inherent (or built-in) metabolic systems that are inducible when subjected to stress. For example, the production of metallothioneins which bind ion-free radicals to prevent metal-induced cellular oxidative stress (Chaps. 9, 20, and 21), the production of secondary metabolites to thwart herbivores (Howe and Jander 2008; Mole 1994), the dispatch of heat shock proteins to prevent enzyme denaturing under temperature extremes (Chaps. 5–7), or the production of anti-microbial proteins to inhibit viral, fungal, and (or) bacterial pathogens (Dangl and Jones 2001; Fritig et al. 1998; Ganz and Lehrer 1999), to name just a few. More broadly, intrinsic stress tolerance can also include constitutive systems such as the processes of cell lignification, the development of trichomes and glandular hairs, or the exudation of resins and waxes which, together, offers mechanical defenses to some of these environmental stressors (Bhuiyan et al. 2009;

1

In their review of “Heavy metal tolerance in plants,” Antonovics et al. (1971), and later Baker and Walker (1990), were first to allude to and distinguish between internal and external tolerance mechanisms. Similarly to the investment of plant resources toward intrinsic versus extrinsic strategies proposed here, the internal and external tolerance mechanisms suggested by Antonovics et al. refer to central (e.g., metabolic) and peripheral (e.g., rhizospheric) processes, respectively, which can impact plant development when faced with critical metal toxicity conditions.

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Duke 1994; Langenheim 2003; Wagner 1991). By contrast, extrinsic stress tolerance refers to plant investment in external systems, particularly symbiotic mutualism, to circumvent environmental stress. In this regard, the symbiotic mutualism encompasses an intimately co-operative relationship between two different species (referred to as symbionts) contributing to the mutual benefit of both individuals (Leung and Poulin 2008). Notably, the balance between the benefits of association among the symbionts is critical for defining the symbiotic mutualism since symbiotic relationships are believed to function along a continuum ranging from parasitism to mutualisms (Boucher et al. 1982; Bronstein 2001; Johnson et al. 1997). From this definition, it can be argued that plants have developed the widest assortment of mutualism in the natural world, whereby host plants typically exchange essential resources (e.g., plant carbohydrates, soil nutrients) and (or) ecological services (e.g., pollination services, shelter) with individuals from another species to reciprocally enhance their tolerance to environmental stressors and thereby increase their survivorship (Boucher 1988; Boucher et al. 1982). Examples of plant mutualism include plant–pollinator interactions with insects, birds, and mammals to ensure plant fertilization and sexual reproduction (Rønsted et al. 2005, 2008; Wiebes 1979), plant– fungus (mycorrhizal) interactions to increase the root system’s resource acquisition capacity (Douds and Johnson 2007; Marschner and Dell 1994), and plant–rhizobial interactions for the fixation of inorganic soil nitrogen (Long 1996, 2001; Young and Johnston 1989). Among these interactions, the mycorrhizal symbiosis is considered to be one of the most widespread and well-studied ecological associations having key implications at the scale of plant physiological and whole-ecosystem function. In this chapter, focused on the arbuscular mycorrhizal (AM) fungi and their symbiotic association with herbaceous plant species, we examine how some plants invest in mycorrhizal symbiosis as an extrinsic stress tolerance strategy in relation to a number of abiotic environmental stressors, such as macro- and micronutrient deficiency, drought, and metal toxicity. It is also suggested that such an investment can contribute

P. Audet

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Fig. 11.2 Defining the mycorrhizosphere and its zone of influence (adapted from Beare et al. 1995)

in shaping plant development by influencing edaphic conditions within the proximal growth environment.

1.2

Mycorrhizae and the AM Symbiosis

Aptly referred to as “fungus roots” (Frank 1885), the mycorrhizae are mostly nonpathogenic soil fungi living intimately with terrestrial plant roots that, together, form a symbiotic mutualism characterized by the direct exchange of plant carbohydrates for soil resources, such as mineral nutrients and water (Allen 1991). The mycorrhizae are ubiquitous organisms having adapted to and successfully colonized nearly all known terrestrial ecosystems by forming symbiotic associations with the broad majority of all plant families (Peterson et al. 2004). For this reason, the mycorrhizal fungi are classified into three primary assemblages depending on their respective morphologies and specific plant hosts: the ectomycorrhizae (primarily associated with Pinaceae, fa*gaceae, Betulaceae, and Salicaceae species), the endomycorrhizae (associated with the majority of angiosperms and some gymnosperms), and the ectendomycorrhizae (primarily associated with

Orchidaceae and Ericaceae species). A common feature among all mycorrhizae is the development of the mycorrhizosphere2 (Fig. 11.2) consisting in the combined zones of influence of the roots (rhizosphere) and extraradical hyphae (hyphosphere), and encompassing a highly active and multilateral interface between the host plants, mycorrhizal fungi, and proximal soil environment (Duponnois et al. 2008; Garbaye 1991). In line with the notion of plant investment in extrinsic systems for the purpose of stress tolerance, plant investment toward the development of the mycorrhizospheric network involves a considerable plant carbon allocation (occasionally representing up to and possibly well over 20% of the plant’s total carbon budget) which is required for

2

Fitzpatrick’s (1984) characterization of the micromorphology of soils indicates that the pedosphere (e.g., soil realm) is constituted of four essential “spheres”: atmosphere (e.g., soil air), biosphere (e.g., litter and microorganisms), lithosphere (e.g., rocks and minerals), and hydrosphere (e.g., soil water). Accordingly, Beare et al. (1995) have subclassified arenas of interaction to identify further interfaces within the pedospheric framework, such as the drilosphere (e.g., worm castings), detritusphere (e.g., saprotrophs), rhizosphere (e.g., plant roots), mycorrhizosphere (e.g., combined roots and extraradical hyphae), etc.

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

actively sustaining the symbiotic infrastructure and maintaining the functional viability of the mycorrhizal symbiont (Douds et al. 2000; Tinker et al. 1994). In exchange, this extrinsic investment provides the host plant with a number of ecological services typically pertaining to the enhancement of the plant’s resource acquisition capability and the stabilization of the proximal soil environment. Falling within the grouping of the endomycorrhizae, the AM fungi and their symbiosis with herbaceous plants are particularly well studied in the field of plant physiology and mycology, and widely recognized for benefiting host plants when subjected to various environmental stressors (Table 11.1). Having originated an estimated 450 million years ago, the AM fungi comprise species of the Glomeromycota phylum which are believed to form associations with up to 90% of all herbaceous plants (Redecker et al. 2000; Remy et al. 1994; Schüßler et al. 2001). As characterized by a unique morphology consisting of intra- and extraradical hyphae, arbuscules, vesicles, and spores (Fig. 11.3), the AM fungi behave in a peculiar manner compared to other mycorrhizal phyla since they penetrate between the cortical cells of vascular plant roots in order to develop an intracellular exchange network (Garcia-Garrido et al. 2009): a behavior believed to be rooted in ancient parasitic origins (Purin and Rillig 2008). Following reciprocal signaling processes between the symbionts resulting in the successful colonization of host roots (Harrison 1999, 2005; Vierheilig and Piché 2002), the intraradical hyphae proliferate within the root architecture to interact with roots cells across a slender periarbuscular zone formed between the fungal arbuscular structures and plant cell membranes. It is across the periarbuscular zone where soil resources (e.g., phosphorus, nitrogen, or mineral nutrients) and plant carbohydrates (e.g., glucose, hexose) are actively exchanged between the symbionts (Hahn and Mendgen 2001). Meanwhile, the extraradical hyphae typically scavenge beyond the root depletion zone to form an expansive mycorrhizospheric network, thereby increasing the host roots’ resource acquisition capabilities and zone of influence compared to the rhizosphere

237

alone (Koide 1991, 2000; Koide and Elliott 1989). With the development of this active and bidirectional symbiotic exchange network, the AM fungi then shift their developmental allocation to the production of extraradical spores and vesicles which are involved, respectively, in fungal reproduction and lipid storage (Bago et al. 2000; Dalpé et al. 2005). Under these circ*mstances, the AM fungi are generally considered to be “true” mutualists due to their host obligate status which requires that they maintain an active symbiosis in order to ensure an influx of plant carbon allocations for the completion their life cycle (Johnson et al. 1997; Jones and Smith 2004). As stated previously, numerous advances have been made over the past decades demonstrating the beneficial role of the AM fungi in plant physiology and soil ecology; this being attributed especially to the dynamic function of the mycorrhizosphere in relation to various edaphic processes (Fig. 11.4). In order to accurately depict such dynamic interactions, the classification of the mycorrhizospheric processes presented here distinguishes specifically between two types of interaction depending on the nature of the benefits of association being either direct or indirect. In the present context, the direct benefits of interaction refer to processes that directly enhance the plant health status as mediated by the dynamics of bidirectional exchange between the symbionts described above (Cushman and Beattie 1991; Schwartz and Hoeksema 1998). For example, the process of mycorrhizal enhanced uptake in which the extraradical hyphae increase the uptake of limiting soil resources in exchange for plant carbohydrates, then enabling the host plant to supplement its nutrient status when subjected to deficiency conditions. Alternatively and occasionally overlooked from a plant physiological perspective, the indirect benefits3 of interaction refer to processes that indirectly enhance the plant growth or survival status by altering the proximal growth environment thereby providing

3

The notion of “indirect benefits” derived from mutualism has previously been used within the context of species community structure.

a

Mycorrhizal exudation

Soil structure stabilization

Enhanced resource acquisition capability

Denotes review publications

Indirect benefit

Mechanism Direct benefit

Metal bioavailability

Soil microbia

Soil retention capacity

Metal bioavailability

Target Essential soil resources (nonmetals, metals, and water)

Promotion of beneficial bacteria (i.e., nitrogen fixation), competitive exclusion of soil pathogens Precipitation of metal–ligands, modulation of soil pH

Metal binding due to negatively charged surface constituents of extraradical hyphae (e.g., carboxyls, hydroxides, oxy-hydroxides, sulfhydryls); Reduction of plant metal uptake to delay phytotoxicity, particularly at high soil exposure levels (e.g., Zn Pb, Cd, Ni) Enhanced soil aggregation properties; increased water and nutrient retention capacity, decrease of nutrient leaching

Mobilization and uptake of trace essential elements having low bioavailability (e.g., Zn, Ni, Co, Cu, Mn, Fe) particularly under nutrient-deficiency conditions Enhanced water use efficiency and drought recovery

Description Preferential uptake of nitrogen (NO3/NH4) and phosphorus (Pi)

Table 11.1 Summary of the impact of AM symbiosis on plant physiology and soil ecology

Augé et al. (2001), Augé (2001)a, Bearden (2001), Bearden and Petersen (2000), Miller and Jastrow (1990) Chapman et al. (2005)a, Barea et al. (1998)a, and Johanson et al. (2003)a Leyval et al. (1997)a, Galli et al. (1994)a, and Gadd (1993)a

Augé et al. (2001), Augé (2001)a, and Montaño et al. (2007)a Leyval et al. (1997)a, Galli et al. (1994)a, Gadd (1993)a, Meharg (2003)a, and González-Guerrero et al. (2009)a

Reference Bolan (1991)a, Chapman et al. (2006)a, Schachtman et al. (1998)a, Smith et al. (2003)a, and George et al. (1995)a Jeffries et al. (2003)a, Koide (1991)a, and Marschner and Dell (1994)a

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

239

Fig. 11.3 Defining structures of an arbuscular mycorrhizal fungus (Glomus intraradices Schenck & Smith) in association with Ri T-DNA carrot roots (Daucus carota L.) grown under aseptic conditions. Shown from (a) to (e) are: vesicles

(Ve), arbuscules (Ar), host roots (HR), spores (Sp), spore clusters (Sc), extraradical hyphae (Eh), and intraradical hyphae (Ih). Roots are stained with an aniline blue 0.02% dye solution and observed under a compound microscrope

more favorable developmental conditions (Bertness and Callaway 1994; Stachowicz 2001; Müller and Krauss 2005). For instance, the process of mycorrhizospheric-enhanced soil aggregation which contributes in stabilizing the proximal growth environment to increase its resource retention capacity. A key aspect of the indirect benefits of interaction is the notion that such processes can benefit host plants as well as

nonassociated species within the mycorrhizosphere’s zone of influence, unlike the direct benefits which suggest an intimate exchange occurring exclusively between the symbionts. By distinguishing between the direct and indirect benefits of interaction, it is intriguing that a combination of such AM-induced mycorrhizospheric processes can complement many intrinsic tolerance mechanisms when subjected to a broad

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Mycorrhizosphere Metal Biosorption and Precipitation of Metal-Ligands

Root Mucilage

240

Enhanced Resource Acquisition

Host Root Hyphal Exudation

+

Extraradical Hyphae

-

+

-

Soil-pH Modulation

+

-

+ -

Soil Matrix Stabilization

Soil Microbial Enrichment

Bulk Soil Fig. 11.4 Summary of potential mycorrhizospheric interactions

range of environmental conditions and abiotic stress. For this reason, it is considered that these processes likely play a key role in enhancing plant stress tolerance, as well as shaping the proximal growth environment to increase the soil’s resilience, in relation to a number of potential ecological stressors.

2

Direct Benefits of Association

2.1

Macro- and Micronutrient Uptake

2.1.1 N Acquisition Plant productivity in temperate agro-ecosystems is most commonly limited by soil nitrogen (N) bioavailability (Vitousek and Howarth 1991; Chapin et al. 2002). Due to its principal role in protein biosynthesis and nucleic acid metabolism, N deficiency typically results in stunted

plant growth and increased leaf senescence thereby detrimentally affecting the plant’s photosynthetic potential and overall growth yield. A particular environmental challenge regarding plant N assimilation exists with regard to the source of N in soils, whether it is in the form of nitrate (NO3−) which is readily assimilated in roots and (or) shoots but easily leached from the rhizosphere or, instead, in the form of ammonium (NH4+) which can be more abundant than the former but requires detoxification prior to its assimilation (Gutschick 1981; Bloom 1997). The primary benefit of mycorrhizal associations pertains to the increase in belowground surface area (e.g., roots and extraradical hyphae) which enhances the host plant’s soil resource acquisition capability compared to the rhizosphere alone. In this regard, two complementary mechanisms describing the role of AM fungi in plant N assimilation have been presented suggesting that the

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

241

NH4+

HXT1 ???

Intraradical Hyphal Carbon Pool

Exudation of Organic Chelators

Amino Acid Synthesis Ornithine aminotransferase

Coenocytic Channel

NH4+ AMT Mep Rh Urease

Amino Acid Synthesis

Extraradical Hyphal Carbon Pool

Ornithine Urea

Arginine

Nitrate reductase

Peri-Arbuscular Interface

Hexose

NO3-

NO3AMT Mep Rh

NH4+ Bidirectional Transfer

NH4+

Glutamine

Arginine

GS-GOGAT Cycle

Intraradical Mycelium

Extraradical Mycelium

Host Root

Soil Environment

Fig. 11.5 AM fungal nitrogen uptake and transfer pathway (adapted from Govindarajulu et al. 2005; modified according to Jin et al. 2005, Chalot et al. 2006, and Cruz et al. 2007). Bidirectional transfer is indicated by solid and dashed lines. Refer to text for abbreviations

mycorrhizosphere contributes first by generally increasing the plant N acquisition capability (both NO3− and NH4+ sources), and second by specifically increasing the uptake of NH4+ as a result of fungal pre-assimilation and detoxification processes (Chapman et al. 2005; Marschner and Dell 1994). Together, these mechanisms are believed to increase the bioavailability of N within and often beyond the rhizosphere’s resource depletion zone to improve plant stress tolerance when subjected to N-deficiency conditions. Among others, Haystead et al. (1988), Faure et al. (1998), and Subramanian and Charest (1997, 1998, 1999) investigated these hypotheses using greenhouse experimental systems in the objective of assessing the nutritional status of plants in relation to the amendment of NO3− and NH4+ fertilizers, and then comparing the N assimilation pathways of AM vs. non-AM plants by measuring the activity of key assimilation enzymes: namely, nitrate reductase (NR), glutamine synthase (GS), glutamate dehydrogenase

(GDH), and glutamate synthase (GOGAT). Collectively, these studies have shown that AM-colonized rye grasses (Lolium perenne), “field” clover (Trifolium repens), and maize (Zea mays) all gained considerable increases in total nitrogen uptake leading to an overall greater amino acid composition compared to non-AM plants. Notably, these physiological effects coincided with increases in the activity of NR, GDH, and GS-GOGAT assimilation enzymes measured in the AM roots and shoots. As predicted, the AM–plant nutritional status was supplemented by increasing the overall uptake of N, especially by increasing the uptake of its less labile form, [NH4+]. More recently, the studies of Toussaint et al. (2004), Govindarajulu et al. (2005), Jin et al. 2005; Chalot et al. (2006), and Cruz et al. (2007) have corroborated these general findings using in vitro culture tools to further characterize the AM fungal uptake, assimilation, and translocation pathways as soil-N travels from the extraradical hyphae to the host root (Fig. 11.5).

242

Accordingly, it has been elucidated that NO3− and NH4+ are actively taken up by extraradical hyphae via transporters of the AMT/Mep/Rh protein superfamily (Khademi et al. 2004). Having reached the cytosol of the extraradical mycelium, NO3− and NH4+ are converted to glutamine and then arginine via the fungal NR, GDH, and GS-GOGAT enzyme cycles. In this form, arginine travels to the intraradical mycelium through cytoplasmic streaming via coenocytic channels to be further broken down through the action of ornithine aminotransferase and urease, thereby releasing ornithine, urea, and ultimately NH4+. Finally, the NH4+ is either catabolized via AM fungal amino acid synthesis or transferred to roots across the periarbuscular interface apparently via ammonium transport proteins. To complete the bidirectional exchange between the symbionts, plant carbohydrates in the form of hexose are transferred to the AM fungus via putative transporters (HXT1, potentially among others – Hahn and Mendgen 2001) and likely mediated via plasma membrane H+-ATPases (GmHA1-5 – Ferrol et al. 2000). Once in the fungal cytosol, the hexose is converted to trehalose, glycogen, and glucose for usage in various fungal metabolisms. As such, the characterization of the AM nitrogen uptake pathway provides key evidence as to the active role of AM fungi in supplementing the plant N nutritional status in the enhancement of plant nutrient stress tolerance.

2.1.2 P Acquisition After soil-N, plant productivity in temperate agroecosystems is limited by phosphorus (P) bioavailability4: a key bioenergetic constituent (e.g., ATP) and cellular structural component (e.g., phospholipids, DNA, RNA). In this regard, P deficiency is prevalent in areas of high rainfall due to extensive 4

Unlike temperate environments, plant productivity in tropical agro-ecosystems is primarily limited by phosphorus bioavailability followed by less labile soil micronutrients due to their slow diffusion rates and subsequently low bioavailability to plants. In addition, the high rates of plant photosynthesis and evapo-transpiration in this ecosystem typically cause the bioavailable nutrient pool to be rapidly assimilated (Brams 1973; Baligar and Bennett 1986; Ewel 1986).

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nutrient leaching and (or) acidic soil conditions conducive to reciprocal antagonisms (also known as phosphorus-induced micronutrient deficiencies) which can cause leaf chlorosis and stunted growth (Cleveland et al. 2002; Mengel and Kirkby 2001). Similarly to the case of N assimilation described above, it has been hypothesized that the AM fungi hold a significant role in plant P acquisition by increasing the plant’s soil resource uptake capacity, particularly by enhancing the uptake of phosphates and inorganic P which typically have slow soil diffusion rates (Marschner 1995; Picone et al. 2003; Saito 2000). In this regard, these physiological mechanisms are among the most thoroughly investigated in the study of the AM symbiosis and have been well reviewed by Bolan (1991), Koide (1991), George et al. (1995), Schachtman et al. (1998), and Smith et al. (2003). The consensus from these studies is that host plants benefit from an investment in AM symbiosis for the supplementation of their P nutritional status, especially when subjected to soil-P deficiency, due to the expansive mycorrhizosphere’s ability to increase soil–surface contact and reduce soil–P diffusion distances. Consistent with the notion of plant investment in extrinsic systems to circumvent environmental stress, the mycorrhizal investment is often inversely correlated with the bioavailability of soil–P such that AM root colonization and symbiotic activity are believed to be highest under low P conditions (Smith et al. 2003, 2004). Consequently, this relationship would suggest that P bioavailability is a key factor dictating the plant’s relative symbiotic investment (or mycorrhizal responsiveness) in order to maximize the reciprocal benefits of association (Graham et al. 1991; Janos 2007; Tawaraya 2003). Under such environmental conditions, soil–P can be actively taken up by the extraradical hyphae and efficiently transferred to host roots (Fig. 11.6 – Schachtman et al. 1998; Smith et al. 2003; Javot et al. 2007). This process is characterized by the activity of extraradical hyphae which increase the solubility of soil-P due to the exudation of organic chelators to then facilitate the uptake of both organic (Porg) and inorganic (Pi) forms of P. Coinciding with these events, a number of AM fungal phosphate

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

243

Hexose

H2PO4-

HXT1 ???

Intraradical Hyphal Carbon Pool

Peri-Arbuscular Interface

ATP, DNA, RNA Synthesis

Extraradical Hyphal Carbon Pool Exudation of Organic Chelators

ATP, DNA, RNA Synthesis

Coenocytic Channel

Pi

Poly-P Polyphosphatase

HPO4-2 HvPT1-8 LePT11-5 LjPT1-3 MtPT1/2/4

NtPT1-4 OsPT1-13 StPT1-5 ZmPT11-6

Pi

GiPT GvPT GmosPT

Bidirectional Transfer

Porg

Porg

Poly-P Polyphophate gluco*kinase

Intraradical Mycelium

Extraradical Mycelium

Host Root

Soil Environment

Fig. 11.6 AM fungal phosphorus uptake and transfer pathway (adapted from Schachtman et al. 1998; modified according to Smith et al. 2003 and Javot et al. 2007). Bidirectional transfer is indicated by solid and dashed lines. Refer to text for abbreviations

transporters have been isolated from the mycelium of a number of AM fungi and found to be upregulated under P-deficiency conditions, such as: Glomus intraradices (GiPT – MaldonadoMendoza et al. 2001), G. mosseae (GmosPT – Benedetto et al. 2005), and G. versiforme (GvPT – Harrison and van Buuren 1995). Once taken up into the cytosol of the extraradical mycelium, the Porg and Pi are converted to polyphosphate complexes (poly-P – e.g., glucose-6-phosphate) through the enzymatic activity of polyphosphate gluco*kinase (Capaccio and Callow 1982; Cox et al. 1980). In this more stable cytosolic form, the poly-P complexes are either stored in fungal vacuoles or transferred to the intraradical mycelium through cytoplasmic streaming via coenocytic channels: a process which is putatively linked with H+-ATPase co-transport (Gauthier and Turpin 1994). Here, the polyphosphate complexes can be broken down by polyphosphatases for use in AM fungal ATP, DNA, RNA, and

(or) phospholipid syntheses, or transferred to the host plant across species-specific phosphate transporters in exchange for plant carbohydrates in the form of hexose. To date, advanced molecular analyses have identified an array of phosphate transporters (Table 11.2) isolated especially in the periarbuscular interface region of various model study organisms, such as barley (Hordeum vulgare), deervetches (Lotus japonica), tomato (Lycopersicon esculentum), alfalfa (Medicago truncatula), rice (Oryza sativa), potato (Solanum tuberosum), and maize (Zea mays). Accordingly, it has been reported that these species also experience an increase in phosphorus assimilation activity (e.g., acid phosphatase, alkaline phosphatase, and H+-ATPase) in their roots which can contribute in increasing P assimilation and P nutritional status following the enhanced uptake and transfer of soil-P from the extraradical hyphae to the host plant (Capaccio and Callow 1982; Dexheimer et al. 1982; Schwab et al. 1991), which was later

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244 Table 11.2 Summary of known AM fungal and plant phosphate transporters (from Javot et al. 2007) Taxon Arbuscular mycorrhizal fungi Glomus intraradices Schenck & Smith Glomus mosseae (T.H. Nicolson & Gerd.) Gerd. & Trappe Glomus versiforme (P. Karst.) S. M. Berch Plant host Hordeum vulgare L.

Lotus japonicus L.

Lycopersicon esculentum L.

Medicago truncatula L.

Oryza sativa L.

Solanum tuberosum L.

Zea mays L.

Nomenclature

Reference

GiPT

Maldonado-Mendoza et al. (2001) Benedetto et al. (2005)

GmosPT

GvPT

Harrison and Van Buuren (1995)

HvPT1 HvPT2 HvPT3 LjPT1 LjPT2 LjPT3 LePT1 LePT2

Smith et al. (1999), Rae et al. (2003), and Glassop et al. (2005)

MtPT1 MtPT2 MtPT4 OsPT1 OsPT2 OsPT3 OsPT4 StPT1 StPT2

Maeda et al. (2006)

LePT3 LePT4

LePT5

OsPT5 OsPT6 OsPT7

OsPT8 OsPT9 OsPT10

StPT3 StPT4

StPT5

ZmPT1 ZmPT2 ZmPT3

corroborated by molecular analyses of phosphate transporter activity (Karandashov and Bucher 2005). As in the case of AM–plant N uptake, the ongoing characterization of the mycorrhizal P uptake and translocation pathways provide key evidence as to the active and intricate role of AM fungi in the enhancement of plant nutrient stress tolerance.

2.1.3 Micronutrient (Metal) Uptake Analogous to the processes of AM–plant N and P acquisition, there is a considerable body of

Daram et al. (1998), Liu et al. (1998a), Rosewarne et al. (1999), and Nagy et al. (2005) Karandashov et al. (2004), Liu et al. (1998b), and Harrison et al. (2002) OsPT11 OsPT12 OsPT13

Paszkowski et al. (2002) and Guimil et al. (2005)

Nagy et al. (2005), Leggewie et al. (1997), Rausch et al. (2001), and Karandashov et al. (2004) Glassop et al. (2005), Wright et al. (2005), and Nagy et al. (2006)

literature describing the beneficial role of the AM symbiosis in micronutrient uptake, particularly soil-metal5 deficiency conditions (Jeffries et al. 2003; Koide 1991; Marschner and Dell 1994).

5

Macro- and micronutrients are alternatively classified according to their physicochemical properties to define them, respectively, as either non-metals (nitrogen, sulfur, phosphorus, boron, chlorine) which have a negative valence, or metals (potassium, calcium, magnesium, iron, manganese, zinc, copper, molybdenum, nickel) which have a positive valence (Foy et al. 1978; Larcher 2003).

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

245

Hexose Essential Metabolic Function

HXT1 ???

Extraradical Hyphal Carbon Pool

Peri-Arbuscular Interface

Intraradical Hyphal Carbon Pool

Exudation of Organic Chelators

Essential Metabolic Function

Coenocytic Channel

GSH-M+

M+ MtZIP2 LeNramp1,3

GintZIP

GintABC1 GintZnT1

Vacuolar Compartmentation

M+

MT-M+ Bidirectional Transfer

Metallothionein Complexation GintMT1

M+ GintZIP ???

M+

M+

Glutathione S-Transferase

Intraradical Mycelium

Extraradical Mycelium

Host Root

Soil Environment

Fig. 11.7 AM fungal micronutrient (metal) uptake and transfer pathway (adapted from Meharg 2003; modified according to Göhre and Pazkowski 2006 and González-Guerrero et al. 2009). Bidirectional transfer is indicated by solid and dashed lines. Refer to text for abbreviations

In this regard, soils of temperate environments (typically classified as alfisols and vertisols) and tropical environments (ultisols and oxisols) can suffer from suboptimal elemental compositions and (or) nutrient imbalances due to long-term weathering and soil erosion resulting in plant zinc (Zn), nickel (Ni), cobalt (Co), copper (Cu), manganese (Mn), and (or) iron (Fe) nutritional deficiencies and (or) mutual antagonisms (Blinkley and Vitousek 1989; White and Zasoski 1999). As described previously, the AM symbiosis contributes in circumventing nutrient deficiency stress by increasing nutrient bioavailability in the mycorrhizosphere due to an increased resource acquisition capability which helps in supplementing the plant nutritional status, especially poorly labile metal nutrients (Eckhard et al. 1994; Liu et al. 2000; Marschner 1998; Rengel et al. 1999; Sharma et al. 1994). A commonality in the mode-of-action seems to exist regarding the general AM–plant metal uptake pathway (Fig. 11.7) as metal nutrients are taken up,

translocated, and transferred from the extraradical hyphae to the host roots, as depicted in reviews by Meharg (2003), Göhre and Pazkowski (2006), and González-Guerrero et al. (2009). These recent studies have reported that the exudation of organic chelators by the extraradical hyphae contributes in solubilizing metal ions in the mycorrhizosphere followed by the mobilization of metal–chelator complexes across fungal transporters, such as GintZIP in Glomus intraradices (González-Guerrero et al. 2009). Free metal ions may also be taken up passively across trans-membrane ion channels depending on the soil metal concentration gradient. In the cytosol, metal ions are typically bound and (or) sequestered by metallothionein proteins or glutathione complexes. Notably, it has been shown that this course-of-action can correspond with an upregulation of GintMT1 (encoding for fungal metallothioneins in G. intraradices – GonzálezGuerrero et al. 2005, 2007; López-Pedrosa et al. 2006) as well as an increase in glutathione

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246

S-transferase activity (González-Guerrero et al. 2009), which could represent critical steps in limiting internal stress due to the production of reactive oxygen species in the fungal cytosol (González-Guerrero et al. 2010a). Subsequently, the production of such less reactive metal complexes enables the AM fungi to store excess metal ions into their vacuolar compartments (via GintZIP, GintABC1, or GintZnT1 membrane transporters), integrate them into their essential metabolic function, or transfer them to the host root across the periarbuscular interface (via MtZIP2 or LeNramp1,3) (González-Guerrero et al. 2005, 2007, 2009, 2010b). The identification of ion transporters common to both plants and fungi which are putatively involved in the regulation of metal uptake (Burleigh et al. 2003; López-Millán et al. 2004) could support the perspective that this process is actively comodulated by both symbionts and provides further evidence as to the fundamentally mutualistic nature of the association, as suggested regarding the symbiotic transfer of P. Altogether, these combined processes characterize the complex role of the mycorrhizosphere in plant mineral nutrition contributing by modulating plant nutrient uptake and thereby enhancing the host plant’s physiological status compared to non-AM plants, particularly when subjected to a number of environmental stress factors.

2.2

Plant Water Relations

Besides the significant role of the AM symbiosis in plant mineral nutrition, there is a considerable body of literature describing the beneficial effects of the AM mycorrhizosphere in plant water relations across a broad range of water stress, for instance, from amply watered to droughted conditions (Augé 2001, 2004). Yet, unlike the dynamics of AM–plant nutrient acquisition described above, the specific mechanisms underlying the direct impact of the mycorrhizosphere on plant stress tolerance under such environmental conditions remain slightly ambiguous. A central question in this regard considers whether the AM symbiosis benefits plants more by enhancing

their intrinsic drought resistance (i.e., survival at low internal water content) or, rather, by increasing their drought avoidance (i.e., maintenance of high internal water content) when subjected to a low external water potential, such as drought and drought recovery conditions. As outlined in recent reviews by Augé (2001, 2004), a wide array of AM–plants in association with a number of Glomus and allied AM species (refer to Augé 2001 for a more comprehensive list of plant and AM fungal species interactions) have been shown to develop a variety of beneficial physiological responses compared to non-AM plants, with such responses ranging from relatively higher stomatal conductance, leaf transpiration, and (or) osmotic potential (Allen et al. 1981; Augé 2000; Augé et al. 2003, 2007, 2008). Among AM–plants, such enhanced metabolic and physiological functions under strained water conditions have subsequently been linked to an increased photosynthetic potential due to a generally larger leaf area, relatively greater water potential (i.e., water content) in roots and shoots, and an increased overall growth status observed under both greenhouse and field conditions (Cho et al. 2006; Khalvati et al. 2005). Altogether, these physiological responses contribute to an increased AM–plant stress tolerance occurring especially, but not exclusively, during both drought and drought recovery conditions. In addition to imposing a direct physiological stress toward host plants as symptomatically expressed by a general loss of turgidity and down regulation of various essential metabolisms (Hsiao 1973; Nautiyal et al. 1994), strained water relations often also cause alterations in nutrient bioavailability in the soil solution which leads to significant nutrient imbalances and potential antagonisms between metal ions. This potential correlationality between environmental stressors (e.g., both water and nutrient deficiency) represents an important challenge faced by experimental investigators in determining the role(s) of AM fungi in plant water relations, particularly when attempting to distinguish the specific mechanisms of AM-enhanced plant stress tolerance (Koide 1993). Similar to the notion of enhanced mycorrhizospheric uptake regarding AM–plant nutrient acquisition, it has

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

been suggested that the mycorrhizosphere should play a fundamental role in enhancing both the nutrient and water acquisition capabilities of host plants by increasing their resource acquisition pool compared to the rhizosphere alone; this, again through the general mechanism of actively scavenging the proximal soil environment for essential resources (Koide 1993). More specifically, extraradical hyphae could contribute directly in circumventing water deficiency conditions by penetrating soil micropores to improve hydraulic conductivity due to the expansive mycorrhizosphere, and thereby enhance plant stress avoidance during drought stress and drought recovery (Miller and Jastrow 1990). Accordingly, analyses comparing AM and non-AM root conductivity among plants under drought and amply watered conditions have also shown improvements in AM–plant water uptake that often coincide with increases in the host plant’s mineral nutrition. Here, an improved N, P, and (or) micronutrient status could further benefit AM–plants by circumventing internal mineral deficiencies that may otherwise detrimentally affect their intrinsic drought resistance mechanisms, such as the accumulation and maintenance of high foliar concentrations of soluble sugars and free polyamines (Augé 2001, 2004). This improved mineral status has also been linked with lower amino acid accumulation in AM than non-AM plants which suggests that the former are generally less metabolically strained under these conditions (Augé 2001). The consensus from these overall findings suggests that AM colonization primarily contributes by bolstering intrinsic plant resistance mechanisms by circumventing internal deficiencies. In addition, water conductance and nutrient uptake are improved due to an increased bioavailable pool of soil resources within the mycorrhizosphere (Cho et al. 2006; Khalvati et al. 2005). Together, these mycorrhizospheric processes can increase AM–plant resilience in relation to stressful water relations by enabling a relatively more stable (or less strained) metabolic function, which is typically manifested through an increased overall photosynthetic potential and relative growth potential during drought stress and drought recovery. While these physiological

247

effects provide a significant environmental advantage to AM vs. non-AM plants when subjected to adverse water conditions, plants having overall increased photosynthetic activities and stomatal conductances also tend to have higher rates of evapo-transpiration. Ironically, this effect can impose further stress on the plants themselves by increasing the rate of soil-drying in the proximal soil environment. In adding further complexity to the role of the AM symbiosis in plant water relations, these plants could be more vulnerable compared to less photosynthetically active plants under such environmental conditions due to complications stemming from accelerated soil-drying. Nevertheless, investigations into the role of the mycorrhizosphere in stabilizing the proximal soil environment could shed light into these matters, especially regarding the impact of extraradical hyphae in soil aggregation and biosorption processes which can buffer a number of edaphic factors such as the water and nutrient retention capacities. In fact, such “indirect” mycorrhizospheric processes could represent equally important components of plant stress tolerance and ecosystem function compared to the “direct” processes presented here.

3

Indirect Benefits of Association

3.1

Soil Structure Stabilization

3.1.1 Soil Aggregation Notwithstanding the direct role of the AM symbiosis in plant resource acquisition, the mycorrhizosphere also provides significant indirect benefits of interaction which can buffer and (or) stabilize the soil matrix. In this regard, processes such as mycorrhizal-induced soil aggregation and metal biosorption (Figs. 11.8 and 11.9) are considered, here, to fall within the category of indirect benefits since plant investment in the mycorrhizosphere can provide key ecological services which are not directly associated with the intimately co-modulated mechanism of resource exchange. Accordingly, it can also be argued that non-associated species could benefit

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248

Root Mucilage

Mycorrhizosphere Soil Micropores

Nu.

Hyphal Exudation

Host Root

H2O

Nu. H2O

H2 O

Nu. H2O

Nu. H2O

Particulate MicroAggregation

H 2O Nu.

Macro-Aggregation

Bulk Soil

Fig. 11.8 Mycorrhizal-induced micro- and macroaggregate formation indicating affinities for binding soil nutrients and water. (adapted from Miller and Jastrow 1990 and Rillig and Mummey 2006)

from these processes due to their proximity to the mycorrhizosphere’s zone of influence and its broad effects on the soil growth environment. Coincidentally, it has been suggested that such edaphic interactions should influence ecosystem function at various hierarchical scales due their biogeochemical implications ranging from the micro- (e.g., soil water and nutrient retention, enhanced resource acquisition) to the macroscopic levels (e.g., whole plant functionality, species abundance and distribution) (Beare et al. 1995; Rillig and Mummey 2006; Rillig et al. 2010). In this regard, and further to the role of the extraradical hyphae in directly improving nutrient uptake and hydraulic conductivity (as discussed previously), the proliferation of extraradical hyphae and their penetration into soil

micropores can significantly enhance the aggregation properties of soils to then improve their overall water and nutrient holding capacities (Piotrowski et al. 2004): a process akin to the enmeshment of roots throughout soils during rhizospheric expansion (Angers and Caron 1998). More specifically, the greater degree of mycorrhizal branching and ramification produces localized compression forces within the proximal soil environment which serve to increase the formation of micro- and macroaggregates (Fig. 11.8) (Miller and Jastrow 1990; Rillig and Mummey 2006). Consequently, mycorrhizospheric expansion can directly increase the rate of soil cluster formation and enhance the overall resilience of the soil structure in relation to stress (e.g., soil drying, flooding, compaction, nutrient leaching).

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

249

Hyphospheric Metal Biosorption

M+

–COOH –OH –O(OH) –SH

Exudation of Organic Chelators

Intraradical Mycelium

Extraradical Mycelium

Host Root

Soil Environment

Metal Complexation and Precipitation Chelator-M+ Complex

Mycorrhizospheric Metal Biosorption Fig. 11.9 Mycorrhizal-induced metal biosorption and metal complexation (adapted from Apak 2002, Gadd 1993, Galli et al. 1994, González-Chavez et al. 2002, and González-Guerrero et al. 2008)

Alternatively, under such conditions, bulk soils tend to have a comparatively lower particulate binding capacity and subsequently lower aggregation potential resulting in the relatively more rapid collapse of their matrix structure. In addition to the physical entanglement of soil aggregates by the mycorrhizosphere, mycorrhizal-induced soil aggregation can also be attributed to the exudation of organic acids by extraradical hyphae (Bais et al. 2006; Bertin et al. 2003; Rovira 1969). Fundamentally, these mucilage exudates – typically consisting in polysaccharides and other extra-cellular polymeric compounds – contribute to nutrient chelation for mineral solubilization within the mycorrhizosphere as well as protection of the extraradical mycelium from desiccation. Further to these essential roles, such organic exudates also adhere to soil particles and permit the physical entanglement of micro- and macroaggregates leading to the development of soil clusters within the mycorrhizosphere. Glomalin and glomalin-related soil proteins, considered to be effective biochemical markers of AM fungal growth and mycorrhizosphere development, are

also believed to bind soil particles in the same manner leading to increased structure stability (Driver et al. 2005; Purin and Rillig 2007). Altogether, this improved potential for soil cluster formation can increase the incidence of micropores within the soil architecture leading to an overall increased colloidal surface area. As a result, the mycorrhizosphere can have a higher affinity for retaining water molecules as well as metal and nonmetal ions compared to bulk soil. Taking into account the processes of hyphal proliferation and chelator exudation, Augé (2004) and Rillig and Mummey (2006) have likened the mycorrhizospheric network to an essential skeletal structure and the production of mycorrhizalderived organic compounds as the “glue” which, together, contribute in holding together the soil matrix. Consequently, from a biogeochemical perspective, the mycorrhizospheric network should play central role in enhancing soil water and nutrient retention. When subjected to environmentally stressful conditions, these enhanced soil stabilization properties can significantly increase the soil’s resilience to then buffer the

250

growth environment for plants and associated soil microorganisms. Notably, under drought stress, the development of soil aggregates increases water and nutrient retention to delay the effects of soil drying (Auge 2004; Rillig and Mummey 2006); meanwhile, these aggregates also increase water infiltration during drought recovery due to the more hydratable (or water stable) soil matrix (Rillig et al. 2010). This mycorrhizal-induced structural advantage benefits plant stress tolerance by increasing the soil’s water storage capacity and increasing its resilience. Likewise, the increased water retention capacity within the mycorrhizosphere can also impact plant stress tolerance in relation to nutrient stress (e.g., reciprocal ion antagonisms leading to deficiency) since soil nutrient bioavailability is closely correlated with soil water potential. As a result, nutrient bioavailability may be increased within the mycorrhizosphere due to a greater retention capacity; meanwhile nutrient losses are decreased due to a reduction in leaching.

3.1.2 Metal Biosorption The metal-binding capacity of soil is primarily dictated by its essential composition, whereby soils having a higher proportion of organic matter (e.g., humic and fluvic acids) typically tend to have a greater retention capacity and redox potential than other soil types (Bohn 1971; McBride 1994). Further to the role of the mycorrhizosphere in stabilizing the soil’s structural matrix, the extraradical hyphae have also been shown to increase the biosorption potential of soils. This is attributed primarily to the preferential binding of metal ions to negatively charged mycelial and root surface constituents (Fig. 11.9), such as carboxyl, hydroxide, oxy-hydroxide, and sulfhydryl groups (Apak 2002; Gadd 1993; Galli et al. 1994; González-Chavez et al. 2002, 2008). Similar analyses of non-mycorrhizal fungi suggest that phenolic polymers and melanins should also be effective metal binding sites even among AM fungi (Baldrian 2003; Fogarty and Tobin 1996). Likewise, the exudation of organic chelators within the mycorrhizosphere (described above) has been shown to result in an enhanced binding capacity due to the formation of metal–ligand

P. Audet

complexes and precipitates in the soil solution. For these reasons, the general processes of metal biosorption, including ion-exchange (i.e., CEC), metal complexation, and metal–ligand precipitation and crystallization occurring on and within the fungal cell wall (Gadd 1993; Galli et al. 1994), represent significant mechanisms regarding the modulation of metal bioavailability within the mycorrhizosphere. As in the case of mycorrhizalinduced soil aggregation, these enhanced metal biosorption properties can improve the soil’s resilience by increasing its nutrient retention capacity, while reducing nutrient losses due to leaching (Giller et al. 1998; Leyval et al. 1997). Notably, there is considerable evidence suggesting that, when essential and nonessential metals occur at exceedingly high exposure levels representing potentially toxic metal conditions, such metal biosorption properties can significantly reduce the bioavailability of metals in the soil solution to reduce plant metal uptake and then delay the onset of metal phytotoxicity (Audet and Charest 2007b). In this regard, a wide array of plant species (refer to Audet and Charest 2007b for broad list plant species) subjected to increasingly high metal concentrations, both essential (e.g., Cu, Fe, Mn, Ni, and Zn) and nonessential elements (e.g., Cd, Co, Cr, and Pb), have repeatedly been shown to incur considerably lower (up to 50%) metal uptake among AM (Gl. caledonium, Gl. intraradices, Gl. mosseae, and a consortium of unidentified Glomus species) than non-AM plants; an effect often coinciding with an increased plant growth and (or) health status. As proposed by Leyval et al. (1997), and later Audet and Charest (2006, 2007a, b, 2008, 2009), Hildebrandt et al. (2007), and Giasson et al. 2008, these findings suggest that mycorrhizal-induced metal biosorption could represent a significant extrinsic plant stress avoidance strategy, whereby excess soil metals are bound and precipitated in the soil solution as well as sequestered in fungal tissues instead of being transferred to host roots. As such, plant investment in this extrinsic stress avoidance mechanism could complement known intrinsic plant detoxification mechanisms, for instance, metallothienin and phytochelatin metabolisms (Cobbett 2000; Cobbett and Goldsbrough 2002) by reducing

11

AM Symbiosis and Other Plant–Soil Interactions in Relation…

Critical Deficiency Symptoms

Toxicity Range Luxury Range

Adequate Range

Deficiency Range

Relative Plant Growth

Plant-Metal Uptake

Critical Deficiency Symptoms

Luxury Range Toxicity Range

Critical Toxicity Symptoms

Dynamics of AM-plant stress tolerance

AM non-AM

Extrinsic Metal Exposure

Relative Plant Growth

Metal Biosorption

Enhanced Uptake

Adequate Range

Deficiency Range

Critical Toxicity Symptoms

Dynamics of AM-plant metal uptake

Plant Metal Uptake

251

Nutrient Supplementation

Metal Stress Avoidance

Enhanced Stress Tolerance

AM non-AM

Extrinsic Metal Exposure

Fig. 11.10 Dynamics of AM–plant metal uptake as characterized by the mycorrhizospheric processes of enhanced uptake and metal biosorption. (adapted from Audet and Charest 2007b, 2008, 2009)

Fig. 11.11 AM–plant relative plant growth in relation to metal exposure corresponding to the “Dynamics of AM-plant metal uptake” (adapted from Audet and Charest 2007b, 2008, 2009)

AM–plant metal uptake and subsequently reducing cellular oxidative stress and other physiological challenges associated with metal toxicity conditions. This entire perspective has been summarized by Audet and Charest (2007b, 2008, 2009) who described plant metal uptake (Fig. 11.10) and relative plant growth (Fig. 11.11) by AM and non-AM plants using a conceptual modeling strategy based on meta-analytical, in vitro, and greenhouse culture systems. When taking into account the many different roles of AM fungi in plant physiology and ecosystem function, the mycorrhizosphere is first believed to increase AM–plant metal uptake to supplement the plant nutritional status under nutrient deficiency conditions. Subsequently, the role of the mycorrhizo-

sphere seems to shift toward the modulation of soil nutrients due to metal biosorption processes which can delay the effects of metal phytotoxicity when subjected to potentially toxic conditions. Here, the combination of the enhanced uptake and metal biosorption mechanisms occurring simultaneously and (or) independently causes a distinct metal uptake profile among AM than nonAM plants in relation to a wide range of metal exposure, albeit depending on the soil’s fundamental composition and inherent properties. As such, AM–plant growth and stress tolerance are often significantly improved as evidenced by an increased growth status whether under trace or toxicity conditions. As for conditions within the proximal growth environment, the mycorrhizal-

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252

induced processes of metal biosorption and metal– ligand complexation are also believed to influence a number of edaphic factors, for instance, the soil pH and redox potential (Christie et al. 2004; Fourest and Roux 1992; Joner et al. 2000; Leyval et al. 1997). When considering the case of phosphorus and nitrogen acquisition by mycorrhizae, the extraradical hyphae are believed to cause the moderate alkalinization of the growth substrate due to their selective depletion of nutrients and specific exudation of organic chelators, unlike roots that tend to acidify it (Bago et al. 1996; Eckhard et al. 1995; Gahoonia and Nielsen 1992; Li et al. 1991; Rufyikiri et al. 2004). Similar outcomes have been reported by Li and Christie (2001) and Audet and Charest (2010 and unpublished results) who investigated the impact of the rhizosphere (roots), mycorrhizosphere (roots and extraradical hyphae), and hyphosphere environments (strictly extraradical hyphae) in relation to increasing soil–Zn exposure levels. Although these facets of plant–AM–soil interactions should require further and more in-depth investigation, these studies indicate preliminarily that the presence of roots and (or) extraradical hyphae should play an essential part in shaping edaphic conditions due (in part) to differential nutrient depletion zones caused by AM and non-AM plants, their exudation of organic chelators, and their retention (or biosorption) of metals within the proximal growth environment. These factors are relevant to the bioavailability of metal and non-metal nutrients in soils since the process of hyphal alkalization could favor metal biosorption and contribute in reducing metal bioavailability and toxicity, whereas root acidification may facilitate leaching by increasing metal solubility (Apak 2002; Bradl 2004; Tack et al. 1996).

3.2 3.2.1

Biotic Interactions

Biodiversity of Beneficial and Nonbeneficial Soil Microflora Although this chapter has focused primarily on identifying the role(s) of the AM symbiosis in benefiting plant tolerance when subjected to a number of abiotic stressors (e.g., macro- and

micronutrient deficiency, drought, metal toxicity), the role of the mycorrhizosphere toward biotic soil interactions is still worthy of mention – albeit discussed only briefly in the present context. In addition to shaping growth parameters such as soil nutrient bioavailability and other edaphic factors, mycorrhizal proliferation and exudation can also significantly impact belowground biodiversity by influencing soil microbial communities within the proximal soil environment (Brussaard et al. 1997; Newsham et al. 1995a, b; Wardle et al. 1998, 2004). In turn, such subsidiary mycorrhizospheric interactions can have important consequences toward aboveground species biodiversity (i.e., plant species abundance and distribution) due to the mutual feedback existing between above- and belowground symbionts (Bever 1999, 2003; Bever et al. 1997; Klironomos 2002; van der Heijden et al. 1998). As such, the AM fungi are believed to develop their own soil microflora apparently due to the exudation of organic compounds and chelators within the mycorrhizosphere (Andrade et al. 1997; Fitter and Garbaye 1994; Frey-Klett et al. 2007; Linderman 1988); this, in addition to their modulation of edaphic factors (as described previously) which can provide more favorable soil pH and nutrient bioavailability conditions for the development of such a microflora (Cavagnaro et al. 2006; Deubel and Merbach 2005; Villegas and Fortin 2001). This is not surprising considering that AM fungal spores are known to harbor their own internal bacterial flora within their sporocarps, meanwhile the extraradical hyphae maintain their own extensive array of bacterial biofilms (Andrade et al. 1997; Bianciotto and Bonfante 2002; Fitter and Garbaye 1994; Frey-Klett et al. 2007). Although the ecological role(s) of these bacteria in AM fungal development have yet to be fully understood, various in vitro analyses have demonstrated a number of interactive behaviors ranging from stimulated spore germination, induced hyphal branching, and enhanced sporulation due to the presence of volatile bacterial metabolites (AzcónAguilar and Barea 1992; Barea 1997; Bianciotto and Bonfante 2002; von Alten et al. 1993). In response, the AM fungi seem to reciprocate this ulterior “symbiotic” association by enriching the

11

AM Symbiosis and Other Plant–Soil Interactions in Relation…

mycorrhizosphere environment with carbon in the form of organic exudates (Antoun and Prevost 2001; Artursson et al. 2006). For this reason, it is possible that such bacteria (also known as mycorrhiza helper bacteria – Garbaye 1994; Frey-Klett et al. 2007) and their exudates are fundamentally involved in AM fungal development and mycorrhizospheric function, and then potentially impacting plant health status due to their role as growth promoters (Bianciotto and Bonfante 2002; Bianciotto et al. 1996a, b; Söderberg et al. 2002). In terms of their potential effects on plant stress tolerance and extrinsic growth conditions within the mycorrhizosphere, the mycorrhiza helper bacteria are generally considered to influence edaphic parameters similar to the extraradical hyphae themselves: for instance, by increasing the solubilization of soil nutrient via chelation and (or) contributing in the modulation of nutrient bioavailability via metal biosorption and soil aggregation processes (Cavagnaro et al. 2006; Deubel and Merbach 2005; Villegas and Fortin 2001). Accordingly, these mechanisms should benefit plant stress tolerance by indirectly improving nutrient bioavailability and uptake, particularly when subjected to suboptimal soil nutrient conditions. Moreover, the AM fungi could also benefit plant stress tolerance in relation to biotic stressors by inhibiting and (or) impeding soilborne pathogens (Azcón-Aguilar and Barea 1996; Barea et al. 1998; Newsham et al. 1995a, b). In this regard, St. Arnaud and Elsen (2005) have meticulously summarized the interaction outcomes of a number of AM fungi cultured under in vitro conditions in the presence of soil bacteria (Table 11.3), other soil fungi (Table 11.4), and nematodes (Table 11.5). This meta-analysis as to the impact of the AM fungi on soil-borne pathogens, and vice versa, suggests that their interaction can be highly variable (i.e., having positive, neutral, or negative outcomes under experiment conditions); however, the AM fungi are still widely recognized for benefiting plant growth in relation to a number of highly persistent and destructive pathogens (Azcón-Aguilar and Barea 1996; Barea et al. 1998; Newsham et al. 1995a, b). Harrier and Watson (2003, 2004) as well as Mukerji and Ciancio (2007) have outlined a num-

253

ber of potential mechanisms (particularly in the context of integrated pest and disease management) in which AM fungi can interact with such soil microorganisms within the proximal growth environment to then enhance plant stress tolerance, which include: the competitive exclusion (or mechanical inhibition) of nonbeneficial soil microorganisms by competing for sites of infection and (or) colonization; the subsequent alteration of root architecture and anatomical structure due to AM colonization; the activation of plant defense responses such as antibiotics and phytoalexins due to AM–plant signaling; the modification of soil physicochemical parameters such as soil pH and nutrient bioavailability as well as alteration in carbon partitioning (or rhizo-deposition); and, finally, the increase in damage compensation due to an enhanced nutrient status. Although some of these perspectives have been described only briefly in this sections, it is evident that such mycorrhizospheric processes leading to the development of a beneficial belowground species biodiversity profile can then indirectly influence the composition of aboveground species biodiversity. Likewise, further experimental investigation to better elucidate specific mechanisms of interaction would certainly highlight the impact of AM fungi toward plant stress tolerance in regards to biotic stress and other biodiversity interactions.

4

Conclusions and Future Perspective

4.1

Assessing AM–Plant Interactions in Plant Stress Tolerance

In this chapter, it has been reported how the AM symbiosis is a widespread ecological association which is deeply rooted in the essential function of the vast majority of herbaceous plant species, as well as the function of soils and associated soil microorganisms. By describing some of the mechanisms underlying these plant physiological and soil ecological functions, it is clear that a number of such AM fungal processes are fundamentally involved in enhancing the stress tolerance of

P. Audet

254 Table 11.3 Interactions between AM fungi and bacteria (from St. Arnaud and Elsen 2005) AM fungi Glomus intraradices G. clarum

Interaction outcomea N P, n,N

Reference Hildebrandt et al. (2002) Xavier and Germida (2003)

G. intraradices

N

Filion et al. (1999)

P N P P P P P P P, n N P N P

Mayo et al. (1986) Hildebrandt et al. (2002) Hildebrandt et al. (2002) Mosse (1962) Mayo et al. (1986) Villegas and Fortin (2001, 2002) Filion et al. (1999) Bianciotto et al. (1996a, b) Villegas and Fortin (2001, 2002) Bianciotto et al. (1996a, b) Villegas and Fortin (2001, 2002) Tylka et al. (1991) Tylka et al. (1991)

S. orientalis S. orientalis

G. versiforme G. intraradices G. intraradices Endogone sp. G. versiforme G. intraradices G. intraradices Gigaspora margarita G. intraradices Gi. margarita G. intraradices G. mosseae Scutellospora heterogama Gi. margarita G. mosseae

P P

S. orientalis Spore-associated bacteria Unidentified soil bacteria

S. heterogama G. versiforme G. mosseae

P, N P P

Tylka et al. (1991) Mugnier and Mosse (1987) and Tylka et al. (1991) Tylka et al. (1991) Tylka et al. (1991) Azcón (1987, 1989)

Bacteria Azospirillum brasilense Bacillus chitinosporus; B. pabuli and other spore-associated bacteria Clavibacter michiganensis ssp. michiganensis Corynebacterium sp. Escherichia coli Paenibacillus validus Pseudomonas sp. Pseudomonas sp. P. aeruginosa P. chlororaphis P. fluorescens P. putida Rhizobium leguminosarum Serratia plymutica Streptomyces avermitilis S. griseus

P Positive, N negative, n neutral Refers to the impact of the bacterial species toward the AM fungus (e.g., promotion of spore germination and hyphal growth)

a

plants and bolstering the resilience of soil in relation to a number of abiotic environmental stressors due especially to the dynamic function of the mycorrhizosphere. When taking into account the significant allocation of plant carbohydrates required for the development and maintenance of an expansive and prolific mycorrhizospheric infrastructure, it has been suggested that the AM symbiosis should represent an extrinsic plant stress tolerance strategy which could inherently complement other intrinsic plant stress tolerance strategies. To further depict the roles of the mycorrhizosphere in ecosystem function, the present description of the AM symbiosis has distinguished between the direct vs. indirect dynamics of interaction. Accordingly, this distinction refers to AM-induced activities which either directly benefit host plant due to intimately comodulated pathways or, alternatively, activities

which indirectly benefit the symbionts (potentially including non-associated species) due to their effects in buffering the proximal growth environment. This perspective is intriguing since it presumes that a number of essential mycorrhizospheric processes could occur simultaneously and (or) independently, and thereby have fundamental ecological functions across different trophic levels. This could account, in part, for the widespread abundance and distribution of mycorrhizal associations within the majority of terrestrial ecosystems, especially those subjected to highly stressful and (or) extreme environments. Still, there are so far very few modeling strategies depicting these combined, multilateral effects especially across a broad or continuous spectrum of stress (e.g., from trace to toxicity conditions or from droughted to amply water conditions). For instance, the case for AM–plant metal uptake is

T. koningii T. pseudokoningii Verticillium albo-atrum V. dahliae Wardomyces inflatus Unidentified soil fungi

McAllister et al. (1996) Benhamou et al. (1994) and Filion et al. (1999) St. Arnaud et al. (1996) Chabot (1991) McAllister et al. (1996) Chabot (1991) Francchia et al. (1998) Chabot (1991) Francchia et al. (1998) Calvet et al. (1992) Chabot (1991) Lioussanna et al. (2003) Chabot (1991)

N N P, N N P, n n n n P, n N n N n

G. intraradices

G. intraradices Gi. margarita G. mosseae Gi. margarita

G. mosseae Gi. margarita G. mosseae

G. mosseae Gi. margarita

G. intraradices

Gi. margarita

F. oxysporum f. sp. Chrysanthemi F.o. chrysanthemi F. solani F. solani Gaeumannomyces graminis Gliocladium roseum Ophiostoma ulmi Paedilomyces farinosus Penicillium decumbens Phytophthora sp.

P. nicotianae

Pythium ultimum

P Positive, N negative, n neutral a Refers to the impact of the bacterial species toward the AM fungus

G. mosseae

Fusarium equiseti

Chabot (1991)

P, n, N

Gi. margarita

Bipolaris sorokiniana

Calvet et al. (1992) McAllister et al. (1996)

N n

G. mosseae G. mosseae

Aspergillus fumigatus A. niger

Fungus Pyrenochaeta terrestris Rhizoctonia solani Rhodotorula mucilaginosa Sclerotinia scletotiorum Thievaliopsis basicola Trichoderma aureoviride T. harzianum T. harzianum T. harzianum T. harzianum

Reference McAllister et al. (1996)

AM fungi G. mosseae

Fungus Alternaria alternate

Interaction outcomea, P, n, N N

Table 11.4 Interactions between AM fungi and other fungi (from St. Arnaud and Elsen 2005)

G. mosseae

Gi. margarita G. mosseae

G. mosseae G. mosseae Gi. Margarita

G. intraradices G. mosseae G. mosseae G. intraradices

G. mosseae

Gi. margarita

Gi. margarita

Gi. margarita G. mosseae

AM fungi Gi. margarita

P

n N

Azcón-Aguilar et al. (1986)

Chabot (1991) Francchia et al. (1998)

McAllister et al. (1996) Francchia et al. (1998) Chabot (1991)

Filion et al. (1999) Calvet et al. (1992) Francchia et al. (1998) Rousseay et al. (1996)

P P n N N, n n n

Calvet et al. (1992)

Chabot (1991)

Chabot (1991)

Chabot (1991) Francchia et al. (1998)

Reference Chabot (1991)

P

n

n

n P

Interaction outcome n

11 AM Symbiosis and Other Plant–Soil Interactions in Relation… 255

P. Audet

256 Table 11.5 Interactions between AM fungi and nematodes (from St. Arnaud and Elsen 2005) Nematode Globodera pallida Radopholus similis Pratylenchus coffeae

AM fungi Glomus sp. G. intraradices G. intraradices

Interaction outcomea P N N

Reference Ryan et al. (2000) Elsen et al. (2001) Elsen et al. (2003)

P Positive, N negative, n neutral a Refers to the impact of the bacterial species toward the AM fungus

noteworthy since the AM fungi apparently hold two antithetical roles in plant metal uptake (i.e., enhanced uptake vs. metal biosorption) having different predicted outcomes depending on the soil metal conditions. From this viewpoint, a few lingering questions (among other) have yet to be fully addressed regarding the fundamental role(s) of the AM symbiosis in plant stress tolerance and ecosystem function, such as: How do these combined mycorrhizospheric processes benefit plant stress tolerance? Under what environmental conditions do they occur? How are they regulated by the host plants, or the AM fungi?

Considering the abundance of high-quality data available within the published literatures which were developed primarily using reductionist strategies, future investigations could benefit from more holistic approaches in order to bridge our current mechanistic understanding of mycorrhizospheric function with other known soil ecological processes to better depict the impact of the AM symbiosis in whole-ecosystem function; in other words, taking into account multitrophic interactions and making attempts to quantify such relationships at the physiological and ecological levels. Such an approach would highlight the interconnectedness of above- and belowground species, as well as proving beneficial in re-assessing the balance between the costs of maintaining the symbiosis vs. the benefits of association among the symbionts. As mentioned previously, this resource allocation balance has been critical for plant physiologists and mycologists alike in defining the symbiotic mutualism since it is widely believed to function along a continuum potentially ranging from parasitism to mutualisms (Bronstein 2001; Johnson et al. 1997; Jones and Smith 2004). And so, an assessment of plant–AM–soil interactions using quantifiable

descriptors could provide an insightful analysis of this continuum of interaction. Another potential topic of concern regards the quantification of the mycorrhizosphere in relation to plant mycorrhizal dependence. With exception to the characterization of AM–plant nutrient uptake and symbiotic transfer using molecular tools, we have yet to quantitatively define the mycorrhizosphere’s and (or) hyphosphere’s actual zones of influence (e.g., their capacity to bind metals or stabilize the soil structure) other than by indirect methodologies. Such a quantification of the mycorrhizosphere could help address issues such as: How prolific is the mycorrhizosphere compared to the rhizosphere? What are its biogeochemical properties and subsequent area of influence? How prolific is the mycorrhizosphere in relation to the plant’s carbon allocation investment?

In turn, it may be feasible to quantitatively assess the mycorrhizal dependency (or resource allocation and extrinsic investment) of plants in relation to a wide-range stressors and broad-spectrum of environmental stress. Altogether, the potential focus-shift of future research objectives toward assessing the combined mycorrhizospheric impact in whole-ecosystem function as well as the quantification of the mycorrhizosphere in relation to plant symbiotic investment could highly benefit the effective integration of mycorrhizal technologies into agro-ecosystem management practices, especially sustainable agriculture and environmental remediation (Brussaard et al. 1997, 2007; Gosling et al. 2006; Jeffries et al. 2003; Mäder et al. 2002) – an intricate topic deserving of more lengthy discussion. Nevertheless, in the present context, it can be concluded that the AM fungi have a quintessential role in plant stress tolerance which could translate well toward enhancing the

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AM Symbiosis and Other Plant–Soil Interactions in Relation…

stress tolerance and stress resistance of whole ecosystems, particularly in the current era of climate change and in relation to anthropogenically derived environmental stressors.

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