Molecular Mechanisms to Cold, Drought, Heat and Salt Stress in Higher Plants (Biotechnology Intelligence Unit1)

BIOTECHNOLOGY I N T E L L I G E N C E U N I T

1

Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki

Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

R.G. LANDES C OM PA N Y

BIOTECHNOLOGY INTELLIGENCE UNIT 1

Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants Kazuo Shinozaki, Ph.D. Laboratory of Plant Molecular Biology Tsukuba Life Science Center The Institute of Physical and Chemical Research (RIKEN) Tsukuba, Japan

Kazuko Yamaguchi-Shinozaki, Ph.D. Biological Resources Division Japan International Research Center for Agricultural Sciences (JIRCAS) Tsukuba, Japan

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

BIOTECHNOLOGY INTELLIGENCE UNIT Cold, Drought, Heat and Salt Stress in Higher Plants R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright ©1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-563-1

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Cold, drought, heat and salt stress in higher plants / [edited by] Kazuo Shinozaki, Kazuko Yamaguchi-Shinozaki. p. cm. -- (Biotechnology intelligence unit) ISBN 1-57059-563-1 (alk. paper) 1. Plants, Effects of stress on—Molecular aspects. 2. Plant molecular genetics. I. Shinozaki, Kazuo. II. Yamaguchi-Shinozaki, Kazuko. III. Series. QK754.C65 1999 571.9'52—dc21 99-33224 CIP

CONTENTS 1. Genetic Approaches to Abiotic Stress Responses .................................... 1 M. Koornneef and A.J.M. Peeters The Genetic Approach in Stress Physiology—General Principals ........ 2 Genetic Differences in the Response to Low Temperatures .................. 4 ABA Related Mutants .............................................................................. 5 Conclusions ............................................................................................. 7 2. Molecular Responses to Drought Stress ................................................ 11 Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki A Variety of Functions of Drought-Inducible Genes .......................... 12 Regulation of Gene Expression by Drought ........................................ 14 Signal Perception and Signal Transduction in Drought Stress Response .................................................................................. 18 Conclusion and Perspectives ................................................................ 25 3. Molecular Mechanisms of Salinity Tolerance ....................................... 29 Hans J. Bohnert, Hua Su and Bo Shen Osmolytes, Osmoprotectants, Compatible Solutes, Osmotic Adjustment ........................................................................................ 30 Cellular Mechanisms of Salt Tolerance—the Fungal Model .............. 31 Molecular Mechanisms of Salt Tolerance in Plants ............................. 37 Metabolic Engineering of Glycophytic Plants for Increased Salt Tolerance .................................................................................... 47 Perspectives ............................................................................................ 48 4. Plant Cold Tolerance ............................................................................... 63 Michael F. Thomashow and John Browse Chilling tolerance .................................................................................. 63 Freezing Tolerance ................................................................................ 69 Conclusions and Perspectives ............................................................... 77 5. Molecular Responses to Heat Stress ....................................................... 83 Fritz Schöffl, Ralf Prändl and Andreas Reindl Heat Shock Proteins and Thermotolerance ......................................... 84 Links to Other Abiotic Stresses ............................................................. 87 Transcriptional Regulation ................................................................... 89 The Regulation of HSF .......................................................................... 90 Conclusions and Perspectives ............................................................... 93 6. Cellular Responses to Water Stress ...................................................... 101 Michael R. Blatt, Barbara Leyman and Alexander Grabov The Stomatal Situation ........................................................................ 102 Transport Mechanics ........................................................................... 103 Transport Coordination in the Face of Stress .................................... 105 Interaction of Signaling Elements ....................................................... 114 Initial Events in ABA Stimulus Perception ........................................ 115 Perspectives and Conclusion .............................................................. 117

Acknowledgments ............................................................................... 118 7. Role of Glycine Betaine and Dimethylsulfoniopropionate in Water-Stress Tolerance ..................................................................... 127 Douglas A. Gage and Bala Rathinasabapathi Stress Protection by Glycine Betaine and DMSP In Vivo and In Vitro ..................................................................................... 128 Biosynthesis of DMSP ......................................................................... 134 Conclusion ........................................................................................... 147 8. Osmotic Stress Tolerance in Plants: Role of Proline and Sulfur Metabolisms ........................................................................................... 155 Desh Pal S. Verma Osmoregulation in Microorganisms .................................................. 155 Osmosensing and Signal Transduction Machinery ........................... 156 Osmotic Stress Tolerance in Plants .................................................... 158 Accumulation of Other Osmolytes ..................................................... 160 Accumulation of Proline in Transgenic Plants Expressing Elevated Levels of P5CS ................................................................................. 161 Proline Accumulation Confers Osmoprotection............................... 163 Role of Sulfur metabolism in Osmotic stress Tolerance ................... 164 A Possible Role of DPNPase in Salt Tolerance .................................. 166 Overexpression of Plant HAL2 Gene Confers Reduction in Free Radical Production and in Heavy Metal Toxicity ....................................... 166

EDITORS Kazuo Shinozaki, Ph.D. Laboratory of Plant Molecular Biology Tsukuba Life Science Center The Institute of Physical and Chemical Research (RIKEN) Tsukuba, Japan Chapter 2 Kazuko Yamaguchi-Shinozaki, Ph.D. Biological Resources Division Japan International Research Center for Agricultural Sciences (JIRCAS) Tsukuba, Japan Chapter 2

CONTRIBUTORS Michael R. Blatt, Ph.D. Laboratory of Plant Physiology and Biophysics University of London, Wye College Wye, England, U.K. Chapter 6

Alexander Grabov, Ph.D. Laboratory of Plant Physiology and Biophysics University of London, Wye College Wye, England, U.K. Chapter 6

Bo Shen, Ph.D. Departments of Plant Sciences The University of Arizona Tucson, Arizona, U.S.A. Chapter 3

Hua Su, Ph.D. Departments of Plant Sciences The University of Arizona Tucson, Arizona, U.S.A. Chapter 3

Hans J. Bohnert, Ph.D. Departments of Biochemistry, Molecular and Cellular Sciences The University of Arizona Tucson, Arizona, U.S.A. Chapter 3

M. Koornneef, Ph.D. Laboratory of Genetics Wageningen Agricultural University Wageningen, The Netherlands Chapter 1

John Browse, Ph.D. Institute of Biological Chemistry Washington State University Pullman, Washington, U.S.A. Chapter 4

Barbara Leyman, Ph.D. Laboratory of Plant Physiology and Biophysics University of London, Wye College Wye, England, U.K. Chapter 6

Douglas A. Gage, Ph.D. Department of Biology Michigan State University East Lansing, Michigan, U.S.A. Chapter 7

A.J.M. Peeters, Ph.D. Laboratory of Genetics Wageningen Agricultural University Wageningen, The Netherlands Chapter 1

Ralf Prändl, Ph.D. Lehrstuhl Allgemeine Genetik Universität Tubingen Tubingen, Germany Chapter 5 Bala Rathinasabapathi, Ph.D. Hort Sciences Department University of Florida Gainesville, Florida, U.S.A. Chapter 7 Andreas Reindl, Ph.D. Lehrstuhl Allgemeine Genetik Universität Tubingen Tubingen, Germany Chapter 5 Fritz Schöffl, Ph.D. Lehrstuhl Allgemeine Genetik Universität Tubingen Tubingen, Germany Chapter 5 Michael F. Thomashow, Ph.D. Department of Crop and Soil Sciences, Department of Microbiology Michigan State University East Lansing, Michigan, U.S.A. Chapter 4 Desh Pal S. Verma, Ph.D. Department of Molecular Genetics and Plant Biotechnology Center Ohio State University Columbus, Ohio, U.S.A. Chapter 8

PREFACE

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he genetic improvement of tolerance of crops to environmental stresses, such as drought, high salinity, low temperature and heat, is an important problem for the future of agriculture. Classical breeding methodologies to select stress tolerant cultivars have already made some progress. Biotechnology has the potential to improve environmental stress tolerance of crops using transgenic plant technology. The limiting factor for developing this technology is the isolation of genes that can improve drought tolerance and the precise understandings of molecular process of stress tolerance and plants’ responses to environmental stresses. Plants respond to environmental stresses, such as drought, high salinity, low temperature and heat, through a number of physiological and developmental changes. Recently, higher plants respond to these stresses at gene expression level. A variety of stress-inducible genes have been cloned and analyzed concerning to their expression and function in stress tolerance and stress responses. Recently, many mutants have been isolated that are resistant or hypersensitive to environmental stresses, and cloning of their genes is now in progress. Molecular and genetic analyses of the regulation of gene expression and signal transduction cascades proceed extensively, and will give us more precise insight on the plants' responses to environmental stresses and their adaptation processes. These stress-related genes are thought to become useful resources to produce stress tolerant crops using gene manipulation. In this book, recent progresses on molecular mechanisms of plant responses and tolerance to drought, salt, cold and heat stresses are reviewed by active researchers in this field. I hope that this book stimulates young students and researchers to become interested in new plant science based on molecular biology and new plant biotechnology.

CHAPTER 1

Genetic Approaches to Abiotic Stress Responses M. Koornneef and A.J.M. Peeters

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lants grow in almost any part of the world and under a wide variety of nutrient and climatic conditions differing in temperature, light quantity and quality and availability of water. Plants that grow in a specific environment are adapted to these different local conditions, and can also cope with changes in these conditions which might be adverse for their growth and development. Adaptation is required because plants cannot escape unfavorable conditions due to their sessile growth habit. This implies that species differ genetically in their adaptation and resistance to abiotic stresses. Although more restricted, genetic variation for the adaptation to abiotic stresses can also be present within species and has been used for plant breeding practice. Examples of how plants deal with extreme temporarily adverse conditions are the so-called resurrection plants, which can lose more than 90% of their water content, but still are able to revive when supplied again with water. Examples of plants that can grow under extreme low temperatures are those that grow in arctic regions or at high altitudes. Plants can be preadapted to stress conditions but often various protection mechanisms are induced by the stress treatments itself. This implies that plants are able to perceive stress signals and that after perception signal transduction events take place. As a consequence, these lead to changes in gene expression, as indicated by the many situations where upregulation of genes is observed after the application of various types of abiotic stress (reviewed by Zhu et al1). Ultimately various cellular mechanisms are set in place, which allow the plant to cope with the stress imposed. These mechanisms are for instance osmoadjustment and osmo-protection, changes in pathways affecting ion and water fluxes, production of protection proteins etc.2,3 In case of osmo-adjustment the osmotic potential of the cell is lowered to favor water uptake and maintenance of turgor. Osmoprotectants stabilize proteins and membranes when present in high concentrations and include a variety of compounds such as amino acids (proline), quaternary ammonium compounds (betaines), polyols (pinitol, mannitol), sugars such as fructans2 and specific proteins such as dehydrins.5 The introduction of genes leading to increased levels of such compounds in transgenic plants has resulted in increased stress tolerance.2,4 The genes used for this were often of microbiological origin. Certain gene products might also be involved in the repair of damage caused by the stress. In addition to the cellular content, membranes also play an important role in adaptation. Especially, the degree of saturation of the membrane lipids is an important factor in this.6 When studying the response to stress, one should take into account that organs can differ in this respect. As an example seeds, and often pollen also, can survive extreme desiccation, whereas the vegetative parts and flowers are susceptible to such Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants, edited by Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki. ©1999 R.G. Landes Company.

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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

conditions. This response allows the seeds to survive in a dry state and is also present in those species that grow under favorable conditions. The acquisition of this desiccation tolerance during seed maturation is very similar to vegetative responses to water deficits. In addition to these cellular mechanisms, plants can control their water status by controlling water uptake and water loss. Water uptake can be regulated by the architecture and physiology of the root system, whereas water loss is regulated not only by controlling morphological modifications to avoid excessive loss of water such as specific surface structures found in succulent plants but also by the strict control of stomatal aperture. When such adaptations result in stress resistance the mechanism has been called “avoidance.” Despite variation in the nature of adverse conditions, it should be emphasized that abiotic stresses can have components in common. Insufficient water supply can result from an excessive loss of water, or an insufficient uptake of water. The latter can also result from a high concentration of osmotic material in water, which usually is salts. Chilling and freezing may also lead to osmotic stress due to reduced water absorption and cellular dehydration. It is likely that for coping with other types of abiotic stresses such as UV light, heat, touch, wounding and hypoxia, plants have different mechanisms available. As an example Reactive oxygen species (ROS) are involved in the damage due to ozone but also have been implicated in the damage that occurs from drought and chilling stress.7 Plant possess a number of mechanisms and enzyme systems to scavenge ROS. However, the protection of ROS targets is also a mechanism to deal with such damage and recently Shen et al7 showed that mannitol can play such a role by protecting the enzyme phosphoribulokinase against oxidative inactivation. Another type of abiotic stress, which has its specific mechanisms and genetics, deals with heavy metals. The latter topic is beyond the scope of this review.

The Genetic Approach in Stress Physiology—General Principals The genetic approach in stress biology is based on finding genetic differences in stress responses and to relate this to the structure and function of the genes involved. The use genetic variation with a “detectable” phenotype is an important tool to identify genes related to stress and stress tolerance, because it allows the identification of the respective genes by various techniques such as map based cloning or tagging. The feasibility of these techniques in model species such as Arabidopsis explains why much of the genetic analysis focuses on this species. Genetic variation can be generated by mutations, but also exists in nature or, in case of crop plants, among cultivated varieties. Genetic variation within species is often of a different type than that found in mutant screens. In contrast to mutants, which can be grown in protective environments to survive even when they are weak growing plants, natural variants need to survive under normal growth conditions and therefore in any case should be well growing plants. In order to understand the underlying biochemical and molecular bases of genetic variation present in nature, it is important that the genetic differences in stress tolerance can be correlated with traits and genes that confer this difference in tolerance. For this it is not sufficient to see a correlation in the parents, but this correlation between traits should be analyzed in segregating populations. However, a situation of close linkage still does not prove that these traits are due to the same gene (pleiotropism). Pleiotropism cannot be distinguished easily from close linkage by segregation analysis, unless very large populations are used. Since stress tolerance genetically behaves as a quantitative trait with large environmental effects on the parameters to be analyzed and often is under polygenic control, detailed genetic analyses are difficult. However, the advent of molecular markers, and the use of

Genetic Approaches to Abiotic Stress Responses

3

specific mapping populations and developments in statistical methods, have improved the so called QTL (quantitative trait loci) analysis very much and make this also suitable for the genetic analysis of stress tolerance.8 The more genes with known functions are placed on the various genetic maps, the more candidate genes for stress tolerance can be identified by the co-localisation of QTLs and candidate genes.9 The relation between the map location of the various dehydrin genes with the map position of a number of genes related to stress tolerance and other physiological traits in cereals is an example of this candidate gene approach.10 However, confirmation of a causal relationship should preferentially come from gene transfer experiments with alleles cloned from the various parental genotypes. In cases where individual QTLs have an effect that is large enough, it may be possible to identify the respective gene by map-based cloning approaches. Additional genetic variation can be generated by the introduction of specific genes which originate from other plant species or even from completely different organisms. The latter has opened completely new ways to modify stress tolerance in crop plants.2,4 Furthermore, genetics and gene transfer technology not only allow the modification of stress resistance but also are important tools as functional tests of components involved in the response of plants to abiotic stresses.2 Since most mutations reflect damages within the gene or its controlling elements, one expects that mutants lacking a function are most frequent. However, even loss of function mutants can result in an increase in the functioning of pathways when repressors of such pathways are mutated. Not all genes can be identified by looking for mutant phenotypes. A reason for this is that for some traits, either the phenotype is relatively subtle, or the phenotype is too general, e.g., reduction of plant size or vigor. To solve the problem of subtle phenotypes more sophisticated screens, for instance by using reporter genes, are being developed and will be described hereafter. Another reason for not finding specific mutants is that for many genes genetic redundancy exists. This means that mutations in such genes do not result in an obviously visible phenotype since the redundant counterpart produces enough product to (partially) substitute the function of the mutated gene. Recently a number of additional methods which include enhancer or gene trapping and reverse genetics11,12 have become available and allow the analysis of phenotypes associated with the (loss of) function of specific genes. Reverse genetics uses gene sequences that are identified by “molecular” approaches and in large scale sequencing projects. When collections of plants with insertions of T-DNA or transposable elements are available, one can use these to identify insertions in the gene of interest. Plants with insertions in such genes are identified by the ability to amplify DNA fragments in polymerase chain reaction (PCR). One PCR primer is based on the tagging-DNA, whereas the second is based on the sequence for which an insertion mutant is sought. In case no mutant phenotype is observed when the respective gene is disrupted, due to the redundancy mentioned above, one can expect mutant phenotype, when (insertion) mutations in duplicated genes are combined by cROSsing. In addition to loss of function mutations, gain of function mutants can be isolated by introducing enhancer sequences next to relevant genes resulting in activation of such genes (activation tagging).13 The study of transgenic plants in which cloned genes are over-expressed often has given clues about the function of the respective genes. The development of appropriate mutant screens is a crucial aspect in any genetic approach. One can focus on the stress trait itself, which might be considered as the end point of a signal transduction chain. However, one can also pay attention to specific components known to be involved in stress responses. Examples of the latter are the search and analysis of mutants affected in abscisic acid (ABA) biosynthesis or ABA response and the analysis of mutants affecting specific stress related genes, either by finding mutations in those genes or by finding mutations that modify the expression of such genes. The ease of

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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

finding mutants also depends on the effectiveness of the screening system. For this, resistance to, for instance, salt seems a more attractive screen than the isolation of salt susceptible genotypes. However, screens of the latter type have been successfully applied in case of salt14 and frost.15 A number of these direct and indirect approaches to select mutants affected in stress responses will be described, together with the results obtained in analyzing “natural” genetic differences. In what way these analyses have led and are expected to lead to a better understanding of the responses to abiotic stress will also be discussed.

Genetic Differences in Salt Tolerance Salt overly sensitive (sos) Arabidopsis mutants were isolated by the inability of their roots to grow on 50 mM NaCl.14 It was shown that these mutants were defective in the highaffinity K+ uptake system. These mutants are hypersensitive to salt stress because relatively high Na+ concentrations inhibit the residual low affinity system of Na+ and K+, which results in potassium deficiency, which is the cause of the growth defect. The mutants also affect Na+ uptake and consequently accumulate less.15 In contrast to sos1 mutant, the sos3 mutant can be rescued by high external Ca2+.16 Attempts to isolate salt tolerant mutants in Arabidopsis resulted in the rss mutants, that express this tolerance only at the seed-germination level.17,18 NaCl tolerance at the germination level was also found to be a characteristic of ABA deficient mutants, which trait in these mutants was considered as reflecting the reduced seed dormancy characteristic of ABA deficient mutants.19 No seed dormancy or ABA related phenotype was reported for the rss mutants, which locus maps at a different position than the three known ABA deficient (aba) mutants. Genetic variation for salt tolerance has been described in many crop plants and their wild relatives. In a number of cases, these have been associated with selective ion uptake.20 Hexaploid wheat (Triticum aestivum) is more salt tolerant than tetraploid durum wheat (T. turgidum). It was shown that in hexaploid wheat a single gene (Kna1) is responsible for accumulating less Na+ and more K+ in expanding and young leaves. This gene is located on chromosome 4D, which is lacking in tetraploid wheats.21 The examples mentioned indicate that ion uptake and ion transport can affect stress tolerance. However, in addition, genetic differences in salt tolerance have also been associated with properties that relate to other osmotic mechanisms. For example, Saneoka et al20 found that near-isogenic maize lines differing in glycine betaines also differed in salt tolerance. Moons et al23 showed that ABA accumulation upon salt stress was much more in a salt tolerant rice cultivar as compared with a sensitive variety. The accumulation of a number of ABA induced LEA dehydrin type proteins was also higher in the tolerant varieties. Whether the latter correlations are causal could not be established, since the various traits were not analyzed in segregating populations. Salt tolerance seemed an attractive system for selection at the cell and tissue culture level. However, success of this approach appeared limited to a few successful examples where salt tolerant plants could also be obtained.24

Genetic Differences in the Response to Low Temperatures The ability of plants to tolerate low temperatures differs much between species. Distinctions are often made between chilling (temperatures 600 mM sorbitol, leading to necrotic lesions in sink leaves.227

example, two key genes, Inps1 and Imt1, are transcriptionally enhanced by salt stress, and higher enzyme amounts lead to increased carbon flux through myo-inositol into pinitol biosynthesis in stressed Mesembryanthemum.8,127-129 Genes involved in the degradation of compatible solutes are down-regulated under osmotic stress. This is, for example, the case for proline oxidase in Arabidopsis thaliana. Stress-dependent lower expression of this enzyme, at least in part, may explain the increases in proline during salinity and drought stress.130 Third, many accumulating compounds are end-products of a branch pathway rather than active intermediates in so far as one enzyme in the pathway catalyzes only the forward reaction. Examples for this point are DMSP synthesis in marine algae,9,131 pinitol synthesis in Mesembryanthemum4,55 and glycinebetaine synthesis.6,132-134 Equally, proline biosynthesis has received much attention, because proline accumulation is a nearly universal reaction of plants to osmotic stress.135-137 Its true role in stress protection is, however, not clear—we consider the accumulation of proline a consequence of the necessity for readjusting carbon nitrogen balance under stress.138 The biosynthesis of ectoine (tetrahydropyrimidine and derivatives), an accumulating osmolyte in bacteria, has received

Molecular Mechanisms of Salinity Tolerance

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Fig. 3.2. Pathways for the synthesis of selected compatible solutes. Biochemical pathways originating from glucose-6-P or sorbitol-6-P whose presence in some stress-tolerant species or after gene transfer into transgenic tobacco is correlated with increased osmotic stress tolerance. Genes/ enzymes used in transgenic experiments are PGM (phosphoglucomutase), INPS (myo-inositol 1-P synthase), IMT (myo-inositol O-methyltransferase), GPDH (sorbitol-6-P dehydrogenase), MtlDH (mannitol-1-P dehydrogenase), TPS (trehalosephosphate synthase). Pase indicates unspecific phosphatases. OEP (ononitol epimerase) is found in Mesembryanthemum, but the gene has not yet been cloned. IMP (myo-inositol monophosphatase) is not regulated in Mesembryanthemum during stress and has not been included in transgenic plants.

attention recently.130,140 Expression of the three enzymes leading to ectoine in bacteria confers significant salinity tolerance. Figure 3.2 shows schematically selected pathways that lead to the synthesis of polyols (mannitol, sorbitol, ononitol and pinitol) and to trehalose synthesis.141 Apart from the pinitol biosynthetic pathway,8,11 the pathways shown are engineered pathways (Table 3.1) and may be different from pathways existing in some plant species naturally. The scheme indicates clearly how the addition of a single gene can be exploited for metabolic engineering.

Water Channels Water channels, aquaporins (AQP), are found in all organisms as members of a super-family of membrane proteins, 26-30 kDa in size, termed MIP (major intrinsic protein).142,143 The proteins are characterized by six membrane-spanning domains and a pore-domain with a characteristic sequence signature, NH3-NPAXT-COOH. Aquaporins enhance membrane permeability to water in both directions depending on osmotic pressure differences across a membrane, but other members of the gene family in yeast and vertebrates encode glycerol-facilitators.143 Other MIPs, animal and plant—among them a nodulationspecific protein, may mediate ion transport and transport of other neutral metabolites, such as urea.144,145

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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

Complexity of Plant Isoforms In human DNA, five MIP genes have been characterized among a total of seven MIP-like genes. They are expressed in different tissues, most highly in erythrocytes, kidney cells and the brain. In contrast, Arabidopsis contains at least 23 MIP-like coding regions.146 Sequence signatures of the Arabidopsis MIP indicate two large sub-families of 10 to 12 proteins each whose members are either plasma membrane-located (PIP) or tonoplast-located (TIP), and one MIP which diverges from the others has not been characterized.146 While some of the genes might encode facilitators for diverse small metabolites or ions, eight MIP proteins have already been identified as aquaporins. Why are there so many plant aquaporins? We discuss four possibilities which might explain the high number. 1. MIP-intrinsic functional variations might allow AQP to be active at different membrane osmotic potentials. Yet, all we know is that the intrinsic water permeability distinguishes four human AQP and one glycerol facilitator by a factor of ~100,147 and that plant AQP can be either sluggish or effective water transporters when expressed in Xenopus oocytes.143 There is no report about a functional plant model that would allow mechanistic studies on AQP. By antisensing with a plasma membrane AQP coding region148 which supposedly targeted all expressed PIP, the decrease in AQP amounts led to a decline in water uptake in plants. Such antisense AQP transgenics increased the root to shoot ratio, suggesting a feedback mechanism between water uptake and root mass (Kaldenhoff R, personal communication). Protoplasts from the antisense-expressing plants did not burst as fast as wild type cells when transferred to hypoosmotic solutions.148 2. Functional differences could have evolved for fine tuning water flux through the plant—with high conductance AQP located in the root cortex and vascular tissues which accommodate bulk fluxes and low conductance channels between mesophyll cells, for example, or even within the cell cytosol and organelles and the vacuole. 3. Without assuming functional diversification, the number of AQP arising through gene duplications could have changed gene and protein expression, half-life, and turnover such that AQP amount shows a gradient that follows the water transport gradient. In this scenario, the gene number—requiring different promoters, RNAstability and translation characteristics and protein half-life regulation—would be determined by the necessity of cell-specific differences in accommodating water flux and not by the water transport function per se. This explanation is similar to the following one, and both find precedence in the presence of, for example, a large number of genes encoding plasma membrane H+-ATPases, AHA, which are differentially expressed throughout the plant.95,149,150 Deletion of several AHA genes did not produce a phenotype under normal growth conditions, but affected growth significantly under diverse growth and stress conditions, low temperature, salt stress and external high acidity, for example (Sussman MR, personal communication). 4. Last, AQP/MIP multiplications and diversifications could have been dictated by the need for a flexible response to environmental changes in water supply or evaporation, demanding the presence of several sets of AQP. This assumes evolution of one set of AQP genes for stress responses and that this set is different from others. It is conceivable that a set of Mip genes exists to take care of the business of cell expansion following meristematic activity—and this function (missing from animals) might require regulatory circuits different from those necessary in genes that perform housekeeping (set 2) and stress-response functions (set 3). Although the data are not complete with respect to AQP protein expression and cell-specificity, alignments of sequences indicate that sub-families of two to four closely related sequences

Molecular Mechanisms of Salinity Tolerance

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exist146,151 which might represent the three sets of genes. MIP associated with cell expansion,152 developmental specificity 153,154 and stress functions151,155-158,257 have been described. Mechanisms of Regulation Most important to the topic here is how MIP gene expression, protein amount and aquaporin activity are controlled during development and under environmental stress. Regulation is by gene expression and protein amount, and possibly also by post-translational modification—but we have very little information on mechanistic details in plants. Weig et al146 used quantitative PCR amplification for the 23 Arabidopsis MIP and found differences in mRNA amounts spanning several orders of magnitude. Differences in RNA amounts for each MIP in roots, leaves, bolts and the flowers and siliques were equally pronounced. No signals were detected for at least three MIP, suggesting that these might be expressed under conditions not found during normal growth or that they are expressed in a few cells only or at very low levels. The analysis of such a large gene family, once all genes are known, can best be done by in situ hybridization, immunocytology with specific antibodies and DNA microarray analysis through which the amount, location and regulation of the genes during development and under different environmental conditions can be monitored. For several MIP in a number of organisms, salt stress altered mRNA amounts have been reported. AQP expression also responds to drought and low temperature, hormone treatment (ABA, cytokinine, GA), light, and pathogen infection.143,157,158 Promoter studies have been performed with several MIP, but cell-specificity is most likely the essential distinguishing factor between AQP and must receive more attention in order to understand water transport in plants. The promoter for Rb7a159 from tobacco conveys root-specificity, leads to differential expression in the root in a cellspecific manner and is induced by nematode feeding.156,159 The Mesembryanthemum MipB promoter showed highest expression of the gene in roots;151 after transfer into tobacco and observation of GUS expression, broader specificity was observed, with highest expression in all meristematic cells and in vascular tissues.160 Even less complete is the information about protein amount, localization and changes during development and under stress conditions. One essential consideration is that the large number of genes and high sequence identity among PIP and TIP, respectively, require excellent controls for avoiding cross-hybridizations between transcripts and immunological cross-reactivity between antisera. For example, generation of anti-peptide antibodies against six Mesembryanthemum MIP resulted in distinguishable signals to different cells.161 However, in the absence of probes for all MIP for this species, it cannot be excluded that some of the antibodies react to more than one MIP whose sequence is not yet known, but shares homology with the selected peptide domain. Regulation has been documented at the level of post-translational modification, mostly in animal systems. Salt stress conditions in kidney cells lead to changes in protein expression, which may be controlled by oligomerization, glycosylation, or phosphorylation.162,163 In addition, the presence in the cell membrane and the half-life of AQP is determined by the hormone vasopressin in animal cells. Increased vasopressin leads to the deposition of AQP from internal stores, endosome vesicles, to the outer membrane, and lower hormone levels lead to cycling of membrane patches through endosomes.164 Clearly, such traffic and its control would constitute the fastest, most economic way of regulating water flux. Similar observations remain to be made with plant MIP, but patches of invaginated plasma membrane regions, termed “plasmalemmasomes” that contain abundant AQP protein have been found in plants,165 possibly the functional equivalent of animal endosomes. Our preliminary experiments indicate that PIP from Mesembryanthemum

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sediments in different gradient fractions depending on whether salt-stressed or unstressed cells were used,161 which might indicate that similar membrane shuttle mechanisms exist in plant cells. Evidence for plant AQP regulation comes from studies which measured AQP phosphorylation.153,166 Regulatory sites for phosphorylation have been mapped in several MIP/AQP. 143 Also, effects of pharmaceutical agents on water flow in Chara cells, for example, point towards an association of water flux and the integrity of the cytoskeleton (see ref. 143). Spinach leaf PIP are reversibly phosphorylated in response to the apoplastic water potential and calcium.166 The discovery and preliminary characterization of AQP in plants has provided more questions than answers. Their existence cannot be questioned and they act as water channels. It is then intuitively obvious that control over their action should be important under stress conditions. Although there are few data available, it is equally clear that regulation during stress is complex, involving transcriptional and post-translational controls which seem to involve synthesis, membrane traffic and reversible insertion into membranes, complex assembly and MIP protein half-life.

Salt Stress and Radical Scavenging Reactive Oxygen Species and Radical Scavenging Systems Production of Reactive oxygen species (ROS) is an unavoidable process in photosynthetic tissues, but ROS are also produced in mitochondria and cytosol. ROS including singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals react with and can damage proteins, membrane lipids, and other cellular components.33,167,168 Some ROS also serve as signaling molecules, 20 for example, in the initial recognition of attack by fungal pathogens and the transmission of signals after a primary infection.169,170 Focusing on chloroplasts, superoxide is abundantly produced from photoreduction of oxygen. Oxygen concentration as high as 300 mM can be photoreduced to superoxide by photosystem I via a Mehler reaction.171,172 The production of superoxide has been estimated to be approximately 30 mmol (mg chl)-1 h-1 in intact chloroplasts,173 and the rate of production in isolated thylakoids was increased 1.5-fold by the addition of ferredoxin and decreased 50% by addition of NADP+.174 Most of this thylakoid lumen-produced superoxide diffuses to the stroma.173 H2O2 in chloroplasts is predominantly generated by disproportionation of superoxide by SODs. In peroxisomes, H2O2 originates directly from glycollate oxidase activity. Hydroxyl radicals derive from an interaction between hydrogen peroxide and superoxide or directly from hydrogen peroxide in the presence of transition metals such as Fe+2 and Cu+ by a Fenton- or Haber-Weiss-reaction. The oxidized metal ions can be re-reduced by superoxide, glutathione, or ascorbate. Trace amounts, lower than the amount present in chloroplasts, of metal ions are needed to catalyze the Fenton reaction.168,173 It has in fact been shown that elevated amounts of iron lead to increased oxidative stress.175 These Reactive oxygen species are scavenged by resident enzyme systems and nonenzymatic antioxidants. 176 Non-enzymatic detoxification mechanisms include morphological features such as waxy surfaces and leaf or chloroplast movement, nonphotochemical quenching processes by various compounds, for example, the violaxanthinzeaxanthin cycle, and photorespiration. Non-enzymatic antioxidants include flavonones, anthocyanins, α-tocopherol, ascorbate (at a concentration of ~10 mM in chloroplasts), glutathione, carotenoids, phenolics and polyols.20,32,168,177 Botanical sources of such antioxidants not only play important roles in plant stress adaptation, but also retard aging and diseases related to oxidative damage in animals.178

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The enzyme systems involved include SODs which catalyze the reaction from superoxide to hydrogen peroxide, and ascorbate peroxidases (APX) responsible for the conversion of hydrogen peroxide to water. Both SOD and APX are represented by isoforms localized to the stroma and the thylakoid membrane. Ascorbate can be regenerated by the ascorbateglutathione cycle. The level of reduced glutathione is maintained by glutathione reductase using NADPH.168,179,180 In addition, catalase has recently been demonstrated as a sink for H2O2 in C3 plants.181 In contrast to the detoxification systems for H2O2 and O2-, an enzyme system that could deal with the short-lived, extremely toxic hydroxyl radical has not been identified and, in fact, might not have evolved.167,168,179,180 The best way of detoxifying hydroxyl radicals is to prevent their formation by reducing the concentration of H2O2 and free metal ions. Once produced, however, protection depends on the presence of antioxidants in the vicinity of the formation site. Together these systems provide sufficient protection under normal growth conditions; in fact, the scavenging systems are able to handle moderate increases of ROS, unless long-term stress exceeds the detoxification capacity.20,179,182 In chloroplasts, oxidative damage includes first a decline in CO2 fixation, and then inhibition of photochemical apparatus, loss of pigments, oxidation of proteins, and lipid peroxidation.183,184 ROS and Environmental Stress Several lines of evidence support the toxicity of ROS during drought,20 chilling stress184 and salt stress.29,30 First, superoxide production is enhanced, as detected by EPR signals in drought stressed wheat and sunflower.185,186 Equally, H2O2 content increased about three-fold during drought and low temperature.187-189 Enhanced production of ROS resulted in an increase in lipid peroxidation, as documented by a more than 5-fold increase of malonaldehyde production in wheat.190 Second, the concentration of free transition iron increased under drought stress,190,191 which stimulated production of hydroxyl radicals in the presence of high concentrations of H 2O 2 via a Fenton reaction. Compared to superoxide and H2O2, hydroxyl radicals oxidize a variety of molecules at near diffusioncontrolled rates. Finally, levels of non-enzymatic radical scavengers, such as ascorbate, carotenoids, flavonoids, sugar polyols, and proline,183 increase and may complement enzyme protection systems. Excellent evidence for a protective effect of ROS scavenging systems has recently been provided by the overexpression of an enzyme with the combined activities of glutathione S-transferase, GST, and glutathione peroxidase, GPX.192 By doubling the GST/GSX activity in transgenic tobacco, the seedlings and plants showed significantly faster growth than wild type during chilling and salt stress episodes. The increased enzyme activities resulted in higher amounts of oxidized glutathione (GSSG) in the stressed plants, indicating that the oxidized form could provide an increased sink for reducing power. Another set of experiments shed light on the relationships between ROS and the accumulation of polyols. When a bacterial gene (mtlD) encoding mannitol-1-phosphate dehydrogenase was modified so that the enzyme was expressed in chloroplasts, transgenic tobacco contained approximately 100 mM mannitol in the plastids. Using transgenic plants, freshly prepared cells and a thylakoid in vitro system, the protective effect exerted by mannitol on photosynthesis characteristics could be shown.29, 30 The presence of mannitol resulted in increased resistance to oxidative stress generated by methylviologen, and cells exhibited significantly higher CO2 fixation rates than controls during stress. After impregnation of tissue and cells with dimethyl sulfoxide, a hydroxyl radical generator, mannitol-containing cells showed a lower rate of methane sulfinic acid production than wild type, indicating that mannitol acted specifically as a hydroxyl radical scavenger. It could be shown that the primary damage was to enzymes of the Calvin cycle and not to

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components of the light harvesting and electron transfer systems,30 a confirmation of earlier reports.22 At present, the interpretation which we favor is that mannitol interferes with either hydroxyl radical production or damage, but it is unknown whether the protective mechanism is by exclusion of hydroxyl radicals from protein surfaces, a chemical interaction between mannitol and hydroxyl radicals, or by inhibiting or reducing the amount of hydroxyl radicals produced in the Fenton reaction.

Plant Ion Uptake and Compartmentation H+-ATPases and Vacuolar Pyrophosphatase Plasma membrane and vacuolar proton transporters play essential roles in plant salinity stress tolerance by maintaining the transmembrane proton gradient that assures control over ion fluxes and pH regulation (Fig. 3.3).101,193 Three proteins/protein complexes exist for this purpose: the plasma membrane (H+)-ATPase (P-ATPase) and two vacuolar transport systems, a (H+)-ATPase (V-ATPase) and a pyrophosphatase (PPiase). The plant P-ATPase is represented by a gene family of more than 10, encoding proteins of ~100 kDa, with homology to the yeast PMAs.95,150 As the main proton pump in the outer cell membrane it is essential for many physiological functions.194 Increased activity of the proton pump has been shown to accompany salt stress. Halophytic plants have been shown to increase pump activity under salt stress conditions more drastically than glycophytes,56,195 but little is know about the regulatory circuits that lead to either increased protein amount or activity during salt stress. The V-ATPase, a multi-subunit complex homologous to organellar, yeast (VMA) and bacterial F0F1-ATPases, has already been shown to be important in plant salinity tolerance. Electrophysiological studies revealed increased activity of this ATPase when cells or tissues from stressed plants were analyzed.196,197 Transcripts for several subunits of the V-ATPase are upregulated following salt shock.198,199 In Mesembryanthemum, V-ATPase activity increases several-fold following stress.200,201 In a Mesembryanthemum cell culture model it has now been shown, based on immunological data, that the V-ATPase (and possibly the P-ATPase) activity does not increase due to more protein being present, but an unknown mechanism stimulates activity 2 to 3-fold.201 The response is specific for NaCl and could not be elicited by mannitol-induced osmotic stress. PPiase genes and tonoplast-located PPiase proteins have been characterized in detail.94 Contrary to previous assumptions, the enzyme has now been authenticated as also residing in the plasma membrane.202 Its function, if any, under salt stress conditions is little known. A few reports have indicated that PPase activity declines under salt stress in some species,203,204 but increases in others.205 Potassium Transporters and Channels One possible passage for sodium across the plasma membrane is through transport systems for other monovalent cations. Among those the most significant is the uptake system for potassium, the most abundant cation in the cytosol, with important roles in plant nutrition, development and physiological regulation. Many studies have focused on identifying components involved in K+-transport. Physiological observations indicating a biphasic uptake of K+ into roots206 gave rise to the assumption that two uptake entities should be involved, a high-affinity system functioning at µM concentrations of external K+ and a low-affinity system active in the mM range of potassium. Several plant K+ transporter and K+ channel genes have been isolated by functional complementation of yeast mutants deficient in K+ uptake58,59,207 or by sequence homology with known K+ transporter or channel genes. 208-210 Electrophysiological studies in heterologous

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Fig. 3.3. Transport proteins implicated in plant salinity stress tolerance. The schematic depiction of a plant cell includes the vacuole, chloroplast (cp), mitochondrion (mt) and cell wall (shaded). Transmembrane proton gradients established by proton-ATPases and pyrophosphatase are indicated (+/-). Under NaCl stress, Na+ and Cl- are sequestered to the vacuole, and K+ and osmolytes are present in high concentrations in the cytosol. Symbols for several membrane-located transporters and channels are identified by the ion or proton transported and by the direction of movement. For organelles (mt and cp) no transporters have been characterized through molecular techniques. A Na+-ATPase, included in the plasma membrane is hypothetical, and a Na+/H+-antiporter in the plasma membrane has not been detected.

expression systems, such as Xenopus oocytes or yeast cells, indicated that some of them may function at both affinity ranges.211 Inward-rectifying potassium channels function in the mM range, following the electrochemical gradient at the plasma membrane and are categorized as low-affinity systems. 212,231 The AKT1- and KAT1-types of plant channels, similar to the Shaker channels in animals, contain a pore-forming region conferring ion selectivity. In contrast to earlier assumptions, these channels are highly selective against Na+,213 and evidence is lacking for specific regulation under salt stress. We think that the potassium channels play a minor role in salinity tolerance. In contrast, K+ transporters which operate at low external potassium may mediate entry of sodium in saline soil. A high-affinity K+-transporter is known from yeast.214 Some of the cloned transporters take up potassium with dual-affinity.209,211 A high-affinity K+ transporter from wheat, HKT1, was indicated as a K+/Na+ symporter86 with high-affinity binding sites for both K+ and Na+. Point mutations, which increased K+ selectivity over Na+, in one of the 12 transmembrane domains of HKT1 conferred increased salt tolerance of yeast. Another line of evidence for the involvement of high-affinity K+ uptake system in salt tolerance came from the study of salt-sensitive mutants. The sos1 mutant of Arabidopsis thaliana was characterized as hypersensitive to Na+ and Li+ and was unable to grow on low

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potassium.92 86Rb uptake experiments showed that sos1 was defective in high-affinity potassium uptake, and it became deficient in potassium when treated with NaCl. Interestingly but not surprisingly, expression of the wheat Hkt1 in sos1 mutant plants alleviated the saltsensitive phenotype (Schroeder JI, Zhu J-K, personal communication). Further support is provided by the expression characteristics of a rice homolog of wheat HKT1 in two varieties that are distinguished by their salinity tolerance. The tolerant variety decreased expression of the root-specific HKT1 and efficiently excludes sodium, while a salt-sensitive variety maintained high expression of the HKT1 in the presence of high NaCl.210,215 Irrespective of the indices pointing to the involvement of HKT1-type transporters, or high-affinity potassium uptake systems in general, in salt tolerance, there are other equally likely scenarios. First, the presence of sodium is known to interfere with potassium uptake, as shown for several of the cloned transport proteins, and protective effects exerted by increased potassium might be based on the nutritional value, and not on a sodium exclusion mechanism. High sodium sensitivity, as for example shown by the sos1 mutant, might be due to growth interference when K+ uptake is reduced by the presence of sodium. In this respect, the improved selectivity of K+ transport systems may increase salt tolerance, while it is not involved in Na + detoxification or osmotic adjustment. Other transport systems, finally, might act in sodium uptake. How, for example, the calciumregulated outward-rectifying K+-channel KCO1,216 or the regulation of other channels and transporters, react under sodium stress conditions is unknown. It has been suggested that sodium might enter through outward-rectifying cation channels.217 Among the many possibilities, evidence for significant sodium currents through a calcium transporter, LCT1, exists,218 and hexose and amino acid transporters may also let sodium pass. Sodium Transport Systems How sodium enters plant cells, how it enters the plant circulatory system to be selectively transported over long distances, and how it is partitioned to the vacuole is not known in detail. Most information is available for the last step in this series: sodium transport from cytosol to vacuole is accomplished by a sodium/proton antiporter. A protein of approximately 170 kD219 is a candidate for this tonoplast-located antiporter based on immunological studies and inhibition of the ameloride-regulated antiport activity in the presence of the antibody. It will be important to characterize the protein in detail and to obtain the gene(s), because, when judged by protein size, the putative antiporter seems to be different from the proteins in bacteria, yeast and vertebrate organisms. Increased sodium/proton antiport activity during salt stress has been measured in several model systems, tissues, cells and isolated vacuoles.96,220,221 The increase parallels an increase in the V-ATPase activity.96,200 Our own data indicate that yet another pathway for sodium uptake may exist. When analyzing the induction of myo-inositol synthesis in Mesembryanthemum, a surprising decline of the rate-limiting INPS (myo-inositol-1-phosphate synthase) enzyme in roots was observed, but the concentration of myo-inositol remained constant in the roots.128,129 This is due to drastically enhanced transport of myo-inositol from the leaves through the phloem. In addition, myo-inositol is recycled to the leaves through the xylem and the myo-inositol amount in xylem vessels is correlated with sodium amounts.129 We have cloned a transcript with homology to vertebrate sodium/myo-inositol and yeast proton/myo-inositol symporters222 and characterized its activity by complementation of a yeast mutant defective in myo-inositol uptake.223 It seems possible that such a symporter is responsible for the excretion of sodium into the xylem, but it is equally possible that sodium/myoinositol symport internalizes sodium from the apoplast of the root. The detection of such a symport mechanism is particularly attractive, considering that the passage of myo-inositol

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through the plant circulatory system connects photosynthesis competence with sodium uptake and transport to mesophyll cells of the leaf. The Essentiality of Calcium Increasing calcium improves salinity tolerance of crop plants. Physiological experiments indicated that the effect is mediated through an increase of intracellular calcium, changes in vacuolar pH and activation of the vacuolar Na+/H+-antiporter.224, 225 The strict control over calcium concentrations in the cytosol and calcium storage in a number of locations (vacuole, mitochondria, endoplasmic reticulum) assign a crucial role to calcium in plant salinity stress responses. Recently, an Arabidopsis mutant, sos3, with hypersensitivity to NaCl has been characterized. The mutant is different from other salt-sensitive mutants92 in that the phenotype can be masked by the external addition of calcium.226 This phenotype represents the first mutant with an altered response to calcium in higher plants. The phenotype reveals the link between calcium and salinity stress tolerance, although the mechanism through which hypersensitivity and remediation by calcium are connected is not known. One attractive hypothesis is that a signaling system that responds to calcium spikes at low calcium concentrations—for example a homolog of the yeast calcineurin-type system—is defective, and that at higher calcium concentrations a second sensing system can support the signal and elicit stress defense responses (Zhu JK, personal communication).

Metabolic Engineering of Glycophytic Plants for Increased Salt Tolerance In increasing numbers, experiments are reported using transgenic plants for testing concepts originating from the correlative evidence of physiological analyses. Table 3.1 summarizes some of these reports. The concepts tested target four aspects of tolerance acquisition: 1. ROS scavenging, 2. Compatible solutes and osmotic adjustment—carbohydrate biosynthesis and synthesis of charged molecules, 3. Ion balance—potassium uptake vs. sodium uptake, and 4. The synthesis of specific, putatively protective proteins. A note of caution must be added with respect to the over-expression and accumulation strategies that have been followed up to now. Too high an accumulation of metabolites, or too efficient scavenging of H2O2, for example, may not be desirable. When analyzing transgenic tobacco plants that accumulated sorbitol to extremely high concentrations in the cytosol, we observed stunted growth and the formation of necrotic lesions that reduced biomass production, although the plants showed increased salinity and salt stress tolerance.227 The importance of radical oxygen scavenging for preventing oxidative stress in plants has been demonstrated by genetic engineering of several enzymes into transgenic plants.179,180,228 Overexpression of superoxide dismutase (Cu/Zn-SOD and Mn-SOD), ascorbate peroxidase, catalase and glutathione reductase in transgenic plants has already been shown to lead to increased resistance to oxidative stress.181,229,230,232-238 The most dramatic protective effect, up until now, was observed after enhancement of the glutathione cycle.192 In contrast, overexpression of an Fe-SOD in transgenic tobacco neither enhanced tolerance to chilling-induced photoinhibition in leaf discs nor increased tolerance to salt stress in whole plants,240 suggesting that isoforms of SOD may have different roles. Noctor and Foyer20 provided a lucid assessment of the relatively marginal protection that has been observed in many transgenic plant studies, whether with respect to ROS scavenging or otherwise. It would certainly be premature to consider the protection

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provided by the overexpression of SOD, ASX, or enzymes of the ascorbate/glutathione cycle as the final word. Protection has typically been observed in strictly controlled environments, and protective effects have often been marginal. We would like to provide one consideration as to why this is to be expected. In the case of ASX, at least six different isoforms exist which are located in mitochondria, in chloroplasts (several, in different sub-compartments/membranes), soluble in the cytosol, and in the cytoplasmic endomembrane system.241 A similarly complex distribution has been seen for SOD isoforms which are found in the cytosol (Cu/Zn-SOD), mitochondria (Mn-SOD) and plastids (Fe-SOD and Cu/Zn-SOD). Transgenic modifications of single enzymes are likely to have a minimal effect because of the multitude of compartments that require protection. Irrespectively, these experiments have clearly shown that—in practically every study— the engineered expressed transgene elicited some protection. It is now necessary to adopt multi-gene transfer strategies that alter several components of the stress tolerance system: 1. Targeting, for example, ROS scavenging enzymes to several compartments; 2. Assembling gene constructs that target sodium exclusion and enhanced potassium uptake; 3. Generating transgenomes in which different pathways are satisfied, for example, ion homeostasis, carbon allocation, and protein protection simultaneously; 4. Generating transgenomes with strategies that take into account cell-, tissue-, organ- and developmental specificity. The last point is particularly important, because little attention has typically been paid to the “when,” “where,” and “how much” of transgene expression in the presently concluded transgenic experiments. Significantly more attention needs to be directed to the promoter elements that drive transgenes. Most attempts have targeted the metabolic engineering of carbon and nitrogen allocation: ectopic enzyme expression leading to the synthesis of uncharged carbohydrates—mannitol, sorbitol, trehalose, fructan, and ononitol—and to glycinebetaine and proline accumulation (Table 3.1). The underlying mechanism is becoming apparent for some of these strategies, e.g., in the hydroxyl radical scavenging function of mannitol. 29,30 The mechanisms of protection underlying the synthesis or presence of chaperones or specific LEA proteins remain to be determined. Within a very short time, all genes that are essential for the salt tolerance phenotype shown by some species and all genes that support damage avoidance in sensitive species will be available. The task remaining, however, is understanding in which metabolic and signaling pathways the gene products function and in which developmental context stress protection is necessary. This task will require new approaches. We consider two approaches: 1. Multi-gene transfer into model species—yeast, Arabidopsis, tobacco and rice are our suggestions; and 2. A focus on metabolic control analysis. The first strategy utilizes the transfer of all genes, controlled by appropriate promoter elements, for one or several biochemical pathways to generate protection which can be analyzed. Through the second approach, a biochemical description of flux in a multitude of pathways, we will be able to gauge the cost of enzymes/pathways that enhance tolerance in comparison to the cost and benefits of resident pathways.

Perspectives

High salinity is a major factor responsible for the loss of crop biomass.242 Salinity caused by irrigation affects many productive agricultural areas. The degeneration of still productive soils will become a more severe problem in the future. Development of drought- and salt-tolerant crops has been a major objective of plant breeding programs for decades in order to maintain crop productivity in semiarid and saline lands. Although

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several salt-tolerant varieties have been released, the overall progress of traditional breeding has been slow and has not been successful.13 The lack of success is mainly due to the quantitative trait character of salinity tolerance which has to be reconciled with another multigenic trait, high productivity, which is the ultimate goal of any breeding program. Marginal progress has equally been grounded in our poor understanding of the mechanisms of salt tolerance, while the collected body of physiological data has focused our attention more on details in a large variety of species and less on the principles. This has changed over the last few years. Biochemical pathways that lead to the production of compatible solutes such as proline, glycine betaine, DMSP, or pinitol have been studied and most of the pathway genes have been characterized.6,7,9,14,15 We have the first glimpses of how the resulting metabolites from such pathways function in protection. Similarly, the principles of how radical oxygen species act and the principles, genes and proteins which deter radical damage have emerged. Membrane channels, transporters and pores are now available through which cells exert control over ion, carbohydrate, amino acid or water fluxes.58,59,61,146,207,243 We owe most of this recent progress to the power of the yeast and Arabidopsis thaliana molecular genetic systems. Finding the genes whose disruptions generate the various mutant phenotypes becomes rapidly easier as additional mapping data and genomic DNA sequences from Arabidopsis are made available.12 Finally, plant stress perception, and inter- and intracellular signaling of salt stress has been advanced greatly. Mutants in signal transduction pathways and components of several signal transduction pathways have been found and are being characterized at present.244-249 Future studies can follow the blueprint of signaling components isolated from yeast 10,66,68,88,250 for finding and characterizing homologs of the essential signaling intermediates in plants. If we accept that a major objective of plant stress research is application, transgenic crops can be engineered not only for expression of novel biochemical characters, but also for stress signal transduction that enhances the stress response inherent to all plants.

Acknowledgments Because of space constraints a number of references could not be included, and we apologize. We thank Ms. Pat Adams for help with the manuscript. Different projects have, off and on, been supported by the US National Science Foundation (Integrative Plant Biology and International Programs), Department of Energy (Biological Energy), and Department of Agriculture (NRI). Additional support has been provided by the Arizona Agricultural Experiment Station, Japan Tobacco Inc., Rockefeller Foundation (New York) and New Energy Development Organization (Tokyo).

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193. Guern J, Mathieu Y, Kurkdjian A et al. Regulation of vacuolar pH in plant cells. Plant Physiol 1989; 89:27-36. 194. Michelet B, Boutry M. The plasma membrane H+-ATPase. Plant Physiol 1995; 108:1-6. 195. Weiss M, Pick U. Primary structure and effect of pH on the expression of the plasma membrane H+-ATPase from Dunaliella acidophila and Dunaliella salina. Plant Physiol 1996, 112:1693-1702. 196. Reuveni M, Bennett AB, Bressan RA et al. Enhanced H+ transport capacity and ATP hydrolysis activity of the tonoplast H+-ATPase after NaCl adaptation. Plant Physiol 1990; 94:524-530. 197. Ayala F, O’Leary JW, Schumaker KS. Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr. in response to NaCl. J Exp Bot 1996; 47:25-32. 198. Loew R, Rockel B, Kirsch M et al. Early salt stress effects on the differential expression of vacuolar H+-ATPase genes in roots and leaves of Mesembryanthemum crystallinum. Plant Physiol 1996; 110:259-265. 199. Tsiantis M, Bartholomew DM, Smith JAC. Salt regulation of transcript levels for the c-subunit of a leaf vacuolar H+-ATPase in the halophyte Mesembryanthemum crystallinum. Plant J 1996; 9:729-736. 200. Barkla BJ, Zingarelli L, Blumwald E, Smith JAC. Tonoplast Na+/H+ antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembryanthemum crystallinum. Plant Physiol 1995; 109:549-556. 201. Vera-Estrella R, Barkla BJ, Bohnert HJ et al. Salt stress in Mesembryanthemum crystallinum suspension cells activates adaptive mechanisms identical to those observed in the whole plant. Planta, in press. 202. Robinson DG. Pyrophosphatase is not (only) a vacuolar marker. Trend Plant Sci 1996; 1:330. 203. Bremberger C, Luettge U. Dynamics of tonoplast proton pumps and other tonoplast proteins of Mesembryanthemum crystallinum L. during the induction of crassulacean acid metabolism. Plant 1992; 188:575-580. 204. Matsumoto H, Chung GC. Increase in proton-transport activity of tonoplast vesicles as an adaptive response of barley roots to NaCl stress. Plant Cell Physiol 1988; 29:1133-1140. 205. Zingarelli L, Anzani P, Lado P. Enhanced K + -stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus. Physiol Plant 1994; 91:510-516. 141. 206. Epstein E (1966) Dual pattern of ion absorption by plant cells and by plants. Nature 212:1324-1327. 207. Schachtman DP, Schroeder JI. Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants. Nature 1994; 370:655-658. 208. Santa-Maria GE, Rubio F, Dubcovsky J et al. The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter. Plant Cell 1997; 9:2281-2289. 209. Kim EJ, Kwak JM, Uozumi N et al. AKUP1: An Arabidopsis gene encoding high-affinity potassium transport activity. Plant Cell 1998; 10:51-62. 210. Golldack D, Su H, Bennett J et al. Differential expression of HKT1-type potassium transporters in salt-sensitive and salt-tolerant rice lines. Manuscript in preparation, 1998. 211. Fu H-H, Luan S. AtKUP1: A dual affinity K+ transporter from Arabidopsis. Plant Cell 1998; 10:63-73. 212. Maathuis FJM, Verlin D, Smith FA et al. The physiological relevance of Na +-coupled K +-transport. Plant Physiol 1996; 112:1609-1616. 213. Bertl A, Anderson JA, Slayman CL et al. Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: Characterization of Kat1, an inwardrectifying K+ channel from Arabidopsis thaliana, and comparison with endogeneous yeast channels and carriers. Proc Natl Acad Sci USA 1995; 92:2701-2705 214. Gaber RF, Styles CA, Fink GR. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol Cell Biol 1988; 8:2848-2859.

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215. Golldack D, Kamasani U, Quigley F et al. Salt stress-dependent expression of a HKT1-type high affinity potassium transporter in rice. Plant Physiol 1997; S114:118. 216. Czempinski K, Zimmermann S, Ehrhardt T et al. New structure and function in plant K+-channels: KCO1, an outward rectifier with a steep Ca2+ dependency. EMBO J 1997; 16:2565-2575. 217. Schachtman DP, Tyerman SD, Terry BR. The K+/Na+ selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species. Plant Physiol 1991; 97:598-605. 218. Schachtman DP, Kumar R, Schroeder JI et al. Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants. Proc Natl Acad Sci USA 1997; 94:11079-11084. 219. Barkla BJ, Apse MP, Manolson MF et al. The plant vacuolar Na+/H+ antiport. Symp Soc Exp Biol 1994; 48:141-153. 220. Blumwald E, Poole RJ. Salt tolerance in suspension cultures of sugar beet: Induction of Na+/H+ antiport activity at the tonoplast by growth in salt. Plant Physiol 1987; 83:884-887. 221. Garbarino J, DuPont FM. NaCl induces Na + /H + antiport in tonoplast vesicles from barley roots. Plant Physiol 1988; 86:231-236. 222. Cammarata PR, Xu GT, Huang L et al. Inducible expression of Na + /myo-inositol cotransporter mRNA in anterior epithelium of bovine lens: Affiliation with hypertonicity and cell proliferation. Exp Eye Res 1997; 64:745-757. 223. Nelson DE, Bohnert HJ. Characterization of the sodium/myo-inositol symporter from Mesembryanthemum crystallinum. Manuscript in preparation, 1998. 224. Colmer TD, Fan TWM, Higashi RM et al. Interactions of Ca2+ and NaCl stress on the ion relations and intracellular pH of Sorghum bicolor roots tips: An in vivo 31P-NMR study. J Exp Bot 1994; 45:1037-1044. 225. Martinez V, Lauchli A. Effects of calcium on the salt-stress response of barley roots as observed by in vivo phosphorus-31 nuclear magnetic resonance and in vitro analysis. Plata 1993; 190:519-524. 226. Liu J, Zhu JK. An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance. Proc Natl Acad Sci USA 1997; 94:14960-14964. 227. Sheveleva E, Marquez S, Zegeer A et al. Sorbitol dehydrogenase expression in transgenic tobacco: High sorbitol accumulation leads to necrotic lesions in immature leaves. Plant Physiol 1998; 117:831-839. 228. Allen RD. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol 1995; 107:1049-1054. 229. Aono M, Kubo A, Saji H et al. Resistance to active oxygen toxicity of transgenic Nicotiana tabacum that expresses the gene for glutathione reductase from E. coli. Plant Cell Physiol 1991; 32:691-697. 230. Aono M, Kubo A, Saji H et al. Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic glutathione reductase activity. Plant Cell Physiol 1993; 34:129-135. 231. Maathuis FJM, Ichida AM, Sanders D et al. Roles of higher plant K+ channels. Plant Physiol 1997; 114:1141-1149. 232. Bowler C, Slooten L, Vandenbranden S et al. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J 1991; 10:1723-1732. 233. Bowler C, Van Montagu M, Inze D. Superoxide dismutase and stress tolerance. Annu Rev Plant Phys Plant Mol Biol 1992; 43:83-116. 234. Gupta AS, Heinen JL, Holaday AS et al. Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci USA 1993; 90:1629-1633. 235. Van Camp W, Wilekens H, Bowler WH et al. Elevated levels of superoxide dismutase protect transgenic plants against ozone damage. Biotechnol 1994;.12:165-168. 236. McKersie BD, Chen Y, de Beus M et al. Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol 1993; 103:1155-1163.

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237. Foyer CH, Souriau N, Perret S et al. Overexpression of glutathione reductase but not glytathione synthetase leads to increase in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiol 1995; 109:1047-1057. 238. McKersie BD, Bowley SR, Harjanto E et al. Water-deficit tolerance and field performance of transgenic alfalfa overexpressing superoxide dismutase. Plant Physiol 1996; 111:1177-1181. 239. Pitcher LH, Zilinskas BA. Overexpression of copper/zinc superoxide dismutase in the cytosol of transgenic tobacco confers partial resistance to ozone-induced foliar necrosis. Plant Physiol 1996; 110:583-588. 240. Van Camp W, Capiau K, Montagu M et al. Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe-superoxide dismutase in chloroplasts. Plant Physiol 1996; 112:1703-1714. 241. Jespersen HM, Kjaersgard IV, Ostergard L et al. From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase. Biochem J 1997, 326:305-310. 242. Boyer JS. Plant productivity and environment. Science 1982; 218:443-448. 243. Sauer N, Stolz J. SUC1 and SUC2: Two sucrose transporters from Arabidopsis thaliana; expression and characrerization in baker’s yeast and identification of the histidine-tagged protein. Plant J 1994; 6:67-77. 244. Nishihama R, Banno H, Shibata W et al. Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells. Plant Cell Physiol 1995; 36:749-757. 245. Kakimoto T. CKI1, a histidine kinase homolog implicated in cytokinin signal transduction. Science 1996; 274:982-985. 246. Ishitani M, Xiong L, Stevenson B et al. Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: Interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 1997; 9:1935-1949. 247. Hirt H: Multiple roles of MAP kinases in plant signal transduction. Trends Plant Sci 1997; 2:11-15. 248. Mizoguchi T, Irie K, Harashida N et al. A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 1996; 93:765-769. 249. Mizoguchi T, Ichimura K, Shinozaki K. Environmental stress response in plants: The role of mitogen-activated protein kinases. Trends in Biotech 1997; 15:15-19. 250. Shinozaki K, Yamaguchi-Shinozaki K. Gene expression and signal transduction in waterstress response. Plant Physiol 1997; 115:327-334. 251. Tarczynski MC, Jensen RG, Bohnert HJ. Expression of a bacterial mtlD gene in transgenic tobacco leads to production and accumulation of mannitol. Proc Natl Acad Sci USA 1992; 89:2600-2604. 252. Tarczynski MC, Jensen RG, Bohnert HJ. Stress protection of transgenic tobacco by production of the osmolyte, mannitol. Science 1993; 259:508-510. 253. Thomas JC, Sepahi M, Arendall B et al. Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis thaliana. Plant Cell Environ 1995; 18:801-806. 254. Pilon-Smits EAH, Ebskamp MJM, Paul MJ et al. Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol 1995; 107:125-130. 255. Hayashi H, Alia, Mustardy L et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J 1997; 12:133-142. 256. Xu D, Duan X, Wang B et al. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 1996; 110:249-257. 257. Jones JT, Mullet JE. Developmental expression of a turgor-responsive gene that encodes an intrinsic membrane protein. Plant Mol Biol 1995; 28:983-996.

CHAPTER 4

Plant Cold Tolerance Michael F. Thomashow and John Browse

P

lants vary greatly in their responses to cold temperatures. At one extreme are many plants from tropical and subtropical regions which suffer injury when exposed to low nonfreezing temperatures. These include economically important plants such as cotton, soybean, maize, rice, and many tropical and subtropical fruits. Such chilling-sensitive plants undergo sharp reductions in growth rate and development at temperatures between 0˚ and 12˚C.1,2 The physical and physiological changes in chilling-sensitive plants that are induced by exposure to low temperatures, together with the subsequent expression of stress symptoms, are termed chilling injury. The symptoms that are associated with chilling injury include reduced or retarded germination and seedling emergence, wilting and chlorosis of leaf tissue, electrolyte leakage and tissue necrosis. In sharp contrast to plants of tropical origin, those from temperate regions are not only chilling-tolerant, but many are able to survive freezing. Herbaceous plants from temperate regions can survive freezing temperatures ranging from -5˚ to -30˚C, depending on the species, while trees from boreal forests routinely survive winter temperatures below -30˚C. Significantly, the maximum freezing tolerance of these plants is not constitutive, but is induced in response to low nonfreezing temperatures (below ~10˚C), a phenomenon known as “cold acclimation.” For instance, rye plants grown at normal warm temperatures are killed by freezing below about -5˚C, but after cold acclimation can survive freezing temperatures down to about -30˚C. What accounts for the differences in cold tolerance among plant species? Why are cucumber and rice plants injured at chilling temperatures while cold-acclimated cabbage and wheat survive freezing below -15˚C? The answers to these questions are of basic scientific interest and have potential practical applications. Cold temperatures limit the geographical locations where crop and horticultural plant species can be grown and periodically cause significant losses in plant productivity. Greater knowledge of the molecular basis of chilling and freezing tolerance could potentially lead to the development of new strategies to improve plant cold tolerance, resulting in increased plant productivity and expanded areas of agricultural production. Here we summarize the current understanding of the molecular basis for chilling and freezing injury and discuss recent advances in the identification of genes involved in cold tolerance.

Chilling Tolerance Role of Membranes in Chilling Injury The majority of chilling-sensitive plants share a similar threshold for the onset of low-temperature damage and exhibit a common assembly of symptoms. These observations Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants, edited by Kazuo Shinozaki and Kazuko Yamaguchi-Shinozaki. ©1999 R.G. Landes Company.

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have been interpreted by many investigators as indicating that there is a single primary lesion, or trigger, that initiates cell damage at some critical temperature and leads to a cascade of secondary events that are the more readily appreciated consequences of chilling damage.2 Several primary lesions have been proposed, but the most widely studied hypotheses involve temperature-dependent changes in membrane lipid structure.3,4 Early suggestions envisioned a mechanism in which the lipids in membranes underwent an overall phase transition from the liquid crystalline (Lα) state to the gel (Lβ) state.1,5 According to this proposal, the transition from liquid crystalline phase to gel phase would result in alterations in the metabolism of chilled cells and lead to injury and death of the chilling-sensitive plants. It was quickly recognized that such a mechanism was an oversimplification6 but it was more than ten years before a more sophisticated version of the membrane hypothesis was articulated. Raison and Wright7 observed that small additions of disaturated phospholipids to preparations of wheat polar lipids could produce entropy changes during differential scanning calorimetry that were quantitatively similar to those observed for polar lipid extracts from chilling-sensitive mung bean plants. These experiments suggested that only a portion of the lipids (4 to 7%) was actually undergoing a phase change in the 0˚ to 12˚C temperature range. Meanwhile, Murata and coworkers demonstrated a strong correlation across different plant species between the degree of chilling sensitivity and the proportion of disaturated phosphatidylglycerol (PG); molecules that contain only 16:0, 18:0 and 16:1-trans fatty acids.8 Chloroplast PG is invariably synthesized with 16:0 at the sn-2 position of the glycerol backbone. Although this 16:0 may be converted to ∆3-16:1-trans, the geometry of this trans-unsaturated fatty acid is very similar to that of saturated fatty acids. For this reason, the level of disaturated PG depends on the extent to which the glycerol-3-phosphate acyltransferase specifically selects 18:1-ACP to the exclusion of 16:0-ACP and 18:0-ACP that are also available as substrates in the chloroplast stroma.4 Invoking disaturated molecular species of PG as the cause of chilling sensitivity was attractive because, in contrast to proposals based on less precise concepts of lipid unsaturation, it provided a mechanism underpinned by a firm biophysical explanation. Thus, preparations of PG purified from three chilling-sensitive plants were observed to enter the Lα to Lβ phase transition at 29˚ to 33˚C, whereas PG from chilling resistant plants did not enter the transition until the temperature was below 15˚C.9 More recently, the molecular-species distribution of PG in tobacco and Arabidopsis plants has been altered by molecular genetic techniques.10,11 Murata et al10 transformed tobacco plants with gene constructs encoding glycerol-3-phosphate acyltransferase from either squash or Arabidopsis. Transgenic plants containing the squash gene contained elevated levels of disaturated PG (76% of total PG) compared with controls (36%) and showed more damage after chilling. Conversely, transgenic plants expressing the Arabidopsis gene contained 28% disaturated PG and showed less chilling damage than control tobacco plants. One of the measures of chilling injury in this study was the extent of photoinhibition of photosynthesis. Subsequent studies by Moon et al12 revealed that there is no difference between the rate at which transgenic and wild type plants undergo chilling-induced photoinhibition. Rather, the principal effect of the variation in the amount of disaturated PG seems to be on the rate at which damaged photosystems can be repaired. The main target for photoinhibition is thought to be the D1 polypeptide of the photosystem II reaction center.13 When this protein is damaged, presumably by side products from the photochemical reactions,14 newly synthesized D1 protein is inserted into the photosystem II complex to restore photochemical activity. Thus, Moon et al12 hypothesize that the altered amount of disaturated PG has an effect on the rate at which damaged D1 protein is removed from the photosystem II complex and replaced by synthesis and insertion of new protein. An important unanswered question is whether the effect of disaturated-PG

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content on D1 turnover extends to other chloroplast membrane proteins. Conceivably, the D1 protein is simply an efficient reporter of a general defect in the assembly or removal of membrane proteins. Transformation of tobacco plants with a suitably modified version of the des9 gene from Anacystis nidulans, which encodes a broad-specificity glycerolipid ∆9 desaturase substantially reduced the level of saturated fatty acids in PG and other membrane lipids.15 Again, the transgenic plants were more tolerant to chilling treatments than control plants. Interestingly, modest increases in overall membrane unsaturation also appear to reduce chilling damage in tobacco. Kodama et al16 used the Arabidopsis FAD7 gene in transgenic tobacco to produce a modest increase (~10%) in the conversion of dienoic to trienoic fatty acids in membrane lipids with no apparent change in the level of disaturated PG. Compared with control plants, the transgenic tobacco exhibited significantly higher leaf expansion rates following a 7 day exposure to 1˚C. This result is somewhat surprising since the change in lipid composition would not be expected to influence the tendency of membrane lipids to undergo a phase transition. In a similar approach, Wolter et al11 restructured the E. coli plsB gene, which encodes a membrane-bound glycerol-3-phosphate acyltransferase, so that the peptide was directed into the chloroplasts of transgenic Arabidopsis that expressed the gene. The bacterial acyltransferase utilizes both 16:0- and 18:1-ACPs as substrates. The resulting transgenic Arabidopsis plants contained 48-54% disaturated PG and were damaged by treatment at 4˚C for 7 days. These findings suggest that disaturated PG species can induce low-temperature sensitivity in a chilling-tolerant plant such as Arabidopsis, although it is not formally possible to rule out the possibility that accumulation of the plsB protein might have contributed to the phenotype observed.11 Notwithstanding this accumulated body of evidence, studies on the Arabidopsis fab1 mutant demonstrated that the level of disaturated PG cannot be the sole determinant of plant chilling sensitivity. fab1 plants contain an increased proportion of 16:0 fatty acids because of a partial defect in 3-ketoacyl-ACP synthase II, the enzyme responsible for elongation of 16:0 to 18:0.17 As a consequence, PG from fab1 leaves contains 43% disaturated molecular species compared with only 9% in PG from the wild type. The proportion of disaturated PG in fab1 falls close to the middle of the range found for chillingsensitive plants and makes the fab1 mutant comparable to species such as castor bean, cucumber, maize and tobacco.18 However, fab1 plants were able to grow and complete their life cycle normally at 10˚C. They were also unaffected (as compared with wild type controls) by more severe chilling treatments that quickly led to the death of cucumber and other chilling-sensitive plants. These treatments included 4˚C for 7 days in the dark, 2˚C for 7 days in the light and freezing to -2˚C for 24 hours.17 Following each of these treatments, mutant plants returned to 22˚C remained indistinguishable from wild type controls and flowered and set seed normally. fab1 mutant plants do eventually show damage when grown continuously at 2˚C with reduced photosynthesis, reduced growth and leaf chlorosis developing gradually from 10 to 35 days of low-temperature treatment. At 2˚C, fab1 plants undergo a process of chloroplast autophagy.19 Therefore, although the fab1 mutant does not exhibit classic chilling sensitivity, the results of Wu et al19 confirm a deleterious effect of high levels of disaturated PG on low-temperature fitness and provide a rationale for the relatively low content of disaturated PG among chilling-tolerant species which may be exposed to low temperatures for extended periods of the life cycle. Indeed, the chilling-induced chlorosis and slow growth of the fab1 mutant could be due to a defect in chloroplast membrane protein turnover or accumulation of the kind described by Moon et al12 However, the side-by-side comparison of the fab1 mutant with naturally chilling-sensitive species that contain similar levels of

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disaturated PG emphasizes the point that factors other than high levels of disaturated PG are responsible for the injury sustained by these and other chilling-sensitive plants. In summary, these results make it clear that reducing the proportion of disaturated PG, and perhaps increasing overall lipid unsaturation, can measurably improve the low-temperature performance of tobacco plants, possibly through facilitating turnover of the D1 protein and thereby allowing faster recovery from photoinhibition. At the same time, disaturated PG cannot be considered the sole cause of chilling sensitivity because high levels of disaturated PG in Arabidopsis fab1 did not produce a typical chillingsensitive phenotype. Nor is the manipulation of membrane lipids the only way to improve low-temperature performance of tobacco plants. Gupta et al20 produced transgenic tobacco plants that overexpress a chloroplastic Cu/Zn superoxide dismutase. Leaf disks from transgenic plants had higher rates of photosynthesis at 10˚C compared with untransformed controls and also exhibited a greater capacity for recovery at 25˚C after photoinhibition at 3˚C for 4 hours. These findings imply that protecting tissues from the effects of oxidative stress may also reduce chilling damage. The work described here on higher plants is complemented by studies in cyanobacteria. In these prokaryotes, a high level of saturated fatty acids is correlated with an inability to grow at low temperatures, either because of reduced processing of D1 protein21 or reduced activity of nitrate uptake.22 A more complete review of studies in cyanobacteria is included in Nishida and Murata.23

Additional Mechanisms of Chilling Injury Although the roles of membrane lipid unsaturation and disaturated PG in chilling sensitivity have been well established, there are also well-documented examples where other processes must be responsible. One of these involves the chilling of tomato plants in the dark, under which condition photoinhibition and considerations of D1 turnover are clearly not relevant. Tomato plants that have been chilled in the dark show greatly reduced photosynthesis rates during subsequent illumination. Understanding the possible cause of this chilling damage started with the observation that many proteins involved in photosynthesis are products of genes whose transcriptional activities cycle under control of the circadian clock. Martino-Catt and Ort24 used genes for the chlorophyll a/b binding protein of photosystem II (Cab) and for ribulose-1,5-bisphosphate carboxylase/oxygenase activase (rca) to demonstrate that chilling stops the circadian clock. They discovered that low temperature has two separate effects on the normal pattern of expression of Cab and rca proteins: 1. Progression of the timing of the circadian clock controlling gene transcription is suspended throughout the period of low-temperature exposure; and 2. Normal turnover of the existing transcripts is suspended. Upon rewarming, the circadian rhythm of transcriptional and translational activity is reestablished, but is out of phase with the actual time of day by the amount of time that the tomato plant was at low temperature. In addition, after rewarming, the messages that were stabilized at low temperature can no longer be translated into protein. From these results, it is reasonable to suggest that a range of gene-expression and other functions controlled by the circadian clock may be affected by chilling in tomato. This would clearly be expected to result in considerable disruption of photosynthesis and other cellular functions. A few plant species have been demonstrated to have a capacity to increase their chilling tolerance in response to treatment at modestly cool temperatures. Dark-grown seedlings of the maize inbred G50 were killed by exposure to 4˚C for 7 days but could be induced to survive this treatment by prior exposure to 14˚C for 3 days.25 Differential screening techniques allowed the isolation of cDNAs representing chilling acclimation responsive genes including

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65 Fig.4.1. The effects of chilling temperature on the growth rates of Arabidopsis lipid mutants. The results shown are for (A) fad5 and fad6 at 5˚C (B); fab1 at 2˚C; and (C) fad2 at 6˚C. Wild type controls are included in each experiment.

cat3, which encodes the mitochondrial catalase 3 isozyme. Hydrogen peroxide levels in the seedlings were increased during acclimation at 14˚C and treatment of seedlings grown at 2˚C with H2O2 induced chilling tolerance and increased both cat3 transcript levels and the activities of catalase 3 and guaiacol peroxidase. From these results, it appears that peroxide has dual effects at low temperatures. During acclimation at 14˚C, its early accumulation signals the production of antioxidant enzymes such as catalase 3 and guaiacol peroxidase. At 4˚C, in nonacclimated seedlings, it accumulates due to low levels of these, and perhaps other, antioxidant enzymes and may cause damage through oxidation of lipids and proteins.26

Genes Required for Chilling Tolerance Much of the discussion of temperature adaptation of plants focuses on finding defects in chilling-sensitive species that can explain why they are damaged by low temperatures. However, it is probably more useful, especially at the genetic level, to identify the traits that are responsible for the chilling tolerance observed in temperate plants. Thus, it is possible to screen mutant populations of a chilling tolerant species such as Arabidopsis for plants that are no longer fully tolerant to low temperature. Mutants with impaired chilling tolerance are defined as those which have a wild type appearance at normal growth temperatures but which show damage when transferred to chilling temperatures. These mutants each contain a mutation that has no effect at normal temperatures but is disruptive at chilling temperatures. In such a screen, only mutational defects that sensitize a mutant to chilling will be identified; mutations associated exclusively with other processes such as freezing tolerance are excluded. Somerville and coworkers initiated such a mutational approach.27 They screened a population of Arabidopsis mutated by ethyl methane sulfonate

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(EMS). About 20 mutants were isolated that showed damage symptoms in response to a brief and mild chilling regime; they had a normal, wild type phenotype at 22˚C, but after a week at 13˚C exhibited visual damage.28,29 An extensive characterization of one mutant, chs1,27-30 reinforces the validity of the mutational approach. The chs1 mutant showed low-temperature-induced chlorosis indicating a lesion in chloroplast maintenance at low temperatures. Subsequent investigations revealed a loss of chloroplast integrity30 and reduced accumulation of proteins localized to the chloroplast.28,29 The detected changes indicate a sequence of chilling-induced damage caused by disrupted protein accumulation in the chloroplast. Nevertheless, it has not been possible to identify the precise biochemical lesion responsible for initiating these changes. A collection of Arabidopsis mutants with defects in membrane lipid unsaturation31 have offered useful perspectives on the role of membrane unsaturation. Five mutant linesfab1 (see above), fad5, fad6, fad2 and the triple mutant—fad3-fad7-fad8—show damage symptoms when grown at 2˚-6˚C.17,32,33 All of these mutant lines are similar to wild type when grown at 22˚C, but their growth rates are lower than wild type at chilling temperatures (Fig. 4.1). However, in all cases (as discussed above for fab1), the low-temperature damage is distinct from typical chilling sensitivity. For example, symptom development is more gradual and damage is not exacerbated following a return to 22˚C. These results make it clear that a suitable membrane lipid composition is required for chilling tolerance, but that it is unlikely that membrane defects are the sole cause of chilling sensitivity. A more extreme chilling screen was used by Tokuhisa et al34 who exposed Arabidopsis plants to 5˚C for up to 42 days and looked for mutants both during the chilling treatment and after return of the plants to 22˚C. A screen of EMS mutagenized plants using this protocol identified 3% of the plants as having chilling-induced phenotypes including chlorosis, reduced growth, necrosis and death. One drawback of EMS mutagenesis is that the mutations are primarily single base pair substitutions. In many cases, these mutations destroy the function of the gene product. However, there are many examples where missense mutations result in an amino acid substitution in the mutated gene such that the altered polypeptide product functions adequately at normal (permissive) temperatures but loses function at low (nonpermissive) temperatures. If such a missense mutation is in an essential gene, the mutation will render a chilling-induced phenotype. Such alleles have been used extensively in yeast35 and E. coli36 to characterize essential housekeeping genes, and have been termed cold-sensitive or cs alleles. To circumvent this problem, Tokuhisa et al34 repeated the screen on a population in which mutations have been generated by T-DNA insertion.37 Insertion mutagenesis produces a high proportion of null alleles and will thus facilitate the identification, in our screen, of genes which are unnecessary at 22˚C but which are essential for proper growth at 5˚C. Just as importantly, the T-DNA insertion can act as a starting-point to clone and characterize the specific chilling-tolerance gene. Over 8,000 lines of mutants generated by T-DNA insertional mutagenesis were screened and about 280 putative mutants were identified. To date, about 200 of these putatives have been rescreened and 21 mutants have been shown to have heritable, chillingimpaired phenotypes. Two of these mutants, which exhibited chilling-induced chlorosis were designated paleface1 (pfc1) and pfc2. A third mutant that was inhibited in leaf expansion at 5˚C was designated stop1 (sop1). By segregation analysis, each of these mutants has been shown to have linkage, within 2-3 centiMorgans between the kanamycin resistance marker in the T-DNA and the chilling-induced phenotype. Therefore, it is highly probable that the T-DNA in each of these lines is inserted in a gene which is required for chilling tolerance. Molecular characterization of the pfc1 mutant has demonstrated a previously unrecognized requirement for ribosomal RNA processing and modification to provide chilling

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tolerance.38 The wild type allele of the mutated gene and a near-full length (>93%) cDNA clone were isolated by using the T-DNA as a tag. The deduced polypeptide has a 50 amino acid transit peptide for chloroplast targeting, an S-adenosylmethionine-binding motif and 34% identity with genes from bacteria and yeast encoding ribosomal RNA methylases which are required for ribosomal RNA processing or translation. The PFC1 transcript was absent from pfc1 plants and biochemical analyses indicated that the expected methylation of adenosines 1518 and 1519 in the small subunit rRNA occurred in the wild type but not in pfc1. Finally, expression of an antisense PFC1 construct in wild type Arabidopsis produced plants which exhibited the same low-temperature chlorosis seen in the pfc1 mutant. These results demonstrate that PFC1 function is specifically required for low-temperature tolerance of Arabidopsis.

Freezing Tolerance Causes of Freezing Injury As temperatures drop below freezing, ice forms primarily in the intercellular spaces (formation of intracellular ice is generally thought to be a fatal event).39 Ice formation is initiated extracellularly largely due to the apoplastic fluid having a higher freezing point than the intracellular fluids, but may also involve the relative levels of ice nucleating agents.40 Accumulation of ice in the intercellular spaces can potentially result in physical disruption of the tissues and cells.41 However, most of the injury is thought to result from the severe cellular dehydration that occurs with freezing.39,42 The chemical potential of ice is less than that of unfrozen water at a given temperature. Thus, when ice forms extracellularly, there is a drop in water potential outside the cell. Consequently, there is movement of unfrozen water from inside the cell to outside the cell. The net amount of water movement depends on both the initial solute concentration of the intracellular fluid and the freezing temperature, which directly determines the chemical potential of the ice. Freezing at -10˚C results in an osmotic potential of about five osmolar and typically, movement of greater than 90% of the osmotically active water out of the cell. Freeze-induced cellular dehydration could have a number of deleterious effects, resulting in cellular damage such as the denaturation of proteins and precipitation of solutes. 39,43 However, the best documented injury occurs at the membrane level.42,44 Detailed analyses by Steponkus and colleagues45,46 have demonstrated that multiple forms of membrane lesions occur in response to freezing. The specific type of membrane damage depends on the freezing temperature and corresponding severity of cellular dehydration. At freezing temperatures between about -2˚C and -5˚C, the predominant form of injury in nonacclimated plants is “expansion-induced lysis”. It results from the cycle of osmotic contraction and expansion that occurs with freezing and thawing. Specifically, when protoplasts from leaves of nonacclimated plants are frozen to about -4˚C, they dehydrate, and as they shrink, endocytotic vesicles bud off from the plasma membrane. When the protoplasts are thawed and water moves back into the cells, the vesicular material is not reincorporated into the plasma membranes, resulting in a decrease in membrane surface area. Consequently, rehydration results in an intolerable osmotic pressure and the cells burst. Freezing of nonacclimated cells to slightly lower temperatures, approximately -5˚ to -10˚C, results in another form of membrane damage, lamellar-to-hexagonal-II phase transitions.45,46 In this case, cells do not burst upon thawing, but instead become osmotically unresponsive due to the membranes losing their semipermeable characteristics. Freezing cold-acclimated cells to even lower temperatures, with consequent lower water potentials and more severe dehydration, results in additional forms of membrane damage, including “fracture jump lesions”.45,46

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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

Mechanisms of Freezing Tolerance Given the central role of membranes in freezing-injury, it is not surprising that multiple mechanisms appear to be involved in increasing the cryostability of membranes during cold acclimation. Steponkus and colleagues45,46 have demonstrated that unlike plasma membranes from nonacclimated plants, plasma membranes from cold-acclimated plants do not suffer expansion-induced lysis or formation of hexagonal II phase lipids. The elimination of these forms of membrane damage involve a number of changes in lipid composition, including increased levels of fatty acid desaturation in membrane phospholipids.45,46 In addition, the accumulation of sucrose and other simple sugars that typically occurs with cold acclimation seems likely to contribute to the stabilization of membranes, as these molecules can protect membranes against freeze-induced damage in vitro.47,48 Finally, as discussed below, there is emerging evidence that certain cold-induced hydrophilic polypeptides help stabilize membranes against freeze-induced injury. Additional mechanisms could also potentially contribute to freezing tolerance, including ones that help prevent or reverse freeze-induced denaturation of proteins or lessen the direct physical damage to cells caused by the accumulation of extracellular ice. Indeed, molecular chaperones have been shown to accumulate during cold acclimation, including a spinach Hsp70,49,50 a soybean Hsp7051 and a Brassica napus Hsp90.52 In addition, there is evidence suggesting that “freeze-inhibitor” sugars might lessen cellular damage by preventing the formation of adhesions between extracellular ice and the cell walls.53 Also, recent studies indicate that many plants accumulate antifreeze proteins during cold acclimation, some of which are present in the apoplastic fluids. 54-56 These proteins probably do not act by preventing ice formation as they are capable of imparting only a few tenths of a degree of thermal hysteresis (i.e., lower the freezing temperature by a few tenths of a degree without affecting the melting point of the solution). However, they could potentially contribute to freezing tolerance by modifying ice crystal structure and/or preventing ice recrystallization. In all of these cases, however, further study is required to clearly establish whether the proteins or sugars contribute significantly to freezing tolerance.

Role of Cold-Responsive Genes in Freezing Tolerance

In 1985, Guy et al57 established that changes in gene expression occur during cold acclimation. Since then, a fundamental question in cold acclimation research has been to determine whether cold-responsive genes have roles in freezing tolerance. To address this issue, researchers have engaged in the isolation and characterization of genes that are induced during cold acclimation.58,59 Many of these cold-responsive genes encode proteins with known activities that could potentially contribute to freezing tolerance. For instance, the Arabidopsis FAD8 gene60 and barley blt4 genes,61 which encode a fatty acid desaturase and a putative lipid transfer protein, respectively, are induced in response to low temperature. These genes might contribute to freezing tolerance by altering lipid composition. As alluded to above, cold-responsive genes encoding molecular chaperones49-52 might contribute to freezing tolerance by stabilizing proteins against freeze-induced denaturation. Cold-responsive genes encoding various signal transduction and regulatory proteins have also been identified, including MAP kinases,62,63 calcium-dependent protein kinases64,65 and 14-3-3 proteins.66 These proteins might contribute to freezing tolerance by controlling the expression of cold-responsive genes or by regulating the activity of proteins involved in freezing tolerance. Whether any of these cold-responsive genes have important roles in freezing tolerance, however, remains to be determined. While many of the cold-responsive genes that have been isolated from cold-acclimated plants encode proteins with known activities, the majority do not. Indeed, most encode

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69

Fig. 4.2. Genomic organization of COR gene families. Coding and intron regions are depicted as filled and open boxes, respectively. Alternative gene designations are listed. Accession numbers for the genes are: COR15a, X64138; COR15b, L24070; KIN1, X51474; COR6.6/KIN2, X55053/ X62281; LTI65/RD29b, X67670/D13044; COR78/LTI78/RD29a, L22567/X67071/D13044; LTI29/ ERD10, X90958/D17714; COR47/RD17, X90959/AB004872. The figure was drawn by Kathy Wilhelm.

extremely hydrophilic polypeptides that are either newly discovered or are homologs of LEA (late embryogenesis abundant) proteins.59,67 LEA genes are induced late in embryogenesis, just prior to seed desiccation, and like many of the cold-responsive genes, are induced in response to dehydration and ABA.68-70 Based largely on these expression characteristics and the close relationship between freezing and dehydration injury, it has been widely speculated that the cold-responsive genes encoding the novel hydrophilic and LEA proteins might contribute to freezing tolerance. Indeed, recent results provide direct evidence that the Arabidopsis COR (cold-regulated) genes contribute to the increase in freezing tolerance that occurs with cold acclimation.71,72

Arabidopsis COR Genes The Arabidopsis COR genes—also designated LTI (low temperature induced), KIN (coldinducible), RD (responsive to desiccation) and ERD (early dehydration-inducible)—comprise four gene families.67 Each family is composed of two genes that are physically linked in the genome in tandem array (Fig. 4.2). The COR78, COR15, and COR6.6 gene pairs encode newly discovered hydrophilic polypeptides, while the COR47 gen pair encodes homologs of LEA group II proteins (also known as dehydrins and LEA D11 proteins).68,69 At least one member of each gene pair is induced in response to low temperature, dehydration and exogenous application of ABA.58

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Molecular Responses to Cold, Drought, Heat and Salt Stress in Higher Plants

To determine whether COR15a might have a role in freezing tolerance, Artus et al.71 constructed transgenic plants that constitutively express the gene and assessed the effects that this had on freezing tolerance. COR15a encodes a 15 kDa polypeptide that is targeted to the stromal compartment of chloroplasts.73,74 The mature 9.4 kDa polypeptide, COR15am, is extremely hydrophilic and, like the other COR polypeptides and many LEA proteins, has the unusual property of remaining soluble upon boiling in aqueous buffer. In the initial experiments, Artus et al71 compared the freezing tolerance of chloroplasts in nonacclimated transgenic and wild type plants. The results indicated that the COR15am-containing chloroplasts in transgenic plants were 1 to 2˚C more freezing tolerant than were the chloroplasts in wild type plants that did not contain COR15am (cold acclimation increased chloroplast freezing tolerance about 6˚C). In additional experiments, they found that the effects of COR15am were not limited to the chloroplasts. Protoplasts isolated from leaves of the nonacclimated transgenic plants that constitutively produced COR15am were about 1˚C more freezing tolerant at freezing temperatures between -5 and -8˚C than were those isolated from nonacclimated wild type plants. Significantly, protoplast survival was measured by vital staining with fluorescein diacetate, a method that reports on retention of the semipermeable characteristic of the plasma membrane. Thus, it could be concluded from the protoplast survival experiments that constitutive expression of COR15a resulted in an increase in plasma membrane cyrostability. The results of Artus et al71 indicate a role for COR15a in freezing tolerance. However, unlike cold acclimation which increases protoplast survival over the range of -2˚ to -8˚C, expression of COR15a only increased survival over the temperature range of -5˚ to -8˚C (if anything, COR15a expression resulted in a slight decrease in protoplast survival between -2˚ and -4˚C). A possible explanation for this finding is that COR15a expression might prevent certain membrane lesions, but not others. As discussed earlier, the predominant form of membrane injury over the range of -2˚ to -4˚C appears to be expansion-induced lysis, while over the range of -5˚ to -8˚C, the predominant form of injury is freeze-induced lamellar-to-hexagonal II phase transitions. Thus, it is possible that constitutive expression of COR15a might defer the incidence of freeze-induced formation of hexagonal II phase lipids to a lower temperature, but have little or no effect on the incidence of expansion-induced lysis. Additional experimentation is required to test this hypothesis. The mechanism by which COR15a stabilizes membranes against freeze-induced injury is not yet known. It seems unlikely that the COR15am protein has enzymatic activity, as it has a very simple amino acid composition and structure: It is rich in alanine (21%), lysine (18%),glutamic acid (15%) and aspartic acid (10%) residues (which comprise greater than 60% of the protein); is devoid of proline, methionine, tryptophan, cysteine, arginine and histidine residues; and is comprised largely of a 13 amino acid sequence that is repeated (imperfectly) four times. This, however, leaves open many possibilities. COR15am may act indirectly to stabilize membranes. For example, it could potentially regulate the activity of proteins that have roles in freezing tolerance, such as enzymes involved in sugar or lipid metabolism. Alternatively, COR15am might interact directly with the chloroplast envelope and increase membrane cryostability in some manner. The location of COR15am within the chloroplast is not necessarily inconsistent with protection of the plasma membrane, as formation of the hexagonal II phase is an interbilayer event that occurs largely between the plasmalemma and the chloroplast envelope. Decreasing the propensity of the chloroplast envelope to fuse with the plasma membrane could result in less damage to the plasma membrane. Experiments to detect a direct effect of COR15am on the stabilization of membranes, however, have yielded equivocal results.75,76

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Fig. 4.3. CBF1 and COR transcript levels in nonacclimated wild-type Arabidopsis plants (RLD) and nonacclimated transgenic Arabidopsis plant lines that overexpress either CBF1 (A6 and B16)72 or COR15a (T8).71 Overexpression of CBF1 and COR15a was accomplished by transforming wild type RLD plants with hybrid gene constructs having the coding sequences for either CBF1 or COR15a under control of the cauliflower mosaic virus 35S promoter.71,72 Total RNA was prepared from leaves of nonacclimated plants and analyzed for CBF1 and COR transcripts by RNA blot analysis using 32P-radiolabeled probes.71,72 Overexpression of CBF1 results in the stimulation of COR gene expression, but does not affect the transcript levels of eIF4A (eukaryotic initiation factor 4A),92 a constitutively expressed gene that is not responsive to low temperature.

Although constitutive expression of COR15a enhances freezing tolerance at both the organelle (chloroplast) and cellular (protoplast) level, the effects are modest.71 Moreover, unlike cold acclimation, COR15a expression alone does not result in a detectable increase in freezing survival of whole plants.72 These findings are not surprising given the results of genetic analyses indicating that freezing tolerance is a multigenic trait involving genes with additive effects.77 Indeed, multiple genes are activated with cold acclimation in Arabidopsis, including at least one member of each of the four COR gene pairs.58 If multiple COR genes act in concert to increase freezing tolerance, then expression of the entire COR gene “regulon” would presumably increase freezing tolerance more than

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expressing COR15a alone. This hypothesis was recently tested by Jaglo-Ottosen et al.72 Expression of the entire battery of COR genes was accomplished by overexpressing the Arabidopsis transcriptional activator CBF1 (CRT/DRE binding factor 1).78 CBF1 binds to a DNA regulatory element, the CRT (C-repeat)/DRE (drought responsive element), that stimulates transcription in response to both low temperature and water deficit.79 The element is present in the promoters of COR15a, COR78, COR6.6, COR47 and presumably other yet to be identified COR genes. Jaglo-Ottosen et al72 found that constitutive overexpression of CBF1 induce expression of the COR genes in nonacclimated Arabidopsis plants (Fig. 4.3) and increased freezing tolerance at the whole plant level, an effect that was not observed by expressing COR15a alone. Thus, it appears that additional members of the Arabidopsis CRT/DRE regulon are freezing tolerance genes that have roles in cold acclimation. Determining which CRT/DRE-regulated genes have roles in freezing tolerance and their functions are now important goals. In addition, a critical point to establish is whether the CRT/DRE-containing COR genes regulate the full array, or only a subset, of the biochemical changes that occur with cold acclimation (alterations in lipid composition, accumulation of sugars, synthesis of anthocyanin, etc.). Other Possible Freezing Tolerance Proteins More than 20 years ago, Volger and Heber80 reported that cold-acclimated spinach and cabbage synthesize polypeptides that are highly effective in protecting isolated thylakoid membranes against freeze-thaw damage in vitro. These putative cryoprotective polypeptides were detected in cold-acclimated plants, but not nonacclimated plants, suggesting that they were encoded by cold-regulated genes. Subsequent studies by Hincha and colleagues81,82 indicated that the cryoprotective polypeptides act to protect membranes against freezeinduced damage by reducing membrane permeability during freezing and increasing membrane expandability during thawing. A significant limitation in all of these studies, however, was that only partially purified protein preparations were used. Thus, it was unclear whether the cryoprotective activity detected was due to a single protein or multiple polypeptides. Interestingly, however, from the enrichment procedures used, it was evident that the polypeptides, like the COR polypeptides, were very hydrophilic and remained soluble upon boiling. A significant advance in the study of the spinach and cabbage cryoprotective proteins was recently made by Sieg et al.83 These investigators purified a single cryoprotective protein from cold-acclimated cabbage that is effective in protecting isolated thylakoids against freeze-thaw damage in vitro. This protein, which was designated “cryoprotectin,” has a mass of 7 kDa, remains soluble upon boiling and appears to be encoded by a cold-inducible gene (the protein is present in cold-acclimated plants, but not in nonacclimated plants). Unfortunately, there is no information on the amino acid sequence of cryoprotectin, and thus, it is unknown whether it is related to any of the hydrophilic polypeptides encoded by the cold-responsive genes described above. Additional investigation should reveal more about the nature of cryoprotectin, its mode of action in vitro, and provide direct evidence whether it has a role in protecting membranes against freezing-injury in vivo. There is evidence accumulating that suggests certain LEA proteins may also contribute to freezing tolerance. The HVA1 gene of barley, which encodes a LEA group III protein (also known as LEA D7 proteins), is expressed in aleurone layers late in embryogenesis and in seedlings in response to low temperature, ABA and water deficit.84 Although there is no direct evidence that HVA1 expression contributes to increased freezing tolerance, recent results indicate that the gene is able to confer tolerance to dehydration stress. Xu et al85 have reported that expression of HVA1 in transgenic rice results in increased tolerance to

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Table 4.1 Phenotypes associated with sfr mutations in cold-acclimated plants.88,89 Mutant Gene

Freezing Sensitivity

Anthocyanin Level (% wt)

sfr1

Young leaves

162

Normal

No changes detected

sfr2

All leaves (severe)

87

Normal

No changes detected

sfr4

All leaves (severe)

8

Reduced amounts of both glucose (