DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Recent developments in understanding molecular responses of plants to
water stress
DEFRA project code
HH3607TX
Contractor organisation
and location
University of the West of England, Bristol
Coldharbour Lane
Frenchay
Bristol
BS16 1QY
Total DEFRA project costs
Project start date
£ 43036.70
01/08/03
Project end date
29/02/04
Executive summary (maximum 2 sides A4)
1. Professor Steven Neill (UWE, Bristol) was commissioned by Defra to undertake a Desk Study on ‘Recent
developments in understanding molecular responses of plants to water stress’. The aim was to summarize
recent molecular research work and suggest potential Defra funding priorities in this research area.
2. The report has been written in an iterative way. To ensure that the report is an accurate assessment of recent
research with balanced and objective outputs, it was informed by input from leading UK scientists with
expertise in water stress molecular biology and physiology.
3. To provide a commercial grower perspective, the report has also been informed by input from HRI and Defra
commodity specialists with expertise in various UK crops.
4. The report begins with an Executive Summary, written in such a way as to be most helpful to Defra scientists
and non-scientists. The Report proper starts with a brief overview of plant responses to water stress, with the
aim of providing some context to the subsequent detailed analyses of molecular responses. These analyses start
with an evaluative summary of effects on gene expression, move on to the intracellular signalling pathways
activated by water stress and then to a consideration of how water stress is actually perceived by plants. The
report then assesses developments in transgenic analyses to determine the impact of various genes on stress
tolerance, and concludes by identifying outstanding research questions and topics of potential relevance to
Defra.
CSG 15 (Rev. 6/02)
1
Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
Scientific report (maximum 20 sides A4)
Recent developments in understanding molecular responses of
plants to water stress
Steven Neill
Centre for Research in Plant Science
Faculty of Applied Sciences
University of the West of England, Bristol
Bristol BS16 1QY
UK
29 February 2004
01. Executive Summary
02. Introduction
03. Focus of this report
04. Experimental approaches
05. Effects of water stress on plants
06. Altered gene expression during and after water stress
07. Regulation of gene expression during water stress
08. Cell signalling during water stress
09. Perception of water stress
10. Functions of water stress-regulated genes – towards
water stress tolerance
11. Outstanding research questions and approaches
12. Potential Defra funding priorities
13. References
14. Appendix 1: UK expert group
15. Appendix 2: HRI expert group
16. Figures 1- 5
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
1. Executive summary
The report – process
1.1. Steven Neill (UWE, Bristol) has been commissioned by Defra to undertake a Desk Study on ‘Recent
developments in understanding molecular responses of plants to water stress’. The aim is to summarize
recent molecular research work and suggest potential Defra funding priorities in this research area.
1.2. The report has been written in an iterative way. To ensure that the report is an accurate assessment of
recent research with balanced and objective outputs, it has been informed by input from leading UK scientists
with expertise in water stress molecular biology and physiology (see Appendix 1).
1.3. To provide a commercial grower perspective, the report has also been informed by input from HRI and
Defra commodity specialists with expertise in various UK crops (see Appendix 2).
1.4. Following the summary, the report begins with a brief overview of plant responses to water stress, with the
aim of providing some context to the subsequent detailed analyses of molecular responses. These analyses start
with an evaluative summary of effects on gene expression, move on to the intracellular signalling pathways
activated by water stress and then to a consideration of how water stress is actually perceived by plants. The
report then assesses developments in transgenic analyses to determine the impact of various genes on stress
tolerance, and concludes by identifying outstanding research questions and topics of potential relevance to
Defra.
The report – content
1.5. Diminishing water supply represents one of the most serious global environmental problems of the 21 st
century. In the UK, water stress is an important factor limiting crop production, the costs of supplying water in
addition to or instead of rainwater are substantial and water use by agriculture is a major issue. There is thus a
pressing need to develop crops that are better able to tolerate periods of water stress and remain productive as
well as crops that have reduced water requirements.
1.6. Molecular and cell biological research into water stress responses of plants is high on the international
research agenda. Most work has been done with the model species Arabidopsis thaliana; other species include
tomato, rice, maize, the resurrection plant Craterostigma plantagineum and Commelina communis, a species
popular for stomatal work.
1.7. It is abundantly clear that water stress signalling is highly complex and that the signalling pathways interact
extensively. Many plant responses to water stress are mediated by the hormone abscisic acid (ABA). ABA is
rapidly synthesised in roots and shoots in response to declining soil water content and re-distributed to the
guard cells, inducing stomatal closure (the basis of Partial Root Drying [PRD] irrigation).
1.8. The expression of hundreds of genes is altered (positively and negatively) both under water stress and
during recovery from wilting. Many of these are ABA-regulated but there are also those that respond to water
stress per se. Water stress-regulated genes have been described as functional and regulatory. The former
encode proteins potentially active in increasing stress tolerance, the latter encode potential signalling proteins.
The biological functions of most of the stress-regulated genes are not yet known.
1.9. The biochemical processes by which water stress alters gene expression have been studied. Some water
stress- and ABA-responsive regulatory DNA sequences and their cognate transcription factors (DNA-binding
proteins) have been identified but others require characterisation. The precise details of how gene expression is
regulated remain to be elucidated.
1.10. Various intracellular signalling pathways activated by water stress and ABA have been identified. Key
components include calcium, inositol phospholipids, reversible protein phosphorylation, RNA metabolism and
processing, hydrogen peroxide and nitric oxide. These latter two compounds have recently been identified as
essential intermediates in ABA-regulated stomatal closure and it is increasingly clear that they are endogenous
plant signals.
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title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
1.11. Little is known of the mechanisms by which cells sense water stress or ABA. Some potential
sensor/receptor proteins have been identified but there are not yet any clear data demonstrating their function.
1.12.Transgenic analyses using genetically modified plants have shown that under- or over-expression of stressinduced genes encoding enzymes that synthesise compatible solutes or ‘detoxification’ enzymes, or of those
encoding signalling proteins such as regulatory enzymes of ABA biosynthesis, ABA- or stress-activated
transcription factors, protein kinases or calcium-binding proteins, can improve water stress tolerance. However,
most of these tolerance assays are simple laboratory survival assays and the effects of these manipulations on
field performance have not been established.
The report – outputs
1.13. Recent years have seen some excellent studies advancing our understanding of the molecular mechanisms
by which plants respond to water stress, with the ultimate goal being to enhance crop production. Although
there are still many unresolved issues, the data generated with model plants such as Arabidopsis and rice can
now be used to assess the importance of specific genes and signalling pathways in determining yield
characteristics related to water stress and to guide research in crop species. The deliverables of such research
will be the identification of genes and pathways whose action impacts directly on yield and survival under
water stress. Subsequent utilisation of these genes in crop plants is not a trivial task. Water stress tolerance is
not a single gene trait, and the complexity of water stress signalling means that ‘one gene for all’ solutions are
not possible. Moreover, there are few examples where direct translation of basic biochemical and physiological
knowledge into yield improvement has been achieved. However, where improvements have been realised, they
have in fact come about through amelioration of the consequences of abiotic stress, such as through improving
water use efficiency (WUE).
Despite the complexity of stress signalling, transgenic studies carried out so far do indicate that water
stress tolerance can be enhanced by the introduction of single genes, such as those encoding detoxification
enzymes, ABA-responsive transcription factors or pivotal enzymes of ABA biosynthesis. The success of these
latter approaches is likely to reflect the subsequent activation of many genes regulated by ABA.
It must be emphasised that molecular approaches to improving water use and stress tolerance do not of
necessity require strategies based solely on genetic modification (GM). In fact, there are alternative non-GM
approaches that, although using GM to identify and analyse useful genes, can then introduce these via non-GM
molecular plant breeding. Additionally, it may be possible to combine genetics and cultural practices to alter
stress signalling and gene expression in plants so as to modify plant metabolism at key stages of the crop cycle
and thereby conserve water and enhance yield.
1.14. I suggest that there is real need to link molecular biology with plant physiology and crop biology. There
is a good understanding of the processes regulating crop growth and yield and some of these processes may
provide targets for intervention. 1.15. I suggest that co-ordinated research efforts will be fruitful – involving the
input of collaborative research teams with different and complementary research expertise – for example, in
molecular biology, physiology, genetics and crop biology.
1.16. It is also important to take a long-term view of research – both in terms of future strategic requirements
and outputs from on-going programmes.
1.17. A key requirement for water stress management in the UK in the short-term is to develop crop plants that
respond to mild soil drying or atmospheric water deficit in ways that ensure yield maintenance. Events at the
‘wet end’ of water stress impact on yield. Thus elucidation of early signalling events – what signal transduction
pathways are altered, which genes are switched on/off/not affected etc, is certainly a research priority.
1.18. In other situations, it might be that plants will be subject to sporadic, relatively severe water stress. In
these situations, it could be beneficial for the plant to respond quickly by stomatal closure (and then to re-open
them after relief of water stress). Here, as in 1.17, identification of the rapid early signalling responses of plants
to mild stress would be very informative, as would an understanding of the control of stomatal re-opening.
1.19. Roots are at the front end of water acquisition yet there are relatively few molecular and cellular data
relating to the effects of water stress on them. Improving the ability of roots to ‘forage’ for water and deliver
water to the shoot system under mild water stress would contribute to continued growth and yield maintenance.
CSG 15 (1/00)
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
Indeed, rapid growth early in the season prior to later-developing serious water stress may well be the key in
some situations. Thus the effects of stress on root biology is another priority – roots as primary sensors and
responders, e.g. continued growth as ‘water foragers’; osmotic adjustment, cell wall strengthening, sources of
ameliorative and negative signals; using transcriptomics, proteomics, signalling etc.
1.20. Given the potential changes in global and UK climates, it is essential to identify mechanisms to improve
water use by plants by identifying traits that contribute to WUE. This will require molecular and physiological
analyses of genes/signalling already shown to affect WUE and in which transgenic plants have altered rates of
water loss.
1.21. Mutant screens to identify genes encoding proteins that affect WUE – e.g. affecting stomatal index,
stomatal aperture, stomatal sensitivities.
1.22. Choice of species. Potato is the UK crop with the largest irrigation input and certainly one identified as a
suitable crop for improvement in terms of water use. Strawberry was also highlighted as a crop with substantial
irrigation and water requirements. Tomato was considered a useful species for experimental studies. I also
suggest that Arabidopsis work can provide data relevant to Defra.
1.23. Microarray analyses. More refined analyses are now required. For example, microarray analyses
throughout the plant life-cycle, to determine gene expression in different tissues under water deficit stress that
develops in a manner likely to better reflect the soil/field situation.
1.24. Proteomics. It is important to look at protein profiles in water-stressed roots, leaves, flowers etc. to
identify proteins or protein modifications of importance.
1.25. Functional analysis (throughout life-cycle etc) of genes already identified as contributors to stress
tolerance
1.26. Other signals affecting water stress - e.g. nitric oxide that has been shown to enhance water stress
tolerance and mediates ABA-induced stomatal closure.
1.27. Coupling of molecular and physiological analyses with cultural practices that may enhance stress
signalling.
1.28. Interactions with other stresses in the field.
1.29. Sentinel plants and precision irrigation. One potentially interesting and useful application of research
aimed at analysing the early signals initiated during mild water stress could be the use of ‘sentinel plants’.
These would be plants that alerted growers in some way to mild water stress and therefore impending watering
requirements.
2. Introduction
2.1. The problem of reduced availability of fresh water is one of the most serious that face society in the 21 st
century. Global climate changes mean that droughts are ever more frequent and, even in countries not
traditionally associated with chronic droughts, water shortage is an increasingly serious issue. On a global scale,
agriculture is estimated to represent nearly 90 % of fresh water use and large areas of land require irrigation to
support crop production (e.g. 1000 km2 in the UK; 214,000 km2 USA, 526,0000 km2 China)49. Drought is a
meteorological condition in which the lack of rainfall results in soil drying. Water uptake by plant roots is
inevitably restricted as the soil water content declines and ultimately plants are damaged and killed, with very
substantial negative effects on crop yield. In fact, drought is probably the single largest abiotic (i.e.
environmental/non-biological) limitation to crop production5. Stress is usually defined as an external factor that
exerts a disadvantageous influence; hence water stress is a term commonly used to mean the influence on
plants of reduced water availability that results in cellular dehydration. Other abiotic stresses can also induce
water stress/dehydration in plants, such as high air temperatures, when water loss from plant leaves via
evaporation can exceed water uptake by plant roots. Low temperatures inhibit water uptake and induce cellular
dehydration and both salt stress and flooding inhibit water uptake by roots. Transpirational water loss from
plants via their stomata has been estimated to account for over 60 % of global fresh water use16,38. The
importance of water stress to crop production and the importance of plants to the global water cycle are
reflected by the high priority given to water stress research by research funders across the world and by the
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
huge number of primary research papers and review articles describing various facets of water stress biology
and ecology2-5,8,9,15-18,22, 24,41-43,53,59.
2.2 Understanding the mechanisms of plant responses to water stress – what damages plants, what protective
and adaptive processes exist - will lead the way towards development of stress tolerant plants. This may be via
traditional breeding, to incorporate traits associated with stress tolerance, or via genetic manipulation, to
introduce genes identified as important for stress tolerance. In addition, increased understanding of the effects
of water stress on key stages of crop production can help shape cultivation practices to increase the efficiency
of water use. The ideal goal is to produce crops with lower water input, and plants that have reduced sensitivity
to water stress and can survive or even thrive under drought conditions, so that commercial water requirements
can be reduced and crops grown in environments currently unsuitable, such that global water use can be redirected away from crop production towards direct use by humans.
3. Focus of this report
3.1. The focus of this report is on molecular responses to water stress in plants, encompassing effects on gene
expression and the cell signalling events that result in altered gene expression profiles in stressed cells and
plants. Recent years have seen huge numbers of papers on various molecular aspects of water stress, with many
review articles appearing in the literature. In order to keep this report to a manageable size, the emphasis is on
molecular responses to water stress resulting from reduced water availability per se, rather than dehydration
induced by other stresses. In addition, some primary articles are not cited directly, but, where possible and
appropriate, by reference to recent reviews.
3.2. Much of the most recent research utilises cutting-edge ‘post-genomics’ and transgenic approaches and
technologies. In addition, most of the research has used the plant Arabidopsis thaliana, a weed species that has
been adopted as the ‘international model plant’ for molecular plant science. Brief explanations of these
experimental approaches are provided in Section 4 (referred to by * in text).
3.3. It is clear that cellular and plant responses to water stress are many and varied. Moreover, these responses
do not occur independently of one another. Rather, the picture is exceedingly complex, and it can be seen that
many of the stress-induced signalling pathways interact, although all the details of these interactions are not yet
known.
3.4. The report starts with a brief overview of plant responses to water stress, with the aim of giving some
context to the subsequent detailed analyses of molecular responses. These start with an evaluative summary of
effects on gene expression, move on to the intracellular signalling pathways activated by water stress and then
on to a consideration of how water stress is actually perceived by plants. The report then assesses developments
in transgenic analyses to determine the impact of various genes on stress tolerance, and concludes by
identifying outstanding research questions and topics of potential relevance to Defra.
4. Experimental approaches
4.1. The majority of molecular plant science research is now done with Arabidopsis thaliana, a plant with many
advantages for molecular biologists. It is small and readily grown in large numbers, amenable to chemical and
insertional mutagenesis, easily genetically transformed and its entire genome has been sequenced 48.
Consequently, most of the recent advances in molecular water stress research have also been achieved with
Arabidopsis, with the anticipation that the findings will guide subsequent work in commercially important
species. Excellent resources are available to the Arabidopsis research community. These include searchable
databases of genes, regulatory DNA sequences, expression data and proteins, and collections of mutants
generated in different ways that can be used for physiological and reverse genetic analyses. It is worthwhile
pointing out here though, that many processes in crop plants that contribute to yield will not be the same in
Arabidopsis, and that the water stress applied experimentally to Arabidopsis plants is usually very different to
that likely to be experienced by crops ‘in the field’. Much guard cell research is now done with Arabidopsis, so
as to combine the power of Arabidopsis molecular and cell biology with single cell studies. Commelina
communis has also been a useful tool for guard cell biologists, as it is easy to prepare epidermal peels for
CSG 15 (1/00)
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
stomatal studies. In addition, significant progress has been achieved via use of the resurrection plant
Craterostigma plantagineum, a plant that exhibits extreme desiccation tolerance in all tissues. Many of the
genes expressed during severe desiccation in this plant are similar to those induced by dehydration in
desiccation-intolerant plants such as Arabidopsis.
4.2 Major advances have been made by the use of mutants altered in their responses to various stimuli. Mutants
can be generated either via chemical mutagenesis of seeds and subsequent recovery of homozygous mutant
plants or by insertional mutagenesis. Here, mutations are induced by insertion into genes of mobile DNA, from
transposable elements or from the T-DNA of the Ti plasmid of the bacterium Agrobacterium tumefaciens.
Mutational analysis is a powerful technique to elucidate the mechanisms controlling biological pathways:
generation of mutant phenotypes provides the route to isolation of the genes encoding the proteins that execute
and regulate the particular pathway under study (e.g. a signalling or biosynthetic pathway activated by water
stress). Mutated genes can then be isolated from DNA prepared from the mutant plants. This task is greatly
facilitated during insertional mutagenesis, as the inserted DNA itself is used as a ‘molecular tag’ to locate and
obtain the mutated DNA.
4.3. Genetic transformation of plants represents the insertion of novel DNA into the host plant (cell)
chromosomes and subsequent expression of the DNA into proteins. Transformation is commonly achieved by
use of the bacterium A. tumefaciens that infects many plant species, including Arabidopsis. During genetic
transformation by A. tumefaciens, T-DNA, part of a large tumour-inducing Ti plasmid in the bacterium, is
transferred into plant cells and stably incorporated into plant chromosomes. An alternative transformation
technology involves micro-projectile bombardment. Here, microscopic spherical particles are coated in DNA
and ‘fired’ into plant cells; some of the DNA carried into the plant cell becomes incorporated into
chromosomes. In both cases, regeneration of transformed cells gives rise to transgenic plants. The inserted
DNA must be linked to flanking plant regulatory DNA sequences - 5’ promoter and 3’ terminator sequences to ensure that the inserted DNA is recognised and expressed appropriately.
4.4. Identification of plant promoter sequences, i.e. those cis-regulatory elements that confer water stressinducibility to water stress-inducible genes, is achieved experimentally using transgenic analyses with reporter
genes. Here, potential promoter sequences are linked to reporter genes prior to transformation of plants or plant
cells. These reporter genes encode proteins whose activity can be easily assayed in transformed cells.
Commonly used reporter genes include -glucuronidase, GUS (cleavage of specific substrates by GUS gives
rise to fluorescent or coloured products), luciferase, LUC (metabolism of appropriate substrates results in
emission of luminescence) and green fluorescent protein, GFP (a protein that fluoresces green). Assay of the
reporter gene activity in different cells or in response to various stimuli indicates the activity of the promoter
under assay. Promoters (cis-elements) can confer tissue-specificity (e.g. genes under control of such a promoter
might only be expressed in shoot cells) and be constitutive or inducible (e.g. the promoter drives expression of
the gene at the same level constantly or confers inducibility to a particular stimulus, e.g. water stress; see
Section 7.1).
4.5.Functional genomics. A major thrust of ‘post-genomics’ research in all organisms is functional genomics.
Functional genomics utilises several approaches, including large-scale analyses of gene expression and protein
content, promoter analyses, biochemical assays, bioinformatics (computer-driven statistical analyses of DNA
sequences and experimental data, e.g. of RNA or protein expression data) and reverse genetics in order to
determine all the functions of genes and other DNA sequences in the genome, in a whole genome context. This
is an important point – genes (and, of course, their protein products) do not function in isolation but in concert
(i.e. it is the specific spectrum of gene expression in the nucleus and protein functions and interactions in the
cell that determine cellular activities).
4.6. Reverse genetics. Traditional genetics can be described as forward - starting with a phenotype and working
forwards to the gene responsible. Reverse genetics is the opposite - starting with a gene and working
backwards to finding the biochemical and biological function(s) of the gene. ‘New’ genes are discovered in
cloning experiments or through genome sequencing projects. In the latter, very large numbers of genes are
cryptic, in that their functions are not yet known (for example, approximately 50% of Arabidopsis genes have
unknown functions). A major challenge of the post-genomic era is to discern the functions of these genes.
Biochemical and biological functions of genes (or strictly speaking, their protein products) can often be inferred
CSG 15 (1/00)
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
from bioinformatic analysis – i.e. comparison with all the known DNA sequences in the available databases
may reveal similarities to sequences with already-known functions. It is essential to point out though, that even
when this is possible, bioinformatic analyses only predict functions based on previous data: it may well be that
proteins have more than one function depending on their cellular context, an important point when considering
the potential functions of genes whose expression is modified under water stress. Reverse genetics employs
transgenic analyses to achieve over-expression or under-expression of the gene under study – physiological,
biochemical and molecular analyses of the resulting transgenic plants indicate potential functions – for
example, over-expression of a water stress-inducible gene may enhance stress tolerance (see Section 10). Overexpression is achieved by expressing the gene under the control of a strong promoter.
4.7. Under-expression can be achieved in two ways. ‘Knock-out mutants’ can be generated in which expression
of the gene is reduced (or even eliminated completely) by insertion into the DNA of exogenous DNA (e.g. from
a transposable element or T-DNA; see 4.2. and 4.3). Collections of such knock-outs are being created and made
publicly available by several research consortia and represent a very powerful resource with which to determine
gene function. An alternative approach is to create ‘knock-down’ mutants via the use of antisense technology
or, more recently and more efficiently, by using the related approach of RNA interference (RNAi). Here, plants
are transformed with modified versions of the genes under study such that double-stranded (ds) RNA is
generated in the host plant cells. This ds RNA is recognised by the host cell machinery, setting off a chain of
events that results in the degradation of the normal single-stranded RNA and thereby eliminates expression of
the gene under study.
4.8. Gene expression and transcriptomics. Gene expression can be quantified by northern (or gel blot) analysis.
Here, mRNA is isolated from cells, fractionated by electrophoresis and subsequently hybridised to DNA
‘probes’ corresponding to the genes whose expression is to be quantified. The degree to which the probe
hybridises to the RNA sample reflects the amount of RNA transcript of the gene in question, and is quantified
by making the probe radioactively or fluoresecence-labelled. RNA gel blots can only be carried out on a geneby-gene basis (e.g. [35]). In order to determine the expression of many genes, ideally all genes in the genome in other words, transcript profiling or transcriptomics - microarray analysis has been developed. In this
technique, DNA sequences representing all (or as many as available, or reflecting genes shown to be induced
via differential cDNA library construction and analysis) are arrayed on a solid support (a microarray slide or
chip) and replica slides hybridised with RNA samples representing all the mRNA present in cells exposed to
various treatments (e.g. control and dehydrated leaves). The RNA probes are labelled with different
fluorescence labels (e.g. red and green) and the amount of hybridisation to each separate DNA sequence on the
slide is quantified and the ratio between control and water stress treatment calculated. This ratio (repeated to
ensure statistical robustness of the data) is a measure of the degree of induction or repression of the particular
gene by water stress. Bioinformatic data handling and analysis identify the DNA sequences from the databases
and cluster the genes into groups on the basis of their expression characteristics (see Section 6.1).
4.9. Proteomics. Transcriptomics provides a picture of the pattern of gene expression in a cell, but only as far as
transcription. In fact, there is not always a good correlation between the amount of mRNA and the
corresponding protein. Moreover, many proteins are post-translationally modified e.g. by phosphorylation or
glycosylation, and compartmentalised within the cells, with potentially more than one sub-cellular population.
Such changes can only be determined by a direct analysis of cellular proteins, or proteomics. Here, cellular
proteins are extracted, fractionated by 2-D electrophoresis and proteins identified by new forms of mass
spectrometry (in particular MALDI-tof and ESI mass spectrometry). Comparison of proteins from control and
water stressed tissue can reveal which proteins are induced and which repressed. Further analyses will be
required to determine the sub-cellular locations (perhaps several) of proteins and, importantly, those other
proteins with which the target protein physically interacts (i.e. the ‘interactome’).
4.10. Metabolomics. The latest wave of post-genomic analyses aims to analyse and quantify all the metabolites
in a cell. For example, it may well be that water stress tolerance is associated with particular metabolites.
Metabolomics uses various spectroscopic techniques to analyse the metabolite profile of cells.
5. Effects of water stress on plants
CSG 15 (1/00)
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
5.1. Water moves through plants from the soil to the leaves and then to the atmosphere via transpiration (Fig.
1). Transpiration is driven by evaporation of water through the stomata, small pores on leaf surfaces bounded
by two guard cells, and almost all the water lost from plants exits via the stomata. The guard cells are multisensory cells that swell and shrink in response to various signals to open and close the stomatal pores and
thereby regulate gas exchange by plants (carbon dioxide in and water vapour out).Thus stomatal aperture is the
major determinant of Water Use Efficiency (WUE), the efficiency with which plants acquire CO2 (and can
therefore fix C via photosynthesis) at the expense of water evaporated. Abscisic acid (ABA) is a plant hormone
that induces stomatal closure and is made by plants in response to water stress. Hence stomatal closure, and the
effects of ABA, are absolutely critical determinants of both plant survival under water stress and of crop
productivity, as carbon dioxide uptake and photosynthesis are severely restricted as stomata close15,38. Water is
essential for most biological processes; water stress results in perturbation of cell biology and induces damage
to cellular structures such as membranes and proteins. Leaf expansion is particularly sensitive to water stress
and the photosynthetic machinery inside chloroplasts is also damaged. The generation of Reactive Oxygen
Species (ROS) is accelerated in water stressed conditions. Although ROS function as signalling molecules at
low concentrations (see 8.16), at high concentrations they cause cellular damage. Particular stages of the crop
cycle are likely to have differing sensitivities to water stress – for example, seedling establishment and
flowering/seed set may be particularly prone (Fig. 1) and it will be important to determine molecular responses
accordingly. Moreover, water stress will induce different effects in different situations. For example, slowly
developing or rapid stresses are likely to induce different effects, and in the field, plants may well experience
several stresses simultaneously.
5.2. Plants exhibit various responses to water stress that can be viewed as protective survival mechanisms.
These can be classed broadly into those that minimise water loss and those that maximise water uptake5. In
addition, there are various cellular responses that permit tolerance of cellular dehydration. Minimising water
loss is achieved by stomatal closure, by reducing light absorbance through changes in leaf angle and by
senescence and shedding of older leaves; water uptake is maximised by increased root growth and by osmotic
adjustment5. Here, increased synthesis of osmotically active solutes decreases cellular water potential and
facilitates continued uptake of water from the soil. Often the solutes synthesised are specialised molecules such
as proline or glycine betaine. These are termed compatible solutes – accumulation of large amounts of these
compounds is compatible with cellular activities, as distinct from other solutes such as ions, which at high
concentrations denature proteins and interfere with cellular processes. Specialised proteins, termed LEA
proteins or dehydrins, also accumulate during water stress. These are thought to be osmoprotective in that they
confer dehydration tolerance to the cytoplasm in which they accumulate5. Other enzymes induced during water
stress can be classed as having ‘repair’ functions – repairing the cellular damage induced by dehydration and
ROS.
5.3. Induction of various responses such as accumulation of solutes and proteins, stomatal closure and increased
rooting all require that the plant and its constituent cells firstly perceive the water stress signal and then alter
their biochemistry and gene expression such that the appropriate intracellular signals and proteins can be
generated to achieve these responses. Hence, the speed, location and patterns of signalling and gene expression
are key factors determining plant survival. ABA is probably the single most important factor in water stress
signalling. It is synthesised de novo during stress and also redistributed, both within leaves and between roots to
shoots (Fig. 1). Roots of course are the organs first likely to experience reduced water availability and root-toshoot signalling is critical. In addition to ABA as a positive signal indicating impending water stress, there may
be other signals, as well as negative ones – i.e. reduction of signals travelling from root to shoot. Shoot-to-root
signalling may also be altered during stress (Fig. 1).
6. Altered gene expression during and after water stress
6.1. There is no doubt that water stress alters profoundly the pattern of gene expression in cells. Recent
advances here have been achieved by the adoption of large-scale analyses of gene expression, mainly using
microarray technology*. Most work has used Arabidopsis because its entire genome has been sequenced and
thus various types of microarray have been constructed, either commercially or by research consortia. Water
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stress induces the expression of hundreds of genes that have been grouped by various authors on the basis of the
predicted function of their encoded proteins. It should be noted though, that in many cases these functions are
just that – i.e. predicted on the basis of their sequence comparison to other DNA/protein sequences in the public
databases that already have known or inferred functions. In addition, a substantial percentage of Arabidopsis
genes have unknown functions. Even for those genes that have already been characterised in previous studies, it
is quite possible that the biological functions of gene products differ from those anticipated on the basis of
earlier work. Nevertheless, useful groupings of stress-induced genes have been performed3,41. These
classifications are a good start towards identifying the clusters of genes that operate as networks (e.g.
metabolic, signalling) during stress responses, towards identifying those genes whose products may be
important or even essential for stress tolerance and for highlighting those that may have regulatory control over
a hierarchy of other genes.
6.2. Lists of water stress and ABA-regulated genes have been made publicly available by several research
groups, either as web-based supplementary adjuncts to papers, or via the relevant laboratory internet
homepages. This information represents an excellent resource for the research community.
6.3. The Shinozaki group (RIKEN, Tsukuba, Japan) has been pre-eminent in plant water stress research,
particularly with respect to dehydration-induced gene expression41,43. This group has constructed an
Arabidopsis cDNA microarray consisting of 7000 cDNA clones obtained from full-length cDNA libraries.
Although construction of this array represents an excellent technical advance, it should be noted that still only a
quarter to a third of the Arabidopsis genome is represented on the microarray. These microarrays have been
used to analyse gene expression in response to water stress, cold and salt stress, and ABA treatment. Hundreds
of genes were found to alter in expression level and classified as functional or regulatory. Examples of those
in the former category include a large number of genes with predicted functions in various cellular processes
such as membrane transport (of water, sugars and ions), carbohydrate and fatty acid metabolism, RNA
biochemistry and synthesis of proteins and molecules that have clear adaptive significance. These include the
LEA proteins and heat shock proteins and those related to the biosynthesis of osmolytes (osmotically active
solutes) such as proline, oligosaccharides and sugars. Other induced genes encode enzymes likely to be
involved in ‘repair’ processes during water stress, such as those involved with the detoxification of reactive
oxygen species and lipid peroxides (likely to arise during the oxidative stress that occurs under stress
conditions), proteinases and ubiquitin-conjugating enzymes likely involved in protein turnover and potential
detoxification-related aldehyde dehydrogenases. These types of genes may not actually be directly induced by
water stress, i.e. loss of turgor per se, but by the resultant cellular damage. Signalling or regulatory genes found
to be induced by water stress include those encoding stress activated transcription factors, protein kinases
(PKs), protein phosphatases (PPs) and other proteins involved in the generation and action of signalling
molecules such as inositol phosphate (IP3) and calcium, Ca2+. Venn diagram analysis by the RIKEN team
reveals genes induced by several stresses as well as those induced only by specific stress stimuli, with a large
overlap between salt/ABA and dehydration/ABA, and some genes only being induced by salt or dehydration,
indicating potentially important differences between ionic and osmotic stress. In total, 6 % of the genes on the
array were up-regulated by dehydration or ABA, indicating that a substantial proportion of the genome is
potentially stress-inducible. Many genes are also down-regulated by water stress. Several of these genes encode
enzymes or proteins associated with photosynthesis, which is restricted during water shortage.
6.4. In another significant development, the RIKEN team have used their full length array to analyse the
expression of genes whose expression is altered during rehydration following dehydration stress41,43. This is
important because plants clearly need to recover after stressful periods, and identification and subsequent
manipulation of genes altered in expression during recovery from stress may well be useful towards
engineering more productive and fitter plants. Rehydration-induced genes included those involved in
photosynthesis, cell wall biochemistry and metabolism of proline and other osmolytes. Proline is synthesised in
response to dehydration in Arabidopsis. Rehydration-repressed genes included many of those up-regulated by
drought stress.
6.5. Other recent papers have also described the effects of various stresses on gene expression. The commercial
Affymetrix GeneChip, that contained probe sets for ~ 8100 genes (< a third of the total genome), was used to
analyse the effects of salt, osmotic and cold stress23. Several important conclusions can be drawn from these
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data. Firstly, in contrast to [41], here, only a small number of genes was found to be regulated by more than one
stress. Secondly, expression profiles altered with time after stress, the % of genes being stress-unique increasing
with time (between 3 and 27 h). Thirdly, there were major differences in gene expression between leaves and
roots. Fourthly, the majority of the genes represented on the chip that were already known to be controlled by
circadian rhythms were stress-responsive, suggesting that one biological function of the circadian clock is to
‘anticipate’ predictable stresses. Fifthly, in these experiments, the roots were exposed to stress, and roots and
leaves then analysed separately. Transcriptomic changes were detected in leaves 3 h after stress imposition on
the roots, indicating rapid signalling between roots and shoots. Finally, and not surprisingly, although hundreds
of stress-regulated genes were identified, their biochemical and biological functions remain to be elucidated.
6.6. Expression profiles from GeneChip experiments have been used to follow the expression of 402
transcription factor (TF) genes (the Arabidopsis genome contains ~ 1500 TF genes)6. This analysis revealed
that expression of many TFs is regulated by several stresses, indicating their potential multifunctional nature
and the complexity of stress responses. For example, 28 of the 43 senescence-related TFs on the chip were
changed in expression by various stresses. Note that senescence of older leaves is a water stress response.
6.7.The Chua lab (New York, USA) has carried out genome-wide expression-profiling in Arabidopsis to
identify hundreds of ABA-regulated genes19. Instead of microarray analysis, this group used a technique called
massive parallel signature sequencing that involves large-scale separation and cloning of mRNAs and
sequencing of short PCR products. Like the RIKEN group, they identified large numbers of genes whose
expression is modulated by ABA. Many of the genes correlated with those identified elsewhere as being water
stress-inducible, again demonstrating that most genes whose expression is altered during water shortage are
regulated by ABA. The genes identified were grouped into classifications similar to those highlighted in [41],
with many genes encoding potential components of ABA signal transduction. Of particular interest were the
large numbers of genes involved in protein turnover and RNA metabolism. This points to the key importance of
these processes in ABA (and stress) signal transduction, findings backed up in other experiments (see 8.13 and
8.14).
6.8. The RIKEN group has now extended its work to include microarray analysis of stress-induced gene
expression in rice, a monocot and major crop species35. A rice cDNA microarray was prepared using cDNA
clones isolated from cDNA libraries prepared from drought-, cold- and salt-stressed plants. Water stress- and
ABA-inducibility was confirmed by RNA gel blot analysis of candidate genes. As in other studies, ABAinduced genes were also induced by water stress. Venn diagram analysis indicated substantial crossinducibility, particularly between salinity and drought. Comparison between the dehydration-induced genes in
Arabidopsis and rice revealed a considerable degree of similarity, but in addition, some genes found to be
induced in rice were not previously seen in Arabidopsis. The relative importance of common and unique genes
to stress tolerance is an area requiring further study.
6.9. It would be of considerable interest to determine which genes are altered in expression in specific cell
types, for example in guard cells at the ‘front line’ of stress defence, or in root cells in sections of the root with
differing activities with respect to water uptake or extension growth. Several studies have shown that individual
ABA–regulated genes (dehydrins) are induced in guard cells during water stress, but so far no large-scale
studies have been reported. Schroeder’s group (San Diego, USA) have carried out a transcriptomic analysis of
ABA-regulated gene expression in Arabidopsis guard cells28 and the anticipated publication is likely to reveal
several genes that might be involved in water stress- and ABA signal transduction in guard cells.
7. Regulation of gene expression during water stress
7.1. Typically, control of gene expression is effected via the interaction of transcription factors (TFs, nuclear
DNA-binding proteins) with cognate regulatory regions of DNA (cis-elements or promoters) physically
associated with the gene. These interactions, in addition to protein:protein interactions and effects on the
complex of general transcription factors associated with the transcription initiation region of genes and the
RNA polymerase II enzyme that synthesises mRNA, induce conformational changes in DNA and associated
proteins that induce or suppress transcription (see Fig. 2).
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7.2. Several drought and ABA–responsive cis-elements have been identified in Arabidopsis, in particular,
dehydration-responsive elements (DRE; core sequence A/GCCGAC) and DRE-binding proteins (DREBs,
members of the ERF/AP2 class of TFs) and ABA-responsive elements (ABARE; core sequence GTGGC) and
ABRE-binding proteins/factors (AREBs/ABFs, members of the bZIP TF class). In addition, MYB, MYC and
other TFs such as SAP and HD-ZIP proteins bind to cis-elements (MYB recognition sequences A/TAACCA
and C/TAACG/TG; MYC recognition sequence CANNTG) in the promoters of ABA-regulated genes2,8,17,43.
DREB homologues have been identified in rice, indicating the similarities between the control of water stressregulated gene expression in monocots and dicots41. Novel drought-responsive cis-elements have been
identified recently, such as those in the promoter of the erd1 gene and in genes induced by proline41,43. Water
stress and ABA-regulation of gene expression is achieved via alterations in the activities and amounts of such
transcription factors. These changes are induced via changes in the conformation of TFs, in turn brought about
via protein:protein interactions, reversible phosphorylation, de novo synthesis and alterations in the rate of
proteolytic turnover.
7.3. Bioinformatic identification of water stress and ABA-responsive cis-elements. Bioinformatic analyses of
the promoters of the drought and ABA-inducible genes identified by large-scale transcriptomic analyses have
revealed potentially novel cis-elements, in addition to those such as DREs and ABREs, previously identified
using promoter analyses*. Essentially, bioinformatic analyses search the DNA sequences 5’upstream of the
inducible/repressible genes for sequences identical or similar to those previously identified, and align the
5’sequences of co-ordinately expressed genes to identify potential common sequence motifs. Putative promoter
sequences can then be subsequently analysed experimentally. The RIKEN group has constructed an
Arabidopsis promoter database that will be useful for such analyses41. Bioinformatic analyses of genes
responsive to dehydration and with expression characteristics similar to erd1 indicate that a substantial number
of genes have similar 5’DNA sequences. Similarly, sequence analysis of the promoters of rehydration-inducible
genes indicated conservation of the ACTCAT sequence previously shown to confer proline-responsiveness41,43.
In a recent microarray study with rice35, bioinformatic analysis of the promoter regions of dehydration- and
ABA-inducible genes revealed the presence of DRE and ABRE sequences in some inducible genes, indicating
again conserved mechanisms of dehydration and ABA signalling across very different plant species. However,
some inducible rice genes did not contain these cis-sequences, indicating that other, as yet uncharacterised, ciselements must regulate the expression of these genes during water stress.
7.4. Over-expression* of the AtMYC23 and AtMYB2 genes in transgenic Arabidopsis resulted in increased
ABA sensitivity. Microarray analysis showed that over-expression of these ABA-responsive TFs led to
enhanced expression of ABA-responsive genes. Bioinformatic analysis of the promoters of these genes showed
that most of them contain both MYC and MYB recognition sequences43.
7.5. Modifications of TF amounts and activities under water stress are discussed in Section 8.
8. Cell signalling during water stress
8.1. Cellular activity essentially reflect the activities of cellular proteins, so that cell signalling, in which a
signal alters the activities of cells, must involve changes in the activities of proteins. Protein activity reflects
protein conformation (3-D shape), this being altered most commonly by ligand binding or addition/removal of a
phosphate group. Typically, external signals are recognised by cells via a sensor or receptor, often, but not
exclusively, situated on the plasma membrane of the cell. Interaction of the signal with its receptor induces a
cascade of intracellular signalling processes that culminates in cellular and ultimately plant responses. These
responses can be very rapid, occurring within minutes, or slower, taking hours or even days. Often, rapid
responses involve change in the activities of enzymes and ion channels within cells. Longer-term responses
often involve changes in gene expression. It is likely though, that short-term changes subsequently evoke
changes in transcription and it may well be that in some cases transcription and RNA metabolism are integral
facets of short term responses. Typical intracellular responses involve generation of second messengers, defined
as intracellular signalling molecules whose concentrations are transiently altered (usually elevated) in response
to an external signal. Their biological activity results from their interactions with downstream signalling
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proteins, such as ion channels or enzymes such as protein kinases (PKs) and protein phosphatases (PPs) that
add or remove phosphate groups from target proteins.
8.2. In this section, recent findings relating to intracellular signalling in response to water stress are summarised
(see Fig. 3). It should be noted that responses undoubtedly differ between cells (e.g. leaf mesophyll cells, guard
cells, roots etc) and that many molecular responses may actually be induced by ABA, synthesised during water
deficit, rather than by water stress per se.
8.3. Mutant screens. The generation, identification and characterisation of mutant plants impaired or otherwise
altered in stress and ABA signalling is a very powerful approach to highlight key genes required for water
stress responses. Arabidopsis can be easily mutagenised and the resulting mutants screened for interesting
phenotypic abnormalities. The trick is to devise a useful screen. Zhu’s group (Tucson, USA) have been at the
forefront of this approach. This lab developed an elegant screen to identify mutants impaired in responses to
stresses such as cold, ABA and osmotic stress. They used transgenic Arabidopsis in which the promoter of the
RD29A gene (already known to be induced by water stress or ABA) was fused to a firefly LUC reporter gene*.
Assay of altered LUC activity (via luminescence monitoring) resulted in the isolation of many exciting mutants
altered in response to various stresses53,59. Another elegant screen developed specifically to identify mutants
altered in transpirational water flow, uses infra-red thermographic imaging. Here, mutants with faster or slower
rates of water loss are cooler or warmer (transpiration cools leaf surfaces). This approach led to the recent
identification of the ost1 (for open stomata 1) mutant30.
8.4. ABA biosynthesis. Although it has long been known that ABA is rapidly synthesised de novo in response
to water stress, it is only recently that the key genes encoding the ABA biosynthetic enzymes have been
characterised40. ABA is synthesised from the 9’cis-xanthophylls 9’cis-neoxanthin and 9’cis-violaxanthin.
Oxidative cleavage of these epoxy-carotenoids occurs in the chloroplast; subsequent conversion of the cleavage
product xanthoxin to abscisic aldehyde and on to ABA takes place in the cytoplasm (Fig. 4). A major
breakthrough occurred when a Zea mays gene (VP14) encoding the first NCED (9’cis-epoxy-carotenoid
dioxygenase) was cloned and NCED genes have now been cloned from different species, including several
from Arabidopsis. Genes encoding the other enzymes of ABA biosynthesis have also been isolated 40. It is clear
that the expression of these genes varies between tissues and in response to various treatments 40,47. Xanthophyll
cleavage is the key regulatory step in ABA biosynthesis and NCED gene expression is induced by water stress,
with AtNCED3 being the gene up-regulated in Arabidopsis leaves. The AtNCED gene family is differentially
expressed in Arabidopsis and in addition, differing interactions between AtNCED proteins and thylakoid
membranes in chloroplasts may also regulate ABA biosynthesis47. Xanthoxin generated in plastids is exported
to the cytosol. The ABA2 gene has recently been cloned and shown to encode a unique short-chain
dehydrogenase/reductase (SDR1) that converts xanthoxin to abscisic aldehyde. Expression of this gene is
restricted spatially and temporally suggesting that dynamic mobilisation of ABA precursors and/or ABA is
physiologically important7. The final step in ABA biosynthesis is catalysed by abscisic acid aldehyde oxidase
(AAO), a molybdenum cofactor (Moco) enzyme that requires sulphuration of the Moco by the Moco sulphurase
enzyme ABA340. Phenotypic analyses of various ABA biosynthesis mutants have demonstrated the
requirements for these enzymes (and thus ABA) in various processes, including novel growth promoting
ones7,53.
8.5. Calcium signalling. Calcium, Ca2+, is a ubiquitous second messenger in plant cells, cytosolic Ca2+
concentrations being transiently increased via import from the extracellular solution or release from
intracellular stores (Fig. 3). It is well established that Ca2+ elevations are associated with ABA-induced stomatal
closure and various data demonstrate that these increases occur in an oscillating manner, different stimuli
eliciting a distinct ‘calcium signature’ that determines the subsequent cellular response15,39. It has been
suggested recently that the encoding of biological information by Ca2+ oscillations may be restricted to guard
cells, and that in most cases it is the increased Ca2+ per se that effects a switch in cellular metabolism, probably
by interaction with a range of calcium-binding proteins39.
8.6. The Ca2+ sensor, CBL1, integrates plant responses to abiotic stress. CBL1 is a calcineurin B-like Ca2+binding protein. Under- and over-expression* of CBL1 in Arabidopsis impairs or enhances stomatal and gene
expression responses to water stress upstream of ABA; CBL1 might therefore represent an integrative node in
water stress signalling1.
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8.7. Another Ca2+-binding protein, SCaBP5, interacts with the protein kinase PKS3 to negatively regulate
global ABA signalling13. Thus SCaBP5 might also be an integrative signalling node. Moreover, PKS3 interacts
with the PP enzymes ABI2 and ABI1, both of which mediate ABA signal transduction, demonstrating yet again
the complexity of ABA and stress responses.
8.8. Little is known of how cells sense extracellular Ca2+. Recent exciting work has identified a calcium sensor
receptor, CAS, that is expressed on the plasma membrane of Arabidopsis guard cells and that mediates Ca2+
uptake induced by extracellular Ca2+ [14].
8.9. Release of Ca2+ from intracellular stores is usually activated by second messengers that bind to calcium
channels on intracellular membranes thereby activating Ca2+ efflux into the cytosol. Recent studies have
demonstrated the importance of several second messenger systems in both guard cells and other cell types 2,17
(Fig. 3). An important question here is how do specific enzymes activate discrete responses in specific cells?
Activation of phospholipase C to generate inositol trisphosphate, IP3, along with subsequent regulation of IP3
metabolism, is required for optimal responses to ABA. PLC may also mediate non-ABA responses2. Other
Ca2+-releasing second messengers include inositol hexaphosphate, IP6, and phosphatidic acid, generated both
via the action of phospholipase D (PLD) on membrane lipids or from diacylglycerol, itself generated via PLC
action. PLD activity is increased rapidly in leaves in response to water stress but not by ABA, although there
are likely guard cell-specific increases2,17. Cyclic ADP ribose (cADPR, presumably generated via an ADPR
cyclase), and sphingosine-1-phosphate (S1P), generated from the plasma membrane lipid sphingosine by
sphingosine kinase, also activate intracellular Ca2+ release. S1P effects require mediation by the heterotrimeric
G protein GPA117.
8.10. In addition to second messenger effects, cellular responses to water stress, and ABA in particular, require
the action of several protein response regulators. The heterotrimeric G protein GPA1 is required for inhibition
of stomatal opening by ABA. It is not, however, required for ABA-induced stomatal closure, indicating that
different spectra of signalling components are involved in various stomatal responses to ABA52. Whether GPA1
has any role in non-stomatal responses to ABA is not known. Small monomeric G proteins are also involved in
ABA signalling. The plasma membrane-located G protein ROP10 is a negative regulator of ABA signalling that
functions early in ABA signal transduction pathways, as a ROP10-deficient mutant displayed enhanced ABA
effects in several responses, including stomatal closure58. Recruitment of proteins to the plasma membrane
typically requires modification by farnesylation, thus providing a potential link with the ERA farnesyltranferase
previously identified as a negative regulator of ABA signalling17. AtRAc1 (RP6) is another small G protein
involved in ABA signalling, linked to actin re-organisation and membrane trafficking required for stomatal
closure17. Related to these potential effects, syntaxin mutants altered in membrane trafficking have been
identified with impaired responses to water stress and ABA17,24.
8.11. Water stress and ABA alter the expression of a number of genes encoding PKs and PPs (Section 6).
Mutations in genes encoding various PP enzymes, including the PP2C enzymes ABI1 and ABI2, result in
altered stress and ABA signalling, with most data indicating that ABI1/2 are negative regulators of ABA
signalling. abi1 and abi2 mutants are ABA-insensitive. Large-scale gene expression analysis revealed that
induction by ABA of the majority (~ 92 %) of ABA-regulated genes in Arabidopsis requires ABI action,
induction being diminished in the abi-1 mutant. The fact that 8 % of the genes were still induced by ABA in
the mutant indicates at least two ABA-signalling pathways leading to gene induction19. Recent work has shown
that the ABI1/2 proteins interact with several components of ABA signalling. These include the PK PKS3 and
the TF ATBH6 that functions as a negative regulator of ABA signalling, indicating that these two PPs have
multiple cellular targets17. Other work indicates that ABI1 functions downstream of cADPR action17,
demonstrating again the complexity of stress signalling. The PP2A RCN1 has been identified as a positive
regulator of ABA signalling24.
8.12. Water stress and ABA activate various PKs. These include calcium-dependent protein kinases (CDPKs)
requiring Ca2+ for their activation, providing a link between Ca2+ and dehydration signal transduction2,17.
Mitogen activated protein kinases (MAPKs) are also activated by water stress and ABA4,24. Recently-identified
PKs that are essential for water stress and ABA signalling are the Arabidopsis OPEN STOMATA1
(OST1)/SUCROSE NON-FERMENTING (SNF)-RELATED PROTEIN KINASE 2E (SRK2E)30,55 and the
Vicia faba ABA ACTIVATED PROTEIN KINASE, AAPK27. The ost1 mutant was isolated via an osmotic
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stress screen and the SRK2E gene analysed using a T-DNA insertion mutant*; both mutations are in the same
gene. OST1/SRK2E is essential for generation of H2O2 induced in guard cells by ABA (see 8.16) and for
modulation of expression of ABA-responsive genes. Thus this kinase acts early in the ABA signal transduction
chain. AAPK is expressed in a guard cell-specific manner and its action is required for ABA-induced stomatal
closure (and see 8.14).
8.13. Novel data from several sources have revealed the importance to ABA and stress signalling of previously
unexpected facets of cell biology. Regulated protein turnover via the ubiquitin-26S proteasome and related
systems is a key component of many cellular processes that has already been shown to be central to auxin
signalling and is now revealed to also be essential in ABA signalling. ABA up-regulates expression of many
genes involved in regulated proteolysis19. Moreover, phosphorylation of the Arabidopsis AREB transcription
factor ABI5 blocks its degradation by the 26S proteasome17. Very recent genetic data indicate that the
ubiquitin-26S proteasome system is essential for normal ABA signalling: the rpn10 mutant defective in the
RPN10 proteasomal subunit is hypersensitive to ABA45. A related but separate protein targeting process
(sumoylation) has also been shown to function in ABA signalling29.
8.14. RNA processing and metabolism. Several recent studies have uncovered the importance of RNA
processing and metabolism to ABA, and therefore water stress signalling. RNA-binding protein genes are
changed in expression during dehydration19,41. The AAPK enzyme identified in V.faba guard cells
phosphorylates a heterogenous RNA-binding protein, AKIP1, that binds to RNA transcripts of the ABAregulated dehydrin gene after phosphorylation27. The nuclear arrangement of AKIP1 is altered by ABA,
becoming associated into nuclear speckles that may be involved in RNA processing24,27. The ABH1 gene
encodes a potential subunit of an RNA cap-binding complex and the abh1 mutant is hypersensitive to ABA.
Similarly, the sad1 mutant is supersensitive to drought and ABA; SAD1 is probably an Sm-like small
ribonucleoprotein involved in RNA metabolism17,24. Another link between ABA and RNA metabolism is
provided by the HYL1 gene, which encodes a nuclear-localised double stranded RNA-binding protein; hyl1
mutants are ABA-hypersensitive17,24.
8.15. Novel mechanisms of gene expression control during stress have been highlighted by characterisation of
mutants impaired in regulation of RNA polymerase II (RNA pol II). The Arabidopsis C-terminal domain
phosphatases (AtCPLs 1 and 3) negatively control the expression of some stress and ABA-regulated genes,
potentially by dephosphorylating the carboxy-terminal domain of RNA pol II17,24 (Fig. 2).
8.16. ABA, hydrogen peroxide (H2O2) and nitric oxide (NO). H2O2 and NO are now recognised as important
endogenous signalling molecules in plants, being involved in a growing list of developmental and physiological
processes. ABA induces generation of both of these molecules in guard cells, and removal of H 2O2 or NO, or
inhibition of their synthesis either chemically or genetically, reduces ABA-induced stomatal closure31,32. A
plasma membrane-located NADPH oxidase enzyme is critical for ABA-induced H2O2 synthesis26. NO is
generated in response to ABA by both nitrate reductase (NR) and a nitric oxide synthase (NOS)10,12; the
contribution of these enzymes in different species, tissues and conditions is unknown. The details of H 2O2 and
NO signalling remain to be elucidated, although in vitro work shows that H2O2 can oxidise and inactivate the
PP enzymes ABI1 and ABI231, and both signals can modulate calcium channels in guard cells11,31. In the case
of NO, this is potentially via the synthesis of the second messengers cGMP and cADPR 11,32. In addition, the
effects of ABA on H2O2 and NO generation in other tissues are not yet clear, not are the effects of water stress
on H2O2 and NO synthesis. In recent reports, ABA and water stress were found to increase NADPH oxidase
activity in maize leaves20 and dehydration-induced ABA synthesis in wheat root tips was observed to require
the synthesis and action of both H2O2 and NO57. These two papers indicate the level of complexity awaiting
unravelling.
8.17. In addition to interactions with H2O2 and NO, ABA also interacts with other hormonal signals such as
ethylene42. ABA is not only a growth inhibitor, under some conditions it also functions in a growth-positive
manner. In some situations ABA may limit ethylene production, but in others the positive effects of ABA on
growth can be uncoupled from ethylene, with there being distinct effects in roots and shoots7,42. There may also
be interactions with hormones such as cytokinins that can induce stomatal closure, but there has been little work
generally on interactive effects.
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8.18. Long-distance signalling. There is no doubt that long-distance signalling plays an important part in plant
responses to water stress (Fig. 1). Water deficit itself is perceived at the cellular level, as are signals arising as a
consequence of water stress, such as ABA, NO, ROS etc, but the overall response of a plant involves
communication between different cells, tissues and organs, and coordination of processes that might result in
survival and/or continued productivity, such as stomatal closure, expression of ‘protective’ genes, continued
root elongation, senescence of older leaves etc. Shoot responses can be initiated before any detectable decline in
leaf water potential, indicating that root-sourced signals, presumably increased or decreased due to soil water
deficit, communicate impending water stress to the shoot9. The best known of these is ABA, synthesised in
roots and transported to the leaves and guard cells via the transpiration stream in the xylem. Water stress may
not simply increase the amount of ABA produced in roots. It could also be that delivery of ABA to the guard
cells is altered, via effects on uptake or metabolic inactivation by various cells en route, and by release and
guard cell-routing of ABA already present in the leaf mesophyll cells9. In fact, the rapid wilting (within 5 min)
observed in the ABA-deficient Arabidopsis aba2 mutant removed from an environment of high humidity, does
indicate the importance of re-distribution of pre-formed ABA to rapid emergency stress-induced stomatal
closure7. Other potential root signals could include pH (drying increases xylem pH), inorganic ions and
ethylene precursors that might be important during the ‘physiological drought’ induced by the anaerobic
conditions resulting from soil flooding9. It is also possible that transported/storage forms of NO are transported
from root to shoot32. Other changes might include reductions in root-sourced cytokinins and alteration in
phloem-delivered signals between the shoot and the root.
8.19. Signalling networks. It is clear that water stress signalling has many similarities to other abiotic stresses,
such as salinity, high and low temperatures. Indeed, part of the reason for this is that these other abiotic stresses
also have cellular dehydration as one of their components. Another cellular response common to several abiotic
stresses is ROS generation. There is ample evidence that signalling pathways do not operate along simple linear
lines, but function as networks, with considerable cross-talk between pathways (i.e. components of one pathway
interact with components of another22) and it is important to recognise this, as well as the realisation that in the
field, plants are often exposed to multiple stresses (e.g. water deficit and high temperature). Stomatal guard
cells are crucial interpreters of environmental and internal signals, integrating such signals into responses that
might optimise gas exchange. Guard cell signalling is an excellent example of network organisation, and in fact
the suggestion has been advanced that guard cell signalling could be viewed as a ‘scale-free network’ with
properties of robustness and flexibility16. In such a flexible organisation, some components are shared between
different signalling pathways (e.g. calcium and certain PKs and PPs). Robustness is apparent when certain
pathways are blocked, but with only limited effects on stomatal responses (i.e. another signal component ‘takes
over’). Catastrophic effects on stomatal signalling result when key shared signalling nodes (e.g. Ca 2+ release)
are interrupted. A full understanding of stomatal signalling requires an integrated approach that seeks to place
all signalling components into a linked network - a good paradigm for water stress signalling generally.
9. Perception of water stress
9.1. In order to activate any protective and adaptive responses to water stress, plants, and thus plant cells,
require mechanisms that alert them to declining water availability (Fig. 3). In fact, the ability to perceive and
react rapidly to water stress may well be a key factor in determining the survival chances of cells, and thus
whole plants. There is still no clear mechanistic explanation of how cells actually perceive water stress,
although such perception is very rapid – RNA transcripts and proteins indicative of the dehydration response
can be detected within 60 min and increased ABA content some time before that2. As water availability
declines, the cell pressure potential declines and the cell and its plasma membrane shrink. It is possible
therefore, that topological changes on the cell surface might in some way be transduced through membranespanning proteins into intracellular responses. Other primary signals might include modifications of plasma
membrane-cell wall connections as the cell protoplast relaxes, and alterations in the conformation of cellular
macromolecules. In yeast, osmotic stress is detected by a cell surface receptor similar to ‘two-component
system’ sensor proteins in bacteria. When active, a plasma membrane histidine kinase sensor protein, Sln1,
undergoes autophosphorylation and phosphate transfer to an aspartate residue within the Sln1 protein. The
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phosphate group is then transferred, via a phosphorelay protein, to the response regulator protein Ssk1. Active
Ssk1 inactivates a downstream MAPK signalling pathway. Under osmotic stress conditions, Sln1 and Ssk1 are
inactivated and thus the MAPK signalling module is turned on, signalling to the nucleus to induce expression of
genes required for stress survival. Plants have a number of hybrid histidine kinase genes similar to Sln1. In
Arabidopsis, one of these, AtHK1, is up-regulated by water stress and can complement the Sln1 mutation in
yeast, suggesting that it might function as an osmosensor in plants4,43. There is also another plasma membrane
protein in yeast, Sho1, that physically interacts with the MAPK cascade and is also involved in osmosensing4. It
is quite possible that more than one osmosensing mechanism exists in plants, with different mechanisms
operating in different tissues. Induction of ABA biosynthesis is one of the earliest molecular responses to water
stress, so it is likely that water stress perception is linked to enhanced transcription of the key NCED ABA
biosynthesis genes.
9.2. Many subsequent molecular responses are regulated by ABA. Consequently, there must be some
mechanism(s) by which cells perceive ABA and increase the activity of various signalling enzymes and
proteins that culminate in altered gene expression (Fig. 3). There are several reports indicating the presence of
ABA receptors both internally and on the cell surface4,17. There is no reason why more than one type of ABA
receptor should not exist, with the possibility that these different receptors mediate specific ABA responses, or
that the different receptors recognise differently-sourced ABA – for example synthesised or released from
intracellular compartments within the responding cell, in contrast to that arriving at the outside of cells via
diffusion or xylem transport. Two recent papers have described potential ABA binding proteins in guard cells.
A 42 kDa ABA-binding protein has been purified from V. faba epidermis. Incubation with a monoclonal
antibody raised against this protein inhibits ABA-induced PLD activation in guard cells, indicating the
involvement of this protein in ABA signalling56. It will be very interesting indeed to determine the sequence of
this protein. Another study used biotinylated ABA to visualise ABA binding sites on the surface of V. faba
guard cells54. Whether or not the ABA binding site visualised in this experiment represents an ABA receptor
and is the same as the ABA-binding protein isolated in [56] remains to be seen. The Arabidopsis genome
contains many genes encoding plasma membrane protein kinase receptor genes, many of which have no known
function. It may well be that the ABA receptor is represented amongst these. An alternative suggestion is that
ABA interacts directly with RNA25 or directly modulates transcription through the small ribonucleoprotein
SAD117,24.
10. Functions of water stress-regulated genes - towards water stress tolerance
10.1. Following identification of genes induced by water stress, or those whose action is likely to be essential
for activation of water stress responses, functional analyses are required to ascertain both the biochemical and
biological functions of the gene – i.e. functional genomics*. Typically, this involves generation of transgenic
plants in which expression of the gene is increased or reduced, via knock-out or knock-down approaches*.
Genes analysed in this way include those classed as functional, i.e. encoding enzymes or proteins likely to
confer water stress tolerance, and regulatory/signalling genes, i.e. those that potentially regulate stress
tolerance. Many of the signalling genes described earlier in this report have been characterised via this reverse
genetics approach*. Attempts to enhance water stress tolerance have met with mixed success, but there are
sufficiently encouraging laboratory results to warrant further experimentation and field studies. It will be
important to assess the long-term adaptive enhancements achieved in these studies, to determine the effects of
altered gene expression on growth and yield; many of the experiments carried out so far have not used crop
plants and sometimes only assess stress tolerance in simple survival assays.
10.2. A number of studies have used transgenic technology to manipulate the amounts of compatible solutes
such as glycine betaine, trehalose, mannitol and fructan accumulated in transgenic plants. In some cases
increased water stress tolerance has been noted, but the effects do not seem to be due to osmotic adjustment, as
relatively low amounts of these compounds accumulate; other mechanisms may therefore be important2,37,51.
10.3. Attempts to improve water stress tolerance by increasing the accumulation of hydrophilic LEA proteins
have met with mixed success, with over-expression of LEA proteins from different species having different
effects2.
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10.4. Over-expression of genes encoding enzymes that scavenge ROS or detoxify compounds likely to be
produced by ROS action is another approach. Some success has been achieved recently by over-expression of a
water stress-inducible aldehyde dehydrogenase gene in Arabidopsis46. Tolerance of several stresses that
induced oxidative stress was achieved.
10.5. Increasing ABA levels by over-expression of an NCED gene decreases transpiration by reducing stomatal
aperture50 and increases water stress tolerance in both Arabidopsis and wild tobacco34,43. These experiments
demonstrate that water use efficiency can be improved by genetic manipulation of ABA levels. Interestingly,
the tobacco study34 used an inducible promoter* to drive expression of the NCED gene and hence increase
ABA content. It appeared that ABA content had to be increased before the onset of stress, implying that the
elevated ABA has to act in the turgid state for a certain time prior to stress in order to enhance plant tolerance an important point for future work.
10.6. Over-expression of stress-inducible transcription factors that regulate stress signalling pathways such as
DREB and AtMYC and AtMYB TFs in Arabidopsis increases water stress tolerance43,51. These are very
promising experiments as manipulation of single genes leads to alterations in the expression of many genes
under hierarchical control of the TF. Moreover, expression under the control of a stress-inducible promoter*
can confine expression to stressful conditions with limited negative effects on metabolism and growth that can
occur when such TFs are expressed constitutively.
11. Outstanding research questions and approaches
11.1. Recent years have seen some excellent studies advancing our understanding of the molecular mechanisms
by which plants respond to water stress, with the ultimate goal being to enhance crop production. Although
there are still many unresolved issues, the data generated with model plants such as Arabidopsis and rice can
now be used to assess the importance of specific genes and signalling pathways in determining yield
characteristics related to water stress and to guide research in crop species. The deliverables of such research
will be the identification of genes and pathways whose action impacts directly on yield and survival under
water stress. Subsequent utilisation of these genes in crop plants is not a trivial task. Water stress tolerance is
not a single gene trait, and the complexity of water stress signalling means that ‘one gene for all’ solutions are
not possible. Moreover, there are few examples where direct translation of basic biochemical and physiological
knowledge into yield improvement has been achieved. However, where improvements have been realised, they
have in fact come about through amelioration of the consequences of abiotic stress, such as through improving
WUE in wheat44.
Despite the complexity of stress signalling, transgenic studies carried out so far do indicate that water
stress tolerance can be enhanced by the introduction of single genes, such as those encoding detoxification
enzymes, ABA-responsive transcription factors or pivotal enzymes of ABA biosynthesis. The success of these
latter approaches is likely to reflect the subsequent activation of many genes regulated by ABA. Given the
current climate relating to genetically modified (GM) crops however, there is probably no short-term market for
GM water stress-tolerant plants, at least in the UK. Public concern and hostility over GM is not such an issue in
other countries, and it may be that UK antipathy will also decline with time. It must be emphasised that there
are alternative non-GM approaches that, although using GM to identify and analyse useful genes, can then
introduce these via non-GM molecular plant breeding33. Additionally, it may be possible to combine genetics
and cultural practices to alter stress signalling and gene expression in plants so as to modify plant metabolism at
key stages of the crop cycle and thereby conserve water and enhance yield. A superb example of a novel watersaving cultivation practice developed from basic research is that of Partial Root Drying (PRD)9. Here, plants are
watered sequentially on only one side, and then the other. The roots on the non-watered side signal water stress,
with resulting metabolic changes, but the watered side supplies sufficient water. The results so far have been
very promising in terms of decreased water use with little yield losses9.
Although there will be some common facets, the interactions of drought with specific crops (and other
abiotic and biotic stresses) will undoubtedly have a range of characteristics. The precise requirements for
improvement are then clearly dependent on the agricultural context. For example, in arid zones where water
stress is a severe constraint, improvements in crop yield might well be realised via introduction of genes that
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improve desiccation tolerance – when yields are already low, small increases will be more significant than in
situations where yields are typically much higher. In such cropping situations, relevant to the UK, commercially
viable yield improvement or maintenance is the desired outcome. In these situations, introduction of genes
whose effects are beneficial only at very low water availabilities, i.e. genes associated with dehydration
survival, may be irrelevant, as commercial production would not be viable in these circumstances. Thus there
are likely to be several research avenues that will have contrasting outputs.
Some outstanding research questions fall within the ‘fundamental’ end of the spectrum, whereas others
are of more obvious relevance to Defra. In this and the next section, I have attempted to collate the input from
all contributors and make some general comments before going on to highlight various aspects of molecular
water stress research as potential funding priorities for Defra.
11.2. I suggest that there is real need to link molecular biology with plant physiology and crop biology. There is
a good understanding of the processes regulating crop growth and yield and some of these processes may
provide targets for intervention. Often the molecular studies are not very representative of ‘real’ plants and ‘real
situations’. For example, the effects of a very severe drying regime may be determined for young seedlings
whereas in the field, the effects of a mild stress on mature plants may be more meaningful. It is important to
identify which traits to target and to test the resulting phenotypes thoroughly. Sincair et al. have demonstrated
nicely how introduction of a gene at the bottom of the ‘yield hierarchy’ may have negligible, or even negative
effects on final yields44.
11.3. I suggest that co-ordinated research efforts will be fruitful – involving the input of collaborative research
teams with different and complementary research expertise – for example, in molecular biology, physiology,
genetics and crop biology. The importance of such multi-disciplinary effort has recently been emphasised
elsewhere44. It is not easy for such research programmes to start ‘bottom-up’ via applications to discrete
research funders, but could be initiated ‘top-down’ by broader-facing research funders such as Defra.
11.4 It is also important to take a long-term view of research – both in terms of future strategic requirements
and outputs from on-going programmes. For example, although there may be little demand for ‘loweryielding/high survival’ crops presently, global climate predictions indicate that there may well be such
requirements in the future. Even with research programmes aimed at maintaining yield under mild stress,
identification of key genes and subsequent introduction and evaluation is a long process – for example,
successful physiological programmes identified in [44] took 15 or more years.
11.5. A key requirement for water stress management in the UK in the short-term is to develop crop plants that
respond to mild soil drying or atmospheric water deficit in ways that ensure yield maintenance. This will
require molecular and cellular analyses of responses to mild water stress. Typically such analyses have not been
carried out – most work imposes a harsh desiccation treatment on test plants so that the resulting data really
relate to desiccation survival rather than to more subtle effects on plant processes. However, it is at the ‘wet
end’ of water stress where stress impacts most significantly on yield. Sometimes the desired modifications to
crops can appear counter-intuitive. For example, potato is a plant sensitive to water stress and in the field
stomatal closure is rapidly initiated under mild stress [Appendix 2]. This means a substantial reduction in C
fixation with knock-on negative consequences for root initiation and extension. As yield reflects total root
biomass, there is an overall reduction in yield. Thus in this situation it would be desirable to reduce stomatal
sensitivity to mild water stress rather than increase it.
11.6. In other situations, it might be that plants will be subject to sporadic, relatively severe water stress. In
these situations, it could be beneficial for the plant to respond quickly by stomatal closure. Here, as in 11.5,
identification of the rapid early signalling responses of plants to mild stress would be very informative. Under
sporadic stress conditions, it is likely to be essential that the stomata reopen rapidly after the dry period, so that
C fixation and biomass production can resume as quickly as possible. This is not often the case – for example,
potato stomata remain closed for some days after restoration of soil water [Appendix 2]. Here, research to
determine the factors that modulate stomatal re-opening (e.g. metabolism of ABA in the mesophyll and guard
cells) is required.
11.7. Roots are at the front end of water acquisition yet there are relatively few molecular and cellular data
relating to the effects of water stress on them. Improving the ability of roots to ‘forage’ for water and deliver
water to the shoot system under mild water stress would contribute to continued growth and yield maintenance,
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Indeed, rapid growth early in the season prior to later-developing serious water stress may well be the key in
some situations. Root extension is affected not just by water stress per se, but by the soil compaction that
accompanies drying soils, often via ABA and ethylene signalling36. In potato, root extension of some cultivars
is less sensitive than in others and final yields reflect total root biomass [Appendix 2]. Root biology is clearly
then a research priority.
11.8. In the longer-term it is essential to identify mechanisms to improve water use by plants by identifying
traits that contribute to WUE. This is certainly possible - for example it is clear from Fig. 5 that a mutation
screening approach really can identify plants that have altered stomatal behaviour and WUE with enhanced
dehydration tolerance. Other approaches could involve incorporation of genes regulating ABA biosynthesis and
action or ones that improve desiccation survival such as those affecting synthesis of compatible solutes or
detoxification enzymes Such alterations will have a metabolic cost and impact on final yield. Thus, rigorous
‘cost-benefit’ analysis will therefore be useful to ascertain if any alterations really do pay dividends in terms of
harvest yield. Such analyses will require careful physiological analyses over the crop life-cycle under a range of
environmental conditions.
12. Potential Defra funding priorities
12. 1. Choice of species. Potato is the UK crop with the largest irrigation input and certainly one identified as a
suitable crop for improvement in terms of water use. Strawberry was also highlighted as a crop with substantial
irrigation and water requirements. Tomato is mainly grown hydroponically as a glasshouse crop and in this case
water represents only a minor fraction of production costs. However, tomato was considered a useful species
for experimental studies. I suggest that field crops such as brassicas, legumes and cereals are also relevant for
research into molecular aspects of water use. Although they may not be the immediate crops discussed
[Appendix 2], I believe it is essential to plan for the future and to recognise that particular species all have
specific relevance and potential advantages (for example, common vs specific rooting strategies, resource
allocation, stomatal behaviour) that can inform related research studies. I also suggest that Arabidopsis work
can provide data relevant to Defra. Although clearly not having ‘yield characteristics’ relevant to most crops, it
has the advantage that it is suitable for mutational and large scale functional genomic analyses (transcriptomics,
proteomics, functional analyses etc) that will identify genes relevant to crop plants.
12.2. Molecular analyses. Arabidopsis is the species of choice here. However, some of the species above can
also be used. This is easy for small-scale gene analyses and will increasingly become possible for larger-scale
work as microarrays are developed for various species. In fact, identification of modified gene and protein
expression in crops such as potato is an attractive prospect even in the absence of pre-prepared microarrays.
12.3. Analysis of mild water stress effects. As described above, events at the ‘wet end’ of water stress impact
on yield. Thus elucidation of early signalling events – what signal transduction pathways are altered, which
genes are switched on/off/not affected, is certainly a research priority.
12.4. Roots. The effects of stress on root biology is another priority – roots as primary sensors and responders,
e.g. continued growth as ‘water foragers’; osmotic adjustment, cell wall strengthening, sources of ameliorative
and negative signals; using transcriptomics, proteomics, signalling etc.
12.5. Microarray analyses. More refined analyses are now required. For example, microarray analyses
throughout the plant life-cycle, to determine gene expression in different tissues under water deficit stress that
develops in a manner likely to better reflect the soil/field situation. Bioinformatic analyses of gene expression
may be ‘functional’ (i.e. attempt to predict survival value of gene products) and/or ‘hierarchical’ (e.g. attempt to
discern clusters of genes whose expression is co-ordinately changed or maintained in different tissues, stresses
etc) so as to then identify functionality and potential hierarchical control mechanisms. This latter approach may
be useful for identifying potential regulatory DNA elements and cognate transcription factors, which may then
be chosen as targets for selective breeding.
12.6. Proteomics. It is important to look at protein profiles in water stressed roots, leaves, flowers etc. to
identify proteins or protein modifications of importance. Proteomics can be done with non-Arabidopsis. It must
be recognised that cellular protein content may not equate with RNA due to post-translational modifications,
translational control etc AND - very importantly, alternative splicing (in which genes are ‘cut’ and reCSG 15 (1/00)
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assembled into alternative coding sequences thereby generating different proteins) may be much more
important in plants than hitherto suspected. Indeed, stress may even enhance this phenomenon21.
12.7. Functional analysis (throughout life-cycle etc) of genes already identified as contributors to stress
tolerance. As discussed above, these data may be of little short-term relevance as far as high yield is concerned,
but may well have future significance. These experiments can be done in Arabidopsis, and, via identification of
potential orthologues, in crop species. Expression can be under the control of constitutive and inducible
promoters – e.g. ABA biosynthesis genes under erd promoters (early response to dehydration).
12.8. Molecular and physiological analyses of genes/signalling already shown to affect WUE and in which
transgenic plants have altered rates of water loss. WUE can be altered via several approaches, e.g. altered
stomatal numbers; over- or under-expression of potential stress sensors, ABA biosynthetic genes, ABA
receptors, genes encoding key components of ABA signalling pathways in stomata – e.g. enzymes of inositol
phospholipid or sphingolipid metabolism, NO or H2O2 signalling, RNA processing, protein turnover.
Transgenic plants should include those with constitutive, tissue-specific (e.g. guard cell) and inducible (e.g.
expression driven by water stress-responsive promoters of varying sensitivity) gene expression – in order to
assess better any metabolic and hence yield costs. These analyses can provide fundamental information on the
biological roles of these genes and their products as well as determining their potential usefulness.
12.9 Mutant screens to identify genes encoding proteins that affect WUE – e.g. affecting stomatal index,
stomatal aperture etc. As already described, such an approach will deliver - for example the mutant in Figure 5
that clearly has altered stomatal behaviour. Whilst such plants themselves may not be directly useful when high
yields are required, they may well be essential for longer-term application in increasingly arid environments.
Moreover, identification of mutant genes in a species such as Arabidopsis means that the equivalent genes can
be identified in crop species and provide a route to manipulating the biological effects of these genes so as to
maximise their impact on water loss whilst minimising impact on C fixation (and hence yield).
12.10. Other signals affecting water stress - e.g. nitric oxide that has been shown to enhance water stress
tolerance and mediates ABA-induced stomatal closure32.
12.11. Coupling of molecular and physiological analyses with cultural practices that may enhance stress
signalling e.g. PRD. Drought stress is a multi-faceted problem and it may be possible to address some problems
agronomically and others genetically. Thus determination of molecular processes occurring during a treatment
regime such as PRD may be informative.
12.12. Interactions with other stresses in the field. Water stress is unlikely to occur alone with no other
stresses – e.g. high temperatures Thus, after laboratory experiments, analyses of gene expression, signalling,
crop performance etc really need to be carried out in the field.
12.13. Sentinel plants and precision irrigation. One potentially interesting and useful application of research
aimed at analysing the early signals initiated during mild water stress could be the use of ‘sentinel plants’.
These would be plants that alerted growers in some way to mild water stress and therefore impending watering
requirements. For example, identification of very sensitive gene promoters activated by mild stress would
facilitate the design of transgenic plants that could, perhaps, fluoresce or undergo a colour change that would be
detected by satellite or other sensing technology. If these plants were spread throughout the crop, it would be
possible to carry out precision irrigation – i.e. in just those parts of the crop becoming drought stressed. As 1520% of irrigation water is wasted (and many crops are sensitive to over-watering and hypoxic soils [Appendix
2]) this approach could mean substantial water use and cost reductions. In addition, use of GM sentinel plants
(suitably modified to prevent viable pollen or seed production) may well be compatible with non-GM crops.
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DEFRA
project code
HH3607TX
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title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
14. Appendix 1: UK Expert Group
Professor Bill Davies (Lancaster University)
Professor Alistair Hetherington (Lancaster University)
Dr Marc Knight (University of Oxford)
Professor Phil Mullineaux (JIC, Norwich)
Dr Ian Taylor (University of Nottingham)
Dr Andrew Thompson (HRI-Wellesbourne)
Chair: Professor Steven Neill (UWE, Bristol)
This group was assembled to represent leading UK expertise in molecular aspects of plant water stress research.
In addition, Prof Davies was invited to join the group as an expert in plant water stress physiology and crop
biology.
A meeting was held on 17 December 2003 at UWE, Bristol to discuss a draft report for Defra on “Recent
developments in understanding molecular responses of plants to water stress” prepared by Steven Neill.
An early draft of this report had been circulated before the meeting and a revised and up-dated version was
provided on the day. The meeting was chaired by Steven Neill. The aims of the meeting were (1) to ensure that
the report was a reasonable and unbiased assessment of the topic with appropriate emphasis on various aspects
and discussion of the key experimental data and (2) to discuss outstanding fundamental research questions and
potential research priorities for Defra.
(1) The report was accepted as a suitable staring point for discussion.
(2) The discussion was a wide-ranging and frank analysis of most of the topics contained in the draft report. In
particular, the focus was on how the most recent research findings, mainly obtained with the model plant
Arabidopsis thaliana, could be translated into information relevant to crop plants.
The discussion was summarised and circulated by email to the group members by Steven Neill. The outcomes
of the meeting and subsequent comments have been incorporated into the final report.
CSG 15 (1/00)
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
DEFRA
project code
HH3607TX
15. Appendix 2: HRI Expert Group
Dr Steven Adams (HRI-Wellesbourne): Protected crops, tomatoes
Dr Mark Else (HRI-East Malling): Fruit crops
Dr Gordon Hanks (HRI-Kirton): Bulbs
Professor Steven Neill (UWE, Bristol)
Dr Mark Stalham (Cambridge University Experimental Farm): Potatoes
Dr Chris Atkinson (HRI-East Malling): Fruit crops
Chair: Dr Andrew Thompson (HRI-Wellesbourne)
This group was assembled by Dr Andrew Thompson (HRI-Wellesbourne) to represent commodity specialists
who would be able to provide input into the report from a more ‘commercial and field perspective’.
A meeting was held at HRI-Wellesbourne on 19 January 2004 to discuss potential Defra research priorities
arising from recent molecular work into water stress responses in plants. A summary of the draft report and
suggested research topics prepared by Steven Neill (informed by input from the UK Expert Group [see
Appendix 1]) was circulated prior to the meeting.
The meeting was chaired by Andrew Thompson. It began with an introduction by Dr Thompson that was
followed by a summary of the draft report and suggested research topics by Prof Neill. This was followed by
introductory remarks from each of the specialists followed by round-table discussions. The meeting was
summarised to all participants’ satisfaction on the day by Prof Neill and the outcomes of the meeting have been
incorporated into the final Defra report.
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Project
title
Recent developments in understanding molecular responses
of plants to water stress
CSG 15 (1/00)
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DEFRA
project code
HH3607TX