B RIEFINGS IN FUNC TIONAL GENOMICS AND P ROTEOMICS . VOL 4. NO 4. 343^354
doi:10.1093/bfgp/eli005
Functional genomics of abiotic stress
tolerance in cereals
Peter Langridge, Nick Paltridge and Geoff Fincher
Advance Access publication date 9 February 2006
Abstract
Abiotic stresses such as extreme temperatures, low water availability, high salt and mineral deficiencies or toxicities
severely diminish productivity of cereal crops. These stresses are becoming increasingly important because of the
declining availability of good quality water, land degradation and community pressures to move away from chemical
intervention in agriculture. Of the major cereals, wheat and barley are grown in the most hostile and consequently
lowest yielding environments. Extensive genetic studies and surveys of landrace and wild germplasm have indicated
extensive variation for abiotic stress tolerance but this has been difficult to exploit due to the relatively poor
background knowledge of the molecular basis for stress in these species. Interconnected signal transduction pathways that lead to multiple responses to abiotic stresses have been difficult to study using traditional approaches
because of their complexity and the large number of genes and gene products involved in the various defensive and
developmental responses of the plant. Functional genomics is now widely seen as providing tools for dissecting
abiotic stress responses in wheat and barley, through which networks of stress perception, signal transduction and
defensive responses can be examined from gene transcription, through protein complements of cells, to the metabolite profiles of stressed tissues.
Keywords: cereals; abiotic stress; cold; drought; salinity
INTRODUCTION
Abiotic stresses such as extreme temperatures, low
water availability, high salt levels and mineral
deficiency and toxicity are frequently encountered
by plants in both natural and agricultural systems. In
many cases, several classes of abiotic stress challenge
plants in combination. For example, high temperatures and scarcity of water are commonly encountered in periods of drought, and can be exacerbated
by mineral toxicities that constrain root growth.
Higher plants have evolved multiple, interconnected strategies that enable them to survive abiotic
stress. However, these strategies are not well
developed in most agricultural crops. Across a
range of cropping systems around the world, abiotic
stresses are estimated to reduce yields to less than half
of that possible under ideal growing conditions [1].
Traditional approaches to breeding crop plants
with improved stress tolerance have thus far met
with limited success—in part because of the difficulty
of breeding for tolerance traits in traditional breeding
programmes. Desired traits can be crossed into crop
species from wild relatives and, for the cereals,
extensive abiotic stress tolerance has been identified
in screens of land races and related wild species. It is
estimated that only 10–20% of the wild variation has
been used in modern wheat varieties (unpublished
data). A similar situation appears to apply to rice and
barley, although not as extreme as in wheat. There is
Corresponding author. Peter Langridge, Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus,
PMB 1, Glen Osmond, SA 5064, Australia. Tel: þ61 8 8303 7368; Fax: þ61 8 8303 7102; Email: [email protected]
Peter Langridge is professor of plant science at the University of Adelaide and chief executive officer of the Australian Centre for
Plant Functional Genomics (ACPFG). His research has focused on development and application of molecular biology to crop
improvement.
Nicholas Paltridge has since worked on a range of research projects in cereal and crop science and is presently employed by the
School of Agriculture and Wine at the University of Adelaide. His current work focuses on the development of more productive cereal
cropping systems in Tibet.
Geoff Fincher is professor of plant science at the University of Adelaide and director of the University’s Waite Agricultural Research
Institute. He is a plant biochemist who has applied enzymological, cell biological, molecular biological, functional genomics and
structural biological techniques in studies of the enzymes involved in cell wall metabolism.
ß The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
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Langridge et al.
considerable interest at present in using the emerging
technologies of genomics as a means to identify key
loci controlling stress tolerance and as a tool to
screening for allelic variation in the wild and land
race gene pools. Delivery of the outcomes of
genomics can be through conventional breeding
although genetic transformation will offer a more
rapid option in some circumstances.
The cereals are our dominant source of food, with
maize, rice and wheat vying for the number one
position. However, the four closely related Triticeae
crops, wheat, barley, rye and triticale, occupy the
lower yielding environments and cover almost twice
the area sown with maize or rice. Although wheat,
rye and barley show levels of tolerance to many
abiotic stresses well above maize or rice, there is
relatively little known about the molecular basis for
abiotic stress tolerance in these species and there is
still ample scope for improvement. Will genomics
provide the tools for understanding stress tolerance in
these species and lead to increased rates of genetic
gain for tolerance?
PHYSIOLOGY, CELL BIOLOGY
AND BIOCHEMISTRY OF
ABIOTIC STRESS
Individual stresses
Drought stress
Crop plants grown under drought conditions are
exposed to a combination of stresses that are
attributable to high temperatures, excessive irradiance, soil resistance to root penetration and low
water potential. Loss of leaf water causes some
passive loss of turgor in guard cells. Abscisic acid
production is also induced and leads to a further loss
of stomatal turgor. The resulting stomatal closure
causes a concomitant decrease in CO2 availability in
the leaves, and hence in assimilate availability to the
plant. Although the photosynthetic machinery has a
range of photoprotective mechanisms to dissipate
excess light energy, the continued exposure of leaves
to excessive excitation energy can lead to photoreduction of oxygen and the generation of highly
toxic reactive oxygen species (ROS) such as superoxides and peroxides [2]. These dangerous compounds cause chemical damage to DNA and
proteins, and can therefore have serious or even
lethal effects on cellular metabolism. Plants have
evolved several strategies to deal with ROS,
including the production of chemical antioxidants
such as ascorbic acid, glutathione and -tocopherol
that directly remove potentially damaging electrons
from the ROS, and enzymic systems such as
peroxidases and superoxide dismutases that scavenge
the electrons enzymically [3–5]. The enzymes often
use metals such as iron, zinc, copper or manganese
as electron acceptors, so the metal ions must be
available if enzymic detoxification of ROS is to
proceed. In this way, oxidative stress may be linked
to mineral deficiencies. In related responses, reactive
aldehydes produced through perturbations in redox
balances can be removed by the action of aldehyde
dehydrogenases and aldose/aldehyde reductases [6],
and superoxide production in mitochondria can be
limited by the alternative oxidase [7].
Another adaptive mechanism for protection
against drought is the maintenance of turgor during
periods of drought by adjusting the osmotic pressure
of cells. There are two main routes whereby this can
be achieved. Firstly, the cell can sequester ions
into cellular compartments. Secondly, specialized
osmolytes such as proline, glycine betaine, mannitol,
trehalose, ononitol and ectoine can be synthesized
to readjust cellular osmotic potential. These osmolytes are also active in scavenging ROS, especially
if they are targeted to the chloroplast [8]. Other
specialized organic molecules can be used to
protect cellular membranes against physical damage,
and proteins against unfolding. Dehydration induces
the partitioning of amphiphilic molecules such
as glycosylated flavonols and hydroquinones
into membranes; these compounds increase
membrane fluidity and depress phase transition
temperatures [9].
Proline and sugars can coat protein molecules,
exclude solute from their surfaces and thereby reduce
the rate of unfolding [9]. During extreme desiccation, tolerant plants synthesize large amounts of nonreducing disaccharides, such as trehalose, which can
substitute for water by satisfying hydrogen bonding
requirements of polar amino acid residues at protein
surfaces, and maintain the folded active states of the
proteins [10]. Some late embryogenesis abundant
(LEA) proteins and dehydrins might act in a similar
fashion [11, 12]. Transgenic rice expressing
wheat LEA genes PMA80 and PMA1959 showed
enhanced drought and salt tolerance in glasshouse
tests [13] and the barley gene HV1A led to enhance
yield in field-grown transgenic wheat under drought
stress [14]. Small heat shock proteins (HSPs) may
also contribute a general protective function in
Functional genomics of abiotic stress tolerance in cereals
Table 1: Recent publications describing the mapping of
QTL controlling tolerance to a range of abiotic stresses
in wheat and barley
Stress
Wheat
Boron tolerance
Sprouting resistance
Preharvest sprouting
Cold tolerance
Vernalization and cold
Flooding tolerance
Al tolerance
Naþ/Kþ discrimination
Water stress tolerance
(paraquat tolerance)
Drought, low temperature, salinity
Nutrient, drought and salt stress
Flag leaf senescence
Agropyrum, drought
Barley
Boron tolerance
Manganese efficiency
Frost at anthesis
Cold
Drought
Seedling desiccation at germination
Location
Reference
7B, 7D
5AL, 6A, 3B, 7B
6B, 7D
5A
5A, 5D
15 QTL
4DL
4D
5A, 5B, 5D
22
23
24
25
26
27
28, 29
30
31
Groups 5 & 7
17 QTL clusters
2B, 2D
3E, 5E, 7E
32
33
34
35
2H, 3H, 4H, 6H
4HS
2H, 5H
36
37
38
over 70 QTL
7 QTL
18 ^21
39
desiccation tolerance [15], presumably through the
maintenance of proteins in a folded state.
During extreme desiccation in the desert resurrection plants Selaginella lepidophylla and Myrothamnus
flabellifolius, high trehalose concentrations replace
essentially all the water in cells and convert the
cytoplasm into a stable, intracellular glass [9, 16].
Trehalose is found only in high concentrations in
a few species that are adapted to extreme drought
stress, although lower levels have been detected
in Arabidopsis, where it is believed to play a regulatory role in carbon metabolism [16] or stress
tolerance [17].
The genetics of drought tolerance have been based
around the use of a range of screens. Table 1 shows
some recent studies investigating the genetic control
of abiotic stress tolerance in wheat and barley.
A series of studies in barley identified around 70
quantitative trait loci (QTL) for different components of drought tolerance including leaf relative
water content, leaf osmotic potential, osmotic
potential at full turgor, water-soluble carbohydrate
concentration, osmotic adjustment and carbon
isotope discrimination [18–21]. These results imply
an extremely complex control of drought tolerance.
345
In summary, adapted plants have evolved a range
of strategies to enable them to survive the multiple
elements of drought stress, and this provides
opportunities to transfer and optimize key protective
strategies into commercially important cereal species,
such as wheat and barley, where drought tolerance
mechanisms are not always well developed.
Cold and frost stress
When ice crystals form in plant tissues, severe
osmotic and mechanical stresses result. Ice generally
forms first in the extracellular space, water moves out
of the cell along the osmotic gradient so created, and
the osmotic stress is thereby imposed. Mechanical
damage includes expansion-induced-lysis, phase
transitions and fracture lesions in membranes, and
physical damage can be caused simply by the
formation of large ice crystals. In addition, freezing
can induce the production of ROS, which damage
membrane components, and can cause protein
denaturation [40].
The similarities between the consequences of
drought and cold stress are clearly evident and, as one
might expect, the plant responses during cold
acclimation or in species adapted to cold climates
are often similar to those observed in drought stress.
Antioxidant defence mechanisms are invoked,
together with the production of heat shock proteins
(HSPs), the synthesis of high concentrations of
intracellular proline and sugars, and the secretion of
increased levels of sugars into the apoplastic space.
The phase transition temperatures of membranes
can be lowered through the action of fatty acid
desaturases and the incorporation of amphiphilic
proteins into membranes [40]. Small, highly stable
proteins characterized by the presence of multiple
amphipathic -helices, similar to the LEA proteins,
are also synthesized at high levels during acclimation
or in adapted species. These are likely to stabilize
membranes, protect proteins from unfolding and, in
some cases, inhibit the recrystallization of ice through
surface patches of hydrophilic amino acid residues.
There are additional suggestions that antifreeze
proteins in winter rye (Secale cereale) might have
evolved specialized roles in protection against cold
stress through modified forms of (1,3)-b-glucanases,
chitinases and thaumatin like proteins of the
pathogenesis-related protein families [43].
The genetic basis for cold and frost tolerance
has been extensively studied in wheat and barley.
Not surprisingly, the timing and nature of exposure
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1H
Langridge et al.
2H
3H
4H
5H
6H
Mel1
Ppd1
Eps4
Bt
Bt
Fr2
Nax
Eps7S
Dhn6
Bt
Eps2
Ppd2
7H
Eps3
VrnH2
VrnH1
Eps
Fr1
Dhn1
Dhn2
Dhn4a
Eps6L.1
Bt
Dhn4b
Dhn5
Dhn3
Eps6L.2
Eps7L
Figure 1: Location of several abiotic stress loci in
barley: Bt ^ boron tolerance [22], Nax ^ sodium exclusion (unpublished), Mel1 ^ manganese efficiency [37],
Eps ^ earliness per se, Ppd ^ photoperiod [41], Vrn ^
vernalization [26], Fr ^ tolerance to frost at anthesis
[38], Dhn ^ dehydrin [42]. Over 70 QTL have been identified for tolerance to aspects of drought, these have not
been included in the map.
require different mechanisms and different genes
appear to be involved. However, in all cases there
is a close relationship between photoperiod control,
vernalization and cold tolerance. For example,
Reinheimer et al. [38] identified two key loci
involved in frost tolerance at anthesis in barley
(Figure 1). One of these loci co-segregated with an
earliness per se (Eps) locus and a vernalization (Vrn
H1) on chromosome 5H. Although there may be a
single locus on 5H involving earliness, vernalization
and frost tolerance, the locus on 2H appears to be
specific for tolerance to frost at anthesis and is a clear
target for positional cloning.
Salt stress
Osmotic and ionic components of salt stress can be
identified and plants subjected to salt stress invoke
two response pathways [17]. The osmotic component has been discussed above under drought and
cold stress. The ionic response is essentially an attempt
by the plant to detoxify cells, because high concentrations of cellular salt interfere with membrane
integrity, enzyme activity and nutrient acquisition.
Reactive oxygen species (ROS) may be generated
and elicit the usual plant reactions. Ionic homeostasis
is normally maintained through the action of various
ion transporters and non-selective ion channels.
The sequestering of Naþ ions from the cytoplasm
into vacuoles appears to be a particularly important
strategy in salt stress management by the plant, as
suggested by the observation that many naturally salttolerant halophytes rely on this strategy. Furthermore,
transgenic tomato plants over-expressing an
Arabidopsis vacuolar Naþ–Hþ antiport protein can
grow and produce fruit in the presence of 200 mM
NaCl [44]. This raises the exciting possibility that salt
tolerance could be engineered into important crop
plants such as wheat and barley through the transfer
and appropriate expression of a single gene.
Loci related to sodium exclusion have been
identified in wheat and barley (Table 1 and
Figure 1) and these are again good targets for
positional cloning.
Mineral toxicity and deficiency
Micro- and macronutrients in soils are key determinants of plant growth and development. In most
cropping systems, at least some of the soil nutrients
will be present at sub-optimal concentrations or will
be bound to soil in such a way as to limit their
availability for plant uptake. For example, most
cereal cropping soils in Australia are Zn-deficient.
Others are deficient in Mn, Cu and, when other
stresses are present, Fe. In other cases, toxic levels
of minerals such as B and Al can limit the yield
potential. Boron is an essential micronutrient that is
phytotoxic at high concentrations, and is of particular
relevance in Australia. Thus, both mineral deficiency
and toxicity represent important abiotic stresses
commonly encountered by crop species. The stresses
can often, but usually in part only, be managed
through fertilizer application. Mineral stresses can
intensify other stresses, especially water stress, when
plants are exposed to both simultaneously.
Multiple and varied functions for micronutrient
minerals have been identified in plants. These
functions include cofactors for many enzymes
(e.g. oxidoreductases), light harvesting and carbon
assimilation processes in photosynthesis, a role in
pectin structure in cell walls, etc. Plant responses to
stress imposed by mineral deficiencies or toxicity are
not always well-defined but are likely to involve, at
least in part, changes in specific plasma membrane
ion transporting pumps, carriers and channels [45].
For example, toxic levels of Al slow root growth
through processes that involve the inhibition of
plasma membrane Hþ-ATPases, the blockage of
plasmodesmata and oxidative damage [46, 47].
Al impairs plant growth on nearly 1 billion of the
world’s 3 billion hectares of cropland, including
about 35 million hectares in the USA; and Al
tolerance genes from rye may prove useful for
adapting wheat to acidic soils. Some varieties of rye
can tolerate seven times more Al than wheat
(RD Graham, personal communication).
Functional genomics of abiotic stress tolerance in cereals
The identification of adapted wheat and barley
lines (Table 1), coupled with the molecular mapping
of characteristics such as Mn efficiency, Zn efficiency, grain Fe and Zn density and B toxicity
tolerance [48, 49, 22, 37], indicate that significant
advances can be made in the management of abiotic
stresses associated with mineral availability in cereal
crops, through a functional genomics approach.
The poor Mn efficiency of released cereal cultivars
exemplifies the difficulty of achieving good adaptation by empirical breeding methods, even though
a major locus is involved [37]. In the first instance,
genes and gene systems controlling B tolerance and
Mn, Zn and Cu efficiency will be identified from
adapted varieties, wild relatives and native grasses,
with the express purpose of integrating them into
elite varieties of barley and wheat.
Stress networks
A striking feature of plant adaptation to abiotic
stresses is that multiple responses, involving complex
networks that are interconnected at many levels,
are activated when abiotic stresses are encountered.
Plants increase their tolerance to ‘environmental
insults’ through both physical and interactive
molecular and cellular changes that are triggered by
the stress [50]. It is not always possible, therefore,
to attribute a particular response to a specific abiotic
stress. For instance, freezing temperatures, low water
availability and high salt concentrations can all cause
a lowering of cellular osmotic potential and thereby
activate osmotic stress responses. These osmotic stress
responses can operate via both an abscisic acid
(ABA)-dependent and an ABA-independent
signalling pathway [51]. In addition to the induction
of osmotic response pathways, salt stress simultaneously activates a second, ionic response, through
which ion transporters shuttle ions between various
cellular compartments in an attempt to maintain
ionic homeostasis [17]. Drought and cold stresses will
similarly activate additional, more specific response
pathways. In another example, drought tolerance
and tolerance to B toxicity are closely related in
cereals, where boron-toxic soils restrict root development. Thus, stresses induced by soil drying might
incorporate stress attributable to water shortage,
osmotic stress and nutrient deficiency.
Plant responses to abiotic stress are affected at
several levels, and these eventually result in slowing
or cessation of growth. Following perception of
the stress conditions, signal transduction pathways are
347
activated and lead to alterations in gene expression,
as measured by the abundance of mRNA species,
in the protein profiles of cells, in the activities of
key enzymes, and in the relative flux through
and between different metabolic pathways. In turn,
the alterations in cellular activity result in molecular
and cellular changes that constitute the network of
abiotic stress responses invoked to protect the plant
against the unfavourable environmental conditions.
Signalling pathways
Perception of abiotic stress conditions by higher
plants leads to the transduction of a signal that relays
information within and between cells. An early event
in many stress responses is the elevation of
cytoplasmic Ca2þ levels, which leads to the activation of signal transduction pathways involving Ca2þdependent protein kinases and Ca2þ-regulated
protein phosphatases [50]. In the case of osmotic
stress, perception is believed to occur through a
plasma membrane histidine kinase [52]. Genes
encoding many of the enzymes have been identified
in Arabidopsis and their functions have been confirmed in loss-of-function mutants. The presence of
C-repeat-binding factors (CBFs) and drought
responsive elements (DREs) and ABA-responsive
elements (ABF/ABREs), in the promoters of genes
for drought, salt and cold signal transduction,
probably represents a point of pathway convergence
that enables responses of the two stimuli to be
coordinated, although there are clearly specific
Ca2þ-dependent pathways for different stresses
[50]. Oxidative stress pathways also appear to
interconnect with the Ca2þ-mediated response
pathways. Many, but not all, abiotic stress responses
can be induced by ABA treatment.
The nature of general stress responses has
opened the option of engineering tolerance through
up-regulation of transcription factors and other regulatory components of the stress response pathway. The
use of HSPs, chaperones and LEA proteins has been
described earlier. Transcription control has also been
used to enhance stress tolerance in plants [53].
Expression of the Arabidopsis, the DREB1A transcription factor under the control of a stress-inducible
promoter (RD29A), led to significantly increased
drought tolerance of glasshouse-grown wheat [54].
Functional genomics approaches
Against this background, how can we launch
effective studies to transfer information from model
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Langridge et al.
species to the cereals and what information do the
cereals have to offer other crops in understanding
and manipulating stress tolerance? On the surface,
wheat and barley have little to offer. The genomes
are large and poorly characterized relative to the
model species. However, the level of abiotic
tolerance shown by the Triticeae is greater than
most other crop species and the diversity in the land
race and wild gene pool for wheat and barley tells us
that still greater tolerance is achievable.
Role of model species
Many of the recent advances in genomics research
have been founded around developments in model
species. Arabidopsis has led the way but, more
recently, resources and information on rice and
maize have gained importance. The near complete
sequencing of the Arabidopsis and rice genomes has
been crucial to the success of these models. For
Arabidopsis, the mutant and tagged populations and
extensive microarray databases [55] have provided
powerful resources for gene discovery and functional
analysis. The microarray information covers a diverse
series of stress including cold, salt, heat, water deficit
and UV treatments [55]. Through these and related
studies in Arabidopsis many genes involved in the
control of abiotic stress tolerance have been
identified and, in some cases, the information has
been transferable to crop plants. A clear example can
be seen with the DREB transcription factors. These
were first identified in Arabidopsis as being involved in
the control of drought and cold responses [56].
Subsequent experiments identified orthologous
genes in the cereals maize [57], rice [58] and wheat
[59] in addition to several other species. In the case of
wheat, the DREB-1A transcription factor has been
used to generate transgenic lines that show increased
drought tolerance in preliminary trials [60]. A further
example is the ornithine amino transferase gene that
was found to confer salt tolerance when overexpressed in Arabidopsis [61]. This gene has been
used to transform wheat and the transgenic lines
are currently being evaluated in field trials in
Australia [62].
Although these two examples indicate that genes
discovered in Arabidopsis can be used directly or via
orthologues to engineer abiotic stress tolerance in
cereals, the key validation of this approach will only
be revealed when the field trials, currently underway,
have been completed. Many of the crucial stress
responses in crop plants relate to their behaviour as a
crop, when growing as part of a plant community
and when faced with multiple stresses. Therefore, the
preliminary results with the Arabidopsis genes are still
well away from real application.
Genome similarity in wheat, barley and other cereals
Wheat, barley and rye are all closely related. Indeed,
stable hybrids can be produced between wheat and
barley (Trithordeum) and wheat and rye (Triticale).
The high level of genome similarity between wheat,
rye and barley has been recognized for some time.
Not only are the sequences of individual genes
similar between the species, but gene order on the
chromosomes is highly conserved (synteny). Indeed
it is possible to replace many wheat chromosomes
with related chromosome from rye or barley. The
gene order across the grasses is generally well maintained and clear relationships can be drawn across the
grass genomes [63, 64]. However, at the single gene
level the order does start to break down. For example,
a detailed comparison of a 20 cM region of 3 DS
of wheat with rice chromosome 1 showed about
a 20% loss of colinearity [65]. There are also certain
classes of genes, such as the race-specific disease
resistance genes, that are of recent evolutionary
origin and these are not usually located at syntenous
regions. However, this problem does not seem to
apply to many of the key loci controlling abiotic stress
tolerance, and the rice genome sequence [66, 67] has
proved to be a powerful tool for positional cloning
of genes from wheat and barley [68, 69].
Mutant populations
There has been a long history of mutation breeding
in the cereals with several important varieties
resulting from selection of mutant phenotypes [70].
However, systematic development of mutant populations as a genomics resource has only commenced
recently. A large barley mutant population constructed for mutation screening based around detection of single base mismatches was described recently
[71] and a similar type of population was used to
identify a series of mutations in the granule-bound
starch synthase I (GBSSI) of wheat [72]. Wheat
appears to be particularly suitable for mutant
screening since it is able to carry a very high
mutation load, presumably due to polyploidy. Slade
etal. [72] identified 246 alleles after screening for each
homologue in only 1920 mutated individuals. There
are currently several projects underway around the
world to develop mutated populations of both
wheat and barley, and diploid progenitors of
Functional genomics of abiotic stress tolerance in cereals
wheat, and many of these will be available as public
resources.
Work has also been underway to develop transposon-tagged populations of barley using the maize
Ac/Ds system [73]. This will provide an important
resource for functional analysis of cloned genes.
Mapping and map-based cloning
The importance of wheat and barley as crop species
and the long history of systematic breeding have
resulted in extensive information on the genetic
control of a wide range of traits and also in the
identification of broad diversity in stress and disease
tolerance. The advent of molecular marker techniques and the links to breeding programs have
resulted in a large expansion of mapping studies.
Importantly, many of the studies have involved field
evaluation of abiotic stress tolerance. The information shown in Table 1 and Figure 1 covers only
recent studies but indicates both the diversity and
complexity of abiotic stress mapping programs.
Molecular markers have also proved important
in surveys of cultivated, land race and wild relatives
of wheat and barley (Table 2). From these studies
extensive variation has been identified and this is
being used directly in several wheat and barley
breeding programs but also allows extension of the
mapping work. The diversity found in abiotic stress
tolerance will provide an important resource for
validation of candidate genes and will also provide a
mechanism for rapid delivery of genomics outcomes
to pragmatic breeding programs. The concept here
is to use candidate genes identified from genomic
studies to assess stress tolerance mechanisms used
in the land race or wild lines that show elevated
tolerance. Where variation is identified in candidates,
Table 2: Recent studies using molecular markers to
survey germplasm for salt and drought tolerance
Reference
Wheat
Salt tolerance in land races
Salt tolerance in wild relative
Drought in wild relatives
Drought in land races
Drought in wild emmer
74
75
76
77
78
Barley
Drought in cultivated
Drought in cultivated, land races and wild
Drought in wild barley
79
80
81
349
either in the structural gene or in expression levels,
the locus can be transferred and tested in nonadapted or cultivated germplasm for assessment.
In this way, the variation is not only used to
validate candidates for stress tolerance but also to
provide a tool for allele discovery. Desirable alleles
can be transferred by conventional breeding and
selection.
Positional cloning does require high resolution
linkage maps and this has involved large segregating
populations. Association mapping may provide an
alternative but this is not sufficiently developed
in wheat and barley for application at present [82].
The availability of the rice genome sequence and
the generally strong synteny between rice and wheat
and barley means that rice can be used to generate
potential markers close to the target locus. Since
most known abiotic stress loci have been identified
as QTL, high resolution mapping is complicated
by difficulties in clear definition of phenotypes.
Breaking the trait down to well-defined components
or elimination of confounding loci from the
populations may help deal with this difficulty.
The extensive mapping of particular traits, such as
drought tolerance, in multiple cereals and using
different populations may provide a means for
locating common loci. For example, components
of drought tolerance are being mapped in almost all
major cereals by several groups around the world.
In wheat, we are consistently seeing QTL on
group 5 and 7 chromosomes appearing in the
analyses. However, the merging of population has
not yet been attempted. Similarly, can one look for
syntenous loci controlling salt tolerance in wheat,
barley, rice and maize and use the comparison to
generate a large ‘virtual’ mapping population?
Computational tools for these types of analyses are
in the process of development and this approach may
be feasible in the not too distant future.
Transcript profiling
Estimates of gene number in the cereals are very
similar to other complex organisms; for example, in
barley gene number estimates range from around
30 000–50 000 [83]. The situation is complicated
by the three genomes of wheat. Unusual patterns of
gene expression can occur in polyploids. Analysis of
gene expression in wheat using expressed sequence tag
(EST) databases showed that homologous genes can
be expressed in one, but are silent in one or both of
the remaining genomes [84]. The tissue specificity of
350
Langridge et al.
expression from homologous genes may also change.
Consequently, a gene in one genome may be
expressed in roots while the homologues are
expressed in leaf tissue.
There are now well over 1 million cereal EST
sequences in the public databases with wheat and
barley dominating. The large number of ESTs and
the diversity of cDNA libraries that have been used
to generate the sequences, has made ‘electronic
Northerns’ a useful method for assessing gene
expression and this provides a good first measure of
transcript abundance.
Several microarray and macroarray platforms have
been generated for the cereals. For wheat and barley,
there are a number of proprietal arrays such as
the 10 000 cDNA array reported by Leader [85].
More recently Affymetrix arrays have been developed for both wheat and barley [86]. In a reference
experiment using a series of well-defined developmental stages, the 22 K Barley 1 Gene Chip
identified 18 481 transcripts showing expression
above background (Druka et al., personal communication). A similar reference experiment has been
conducted using the Affymetrix Wheat Gene Chip.
The wheat chip carries 61 127 probe sets representing 55 052 transcripts. However, fewer than
30 000 are from high quality sequence data and it is
not yet clear how the remaining probe sets will be
used.
Currently, there are few published reports on the
use of barley or wheat chips for studying altered gene
expression in response to abiotic stress. Transcript
profiling of stressed rice and maize has been
conducted. For example, Hazen et al. [87] screened
a 21 000 rice Affymetrix array with RNA from
drought stressed rice to identify 662 differentially
expressed genes. A further important resource for
transcript profiling can be found at the Rice MPSS
site [88]. This contains the results of an extensive
screen of rice transcript signatures for different
developmental stages and for cold-, drought- and
salt-stressed rice plants. The MPSS data gives a very
broad picture of transcript profiles in the target
tissues.
Proteomics and metabolomics
Although there have been no reports on the
application of proteomics to the study of abiotic
stress tolerance in wheat and barley, proteome
studies have been conducted of wheat leaf [89],
grain [90] and lemma [91]. These results have
indicated the feasibility of differentiating and identifying large numbers of proteins from defined tissues
of wheat. The leaf proteomic study resolved 541
proteins of which 55 were sequenced [89].
A proteomic study of drought- and salt-stressed
rice plants found that around 3000 proteins could be
detected in a single gel and over 1000 could be
quantified [92]. This study found 42 proteins that
changed in abundance or position in response.
Several of the key proteins were identified and are
the subject of further studies.
The importance of metabolite changes during
plant responses to abiotic stress suggests that detailed
metabolite profiling may provide valuable insights
into stress response mechanisms. Metabolomics is a
relatively new area of research and there are no
published reports on its application to stress tolerance
in cereals. However, a recent report on rice found
that 88 main metabolites could be successfully
quantified from the extract of rice leaves [93]. The
compounds identified covered pathways of sugar
and amino acid metabolism, hence these types of
analyses should prove valuable for assessing stress
responses.
CONCLUSIONS
The outline above indicates the complexity of
abiotic stress responses but also shows that much of
the information generated in model species is likely
to have application in the cereals. Key processes of
stress tolerance including the signalling pathway
components such as transcription factors, HSPs,
chaperones and LEA proteins, ROS scavenging
and synthesis of osmoprotectants, ion and water
transporters, and a range of related processes appear
to be common across plant species. Indeed many
are also found in animals. These provide targets
for research in the cereals. However, the cereals will
also provide insights that have not been revealed in
some of the model species that have formed the basis
for much recent study.
The main advantage of cereals, particularly wheat
and barley, is the strong genetic information on
abiotic stress tolerance. While some of the QTL
studies appear complex, such as the drought mapping
work in barley [18], there are clear target regions
where QTL are clustering. In wheat, chromosome
groups 5 and 7 appear important while in barley 2H
Functional genomics of abiotic stress tolerance in cereals
and 5H seem to play key roles. The possibility of
combining QTL data from across the cereals is
particularly attractive and several projects have been
initiated to attempt this.
The rapid advances in genotyping technology,
have meant that high quality maps can be rapidly
generated. This has exposed the weaknesses of poor
phenotyping in many studies and the limitations in
the sizes and structures of some mapping populations. Addressing these problems will be an
important undertaking over the next few years.
Improvements in techniques for evaluation of stress
responses including the automation of some measurements, will improve the throughput and,
hopefully, the accuracy of phenotyping.
A second advantage offered by wheat and barley
is the extensive collection of land race and wild
germplasm. It is estimated that less than 15% of the
available variation has been captured in modern
wheat varieties and around 40% for barley (unpublished data). Many of the diversity screens of these
species have identified levels of stress tolerance
well beyond that seen in current varieties. It is also
important to remember that these species already
show a far higher degree of tolerance to many abiotic
stresses than other crop plants. This is the key reason
why wheat and barley dominate cropping in low
yielding environments. The availability of this
additional variation opens up the opportunity for
exploring stress tolerance mechanisms that may not
be fully developed in model species and also provides
scope for allele discovery and the use of allelic
diversity for functional analysis.
The complexities of abiotic stress responses
essentially preclude the precise experimental dissection of individual abiotic stresses, and suggest that
further studies of individual stresses might not be the
best approach. Genomics, proteomics and metabolomics, coupled with a strong bioinformatics
capability, now enable a ‘broad’ approach to be
taken in the study of plant responses to abiotic
stresses. Thus, the entire system of networks of
signalling pathways and key interconnecting processes that lead to the multiple defensive responses
can be described in detail. New technologies,
including improvements to positional cloning,
mean that an understanding of plant responses to
abiotic stresses and the basis for diversity may well be
achievable for the first time. This understanding can
be used for the manipulation of the responses, or
their transfer to important cereal crop species
351
through either conventional, marker assisted or
transgenic approaches.
Key Points
Cereal crops are often exposed to multiple environmental stresses simultaneously and, in many cases, common responses are
elicited by the plants to deal with these abiotic stresses.
Cereal germplasm collections, landraces and wild relatives of the
common cereals provide extensive natural variation that can be
exploited in crop improvement programs.
The long history of breeding, genetics and physiology of cereal
crops provides a unique resource for genomics studies in these
species.
Functional genomics technologies provide tools for the detection and definition of cellular networks through which stress
perception, signal transduction and defensive responses are
mediated.
References
1.
Boyer JS. Plant productivity and environment. Science 1982;
218:443–8.
2. Niyogi KK. Photoprotection revisited: genetic and molecular approaches. Ann Rev Plant Physiol Plant Mol Biol 1999;
50:333–59.
3. Alscher RG, Donahue JL, Cramer CL. Reactive oxygen
species and antioxidants: relationships in green cells. Physiol
Plant 1997;100:224–33.
4. Foyer CH, LopezDelgado H, Dat JF, et al. Hydrogen
peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol Plant 1997;
100:241–54.
5. Holmberg N, Bulow L. Improving stress tolerance in plants
by gene transfer. Trends Plant Sci 1998;3:61–6.
6. Kirch HH, Nair A, Bartels D. Novel ABA- and
dehydration-inducible aldehyde dehydrogenase genes isolated from the resurrection plant Craterostigma plantagineum
and Arabidopsis thaliana. PlantJ 2001;28:555–67.
7. Purvis AC. Role of the alternative oxidase in limiting
superoxide production by plant mitochondria. Physiol Plant
1997;100:165–70.
8. Shen B, Jensen RG, Bohnert HJ. Increased resistance
to oxidative stress in transgenic plants by targeting mannitol
biosynthesis to chloroplasts. Plant Physiol 1997;113:1177–83.
9. Hoekstra FA, Golovina EA, Buitink J. Mechanisms of plant
desiccation tolerance. Trends Plant Sci 2001;6:431–8.
10. Crowe JH, Oliver AE, Hoekstra FA, et al. Stabilization
of dry membranes by mixtures of hydroxyethyl starch
and glucose: the role of vitrification. Cryobiol 1997;35:
20–30.
11. Close TJ. Dehydrins: emergence of a biochemical role of a
family of plant dehydration proteins. Physiol Plant 1996;97:
795–803.
12. Dure L, Crouch M, Ho THD, et al. Common amino acid
sequence domains among LEA proteins of higher plants.
Plant Mol Biol 1989;12:475–86.
352
Langridge et al.
13. Cheng ZQ, Targolli J, Huang XQ, et al. Wheat LEA
genes, PMA80 and PMA 1959, enhance dehydration
tolerance of transgenic rice (Oryza sativa L.). Mol Breed
2002;10:71–82.
14. Bahieldin A, Mahfouz HT, Eissa HF, et al. Field evaluation
of transgenic wheat plants stably expressing the HVA1 gene
for drought tolerance. Physiol Plant 2005;123:421–7.
15. Wehmeyer N, Vierling E. The expression of small heat
shock proteins in seeds responds to discrete developmental
signals and suggests a general protective role in desiccation
tolerance. Plant Physiol 2000;122:1099–108.
16. Leyman B, Van Dijck PP, Thevelein JM. An unexpected
plethora of trehalose biosynthesis genes in Arabidopsis
thaliana. Trends Plant Sci 2001;6:510–3.
17. Zhu JK. Plant salt tolerance. Trends Plant Sci 2001;6:66–71.
18. Diab AA, Teulat-Merah B, This D, et al. Identification of
drought-inducible genes and differentially expressed sequence tags in barley. TheorAppl Genet 2004;109:1417–25.
19. Teulat B, Borries C, This D. New QTLs identified for
plant water status, water-soluble carbohydrate and osmotic
adjustment in a barley population grown in a growthchamber under two water regimes. TheorAppl Genet 2001;
103:161–70.
20. Teulat B, Merah O, Sirault X, et al. QTLs for grain carbon
isotope discrimination in field-grown barley. Theor Appl
Genet 2002;106:118–26.
21. Teulat B, Zoumarou-Wallis N, Rotter B, et al. QTL for
relative water content in field-grown barley and their
stability across Mediterranean environments. Theor Appl
Genet 2003;108:181–8.
22. Jefferies SPP, Pallotta MA, Paull JG, et al. Mapping and
validation of chromosome regions conferring boron toxicity
tolerance in wheat (Triticum aestivum).TheorAppl Genet 2000;
101:767–77.
23. Zanetti S, Winzeler M, Keller M, et al. Genetic analysis of
pre-harvest sprouting resistance in a wheat x spelt cross.
Crop Sci 2000;40:1406–17.
24. Roy JK, Prasad M, Varshney RK, et al. Identification of
a microsatellite on chromosomes 6B and a STS on 7D of
bread wheat showing an association with preharvest
sprouting tolerance. TheorAppl Genet 1999;99:336–40.
25. Vagujfalvi A, Crosatti C, Galiba G, et al. Two loci on wheat
chromosome 5A regulate the differential cold-dependent
expression of the cor14b gene in frost-tolerant and frostsensitive genotypes. Mol Gen Genet 2000;263:194–200.
26. Snape JW, Sarma R, Quarrie SA, et al. Mapping genes for
flowering time and frost tolerance in cereals using precise
genetic stocks. Euphytica 2001;120:309–15.
27. St Burgos M, Messmer MM, Stamp PP, et al. Flooding
tolerance of spelt (Triticum spelta L.) compared to wheat
(Triticum aestivum L.) – a physiological and genetic approach.
Euphytica 2001;122:287–95.
28. Riede CR, Anderson JA. Linkage of RFLP markers to
an aluminum tolerance gene in wheat. Crop Sci 1996;36:
905–9.
29. Luo MC, Dvorak J. Molecular mapping of an aluminum
tolerance locus on chromosome 4D of Chinese Spring
wheat. Euphytica 1996;91:31–5.
30. Allen GJ, Jones RGW, Leigh RA. Sodium-transport
measured in plasma-membrane vesicles isolated from
wheat genotypes with differing Kþ/Naþ discrimination
traits. Plant Cell Environ 1995;18:105–15.
31. Altinkut A, Gozukirmizi N. Search for microsatellite
markers associated with water-stress tolerance in wheat
through bulked segregant analysis. Mol Biotech 2003;23:
97–106.
32. Cattivelli L, Baldi PP, Crosatti C, et al. Chromosome
regions and stress-related sequences involved in
resistance to abiotic stress in Triticeae. Plant Mol Biol 2002;
48:649–65.
33. Quarrie SA, Steed A, Calestani C, et al. A high-density
genetic map of hexaploid wheat (Triticum aestivum L.) from
the cross Chinese Spring X SQ1 and its use to compare
QTLs for grain yield across a range of environments.
TheorAppl Genet 2005;110:865–80.
34. Verma VOL, Foulkes MJ, Worland AJ, et al. Mapping
quantitative trait loci for flag leaf senescence as a yield
determinant in winter wheat under optimal and droughtstressed environments. Euphytica 2004;135:255–63.
35. Farshadfar E, Mohammadi R, Farshadfar M, et al. Locating
QTLs controlling field and laboratory predictors of drought
tolerance in Agropyron using multiple selection index.
Cereal Res Comm 2004;32:17–24.
36. Jefferies SPP, Barr AR, Karakousis A, et al. Mapping of
chromosome regions conferring boron toxicity tolerance
in barley (Hordeum vulgare L.). Theor Appl Genet 1999;98:
1293–303.
37. Pallotta MA, Graham RD, Langridge PP, et al. RFLP
mapping of manganese efficiency in barley. TheorAppl Genet
2000;101:1100–8.
38. Reinheimer JL, Barr AR, Eglinton JK. QTL mapping of
chromosomal regions conferring reproductive frost tolerance in barley (Hordeum vulgare L.). Theor Appl Genet 2004;
109:1267–74.
39. Zhang F, Chen G, Huang Q, et al. Genetic basis of barley
caryopsis dormancy and seedling desiccation tolerance at the
germination stage. TheorAppl Genet 2005;110:445–53.
40. Thomashow MF. Plant cold acclimation: freezing tolerance
genes and regulatory mechanisms. Ann Rev Plant Physiol
Plant Mol Biol 1999;50:571–99.
41. Laurie DA, Pratchett N, Bezant JH, et al. RFLP mapping
of 5 major genes and 8 quantitative trait loci controlling
flowering time in a winter x spring barley (Hordeum
vulgare L.) cross. Genome 1995;38:575–85.
42. Campbell SA, Close TJ. Dehydrins: genes, proteins, and
associations with phenotypic traits. New Phytologist 1997;137:
61–74.
43. Yu XM, Griffith M. Winter rye antifreeze activity increases
in response to cold and drought, but not abscisic acid.
Physiol Plant 2001;112:78–86.
44. Zhang HX, Blumwald E. Transgenic salt-tolerant tomato
plants accumulate salt in foliage but not in fruit. Nature
Biotech 2001;19:765–8.
45. Barbier-Brygoo H, Gaymard F, Rolland N, et al. Strategies
to identify transport systems in plants. Trends Plant Sci 2001;
6:577–85.
46. Sivaguru M, Fujiwara T, Samaj J, et al. Aluminum-induced
1->3-beta-D-glucan inhibits cell-to-cell trafficking of
molecules through plasmodesmata. A new mechanism
of aluminum toxicity in plants. Plant Physiol 2000;124:
991–1005.
47. Ezaki B, Katsuhara M, Kawamura M, et al. Different
mechanisms of four aluminum (Al)-resistant transgenes for
Al toxicity in Arabidopsis. Plant Physiol 2001;127:918–27.
Functional genomics of abiotic stress tolerance in cereals
48. Graham RD, Welch RM, Bouis HE. Addressing micronutrient malnutrition through enhancing the nutritional
quality of staple foods: principles, perspectives and knowledge gaps. AdvAgron 2001;70:77–142.
49. Huang CY, Barker SJ, Langridge PP, et al. Zinc deficiency
up-regulates expression of high-affinity phosphate transporter genes in both phosphate-sufficient and -deficient barley
roots. Plant Physiol 2000;124:415–22.
50. Knight H, Knight MR. Abiotic stress signalling pathways’,
specificity and cross-talk. Trends Plant Sci 2001;6:262–7.
51. Ishitani M, Xiong LM, Stevenson B, et al. Genetic
analysis of osmotic and cold stress signal transduction
in Arabidopsis’, interactions and convergence of abscisic
acid-dependent and abscisic acid-independent pathways.
Plant Cell 1997;9:1935–49.
52. Urao T, Yakubov B, Satoh R, et al. A transmembrane
hybrid-type histidine kinase in arabidopsis functions as an
osmosensor. Plant Cell 1999;11:1743–54.
53. Vinocur B, Altman A. Recent advances in engineering plant
tolerance to abiotic stress: achievements and limitations.
Current Opin Biotech 2005;16:123–32.
54. Pellegrineschi A, Reynolds M, Pacheco M, et al. Stressinduced expression in wheat of the Arabidopsis thaliana
DREB1A gene delays water stress symptoms under greenhouse conditions. Genome 2004;47:493–500.
55. The Arabidopsis Information Resource, http://www.
arabidopsis.org/Carnegie, Inst, Washington [25 October
2005, date last accessed].
56. Liu Q, Kasuga M, Sakuma Y, et al. Two transcription
factors, DREB1 and DREB2, with an EREBP/AP2 DNA
binding domain separate two cellular signal transduction
pathways in drought- and low-temperature-responsive gene
expression, respectively, in Arabidopsis. Plant Cell 1998;10:
1391–406.
57. Qin F, Sakuma Y, Li J, et al. Cloning and functional analysis
of a novel DREB1/CBF transcription factor involved in
cold-responsive gene expression in Zea mays L. Plant Cell
Physiol 2004;45:1042–52.
58. Dubouzet JG, Sakuma Y, Ito Y, et al. OsDREB genes in
rice, Oryza sativa L., encode transcription activators that
function in drought-, high-salt- and cold-responsive gene
expression. PlantJ 2003;33:751–63.
59. Shen YG, Zhang WK, He SJ, et al. An EREBP/AP2-type
protein in Triticumaestivum was a DRE-binding transcription
factor induced by cold, dehydration and ABA stress. Theor
Appl Genet 2003;106:923–30.
60. Pellegrineschi A, Hoisington D. Results of Transgenic
Wheat Trial Look Promising, http://www.cimmyt.org/
english/wps/news/dreb.htm [25 October 2005, date last
accessed].
61. Roosens NH, Thu TT, Iskandar HM, et al. Isolation of the
ornithine-delta-aminotransferase cDNA and effect of salt
stress on its expression in Arabidopsis thaliana. Plant Physiol
1998;117:263–71.
62. DIR 053/2004 – Field trial of genetically modified salt
tolerant wheat on saline land, http://www.ogtr.gov.au/ir/
dir053.htm, Office of the Gene Technology Regulator [30
January 2006, date last accessed].
63. Devos KM. Updating the ‘Crop circle’. Current Opin Plant
Biol 2005;8:155–62.
64. Ware D, Stein L. Comparison of genes among cereals.
Current Opin Plant Biol 2003;6:121–7.
353
65. Sutton T, Whitford R, Baumann U, et al. The Ph2 pairing
homoeologous locus of wheat (Triticum aestivum), identification of candidate meiotic genes using a comparative genetics
approach. PlantJ 2003;36:443–56.
66. Yu J, Hu SN, Wang J, et al. A draft sequence of the
rice genome (Oryza sativa L. ssp indica). Science 2002;296:
79–92.
67. Goff SA, Ricke D, Lan TH, et al. A draft sequence of the
rice genome (Oryza sativa L. ssp japonica). Science 2002;296:
92–100.
68. Feuillet C, Travella S, Stein N, et al. Map-based isolation of
the leaf rust disease resistance gene Lr10 from the hexaploid
wheat (Triticum aestivum L.) genome. Proc Natl Acad Sci USA
2003;100:15253–8.
69. Yan L, Loukoianov A, Tranquilli G, et al. Positional cloning
of the wheat vernalization gene VRN1. Proc Natl Acad Sci
USA 2003;100:6263–8.
70. Ahloowalia BS, Maluszynski M. Induced mutations –
a new paradigm in plant breeding. Euphytica 2001;118:
167–73.
71. Caldwell DG, McCallum N, Shaw PP, et al. A structured
mutant population for forward and reverse genetics in
Barley (Hordeum vulgare L.). PlantJ 2004;40:143–50.
72. Slade AJ, Fuerstenberg SI, Loeffler D, et al. A reverse
genetic, nontransgenic approach to wheat crop improvement by TILLING. Nature Biotech 2005;23:75–81.
73. Cooper LD, Marquez-Cedillo L, Singh J, et al. Mapping
Ds insertions in barley using a sequence-based approach.
Mol Genet Genom 2004;272:181–93.
74. Liu X, Shi J, Zhang XY, et al. Screening salt tolerance
germplasms and tagging the tolerance gene(s) using
microsatellite (SSR) markers in wheat. Acta Botanica Sinica
2001;43:948–54.
75. Wang RRC, Li XM, Hu ZM, et al. Development of
salinity-tolerant wheat recombinant lines from a wheat
disomic addition line carrying a Thinopyrum junceum
chromosome. IntlJ Plant Sci 2003;164:25–33.
76. Tyankova N, Zagorska N, Dimitrov B. Study of drought
response in wheat cultivars, stabilized wheat-wheatgrass
lines and intergeneric wheat amphidiploids cultivated
in vitro. Cereal Res Comm 2004;32:99–105.
77. Moghaddam AE, Trethowan RM, William HM, et al.
Assessment of genetic diversity in bread wheat genotypes for
tolerance to drought using AFLPs and agronomic traits.
Euphytica 2005;141:147–56.
78. Peleg Z, Fahima T, Abbo S, et al. Genetic diversity for
drought resistance in wild emmer wheat and its ecogeographical associations. Plant Cell Environ 2005;28:176–91.
79. Rizza F, Badeck FW, Cattivelli L, et al. Use of a water stress
index to identify barley genotypes adapted to rainfed and
irrigated conditions. Crop Sci 2004;44:2127–37.
80. Forster BPP, Ellis RPP, Moir J, et al. Genotype
and phenotype associations with drought tolerance
in barley tested in North Africa. Ann Appl Biol 2004;144:
157–68.
81. Nevo E, Beharav A, Meyer RC, et al. Genomic
microsatellite adaptive divergence of wild barley by
microclimatic stress in ‘Evolution Canyon’, Israel. Biol J
Linnean Soc 2005;84:205–24.
82. Powell W, Langridge P. Unfashionable species flourish in
the 21st Century. Genome Biol 2004;5:233.
354
Langridge et al.
83. Zhang HN, Sreenivasulu N, Weschke W, et al. Large-scale
analysis of the barley transcriptome based on expressed
sequence tags. PlantJ 2004;40:276–90.
84. Mochida K, Yamazaki Y, Ogihara Y. Discrimination of
homologous gene expression in hexaploid wheat by
SNP analysis of contigs grouped from a large number of
expressed sequence tags. Mol Genet Genom 2004;270:371–7.
85. Leader DJ. Transcriptional analysis and functional genomics
in wheat. J Cereal Sci 2005;41:149–63.
86. Close TJ, Wanamaker SI, Caldo RA, et al. A new resource
for cereal genomics: 22K barley GeneChip comes of age.
Plant Physiol 2004;134:960–8.
87. Hazen SPP, Hawley RM, Davis GL, et al. Quantitative trait
loci and comparative genomics of cereal cell wall composition. Plant Physiol 2005;132:263–71.
88. Meyers B. Rice MPSS site, http://mpss.udel.edu/rice/,
University of Delaware [25 October 2005, date last accessed].
89. Bahrman N, Negroni L, Jaminon O, et al. Wheat leaf
proteome analysis using sequence data of proteins separated
by two-dimensional electrophoresis. Proteomics 2004;4:
2672–84.
90. Skylas D, Van Dyk D, Wrigley CW. Proteornics of wheat
grain. J Cereal Sci 2005;41:165–79.
91. Woo SH, Kimura M, Higa-Nishiyama A, et al. Proteome
analysis of wheat lemma. Biosci Biotech Biochem 2003;67:
2486–91.
92. Salekdeh GH, Siopongco J, Wade LJ, et al. A proteomic
approach to analyzing drought- and salt-responsiveness in
rice. Field Crops Res 2002;76:199–219.
93. Sato S, Soga T, Nishioka T, et al. Simultaneous determination of the main metabolites in rice leaves using
capillary electrophoresis mass spectrometry and capillary
electrophoresis diode array detection. Plant J 2004;40:
151–63.