BACKGROUND
the leaf chlorophyll content of plants determines their capacity to absorb energy from sunlight and is therefore considered a measure of photosynthetic potential
<cit> .
plants typically respond to water stress by reducing the leaf chlorophyll content, and hence their photosynthetic activity
<cit> .
genotypes that tolerate water stress tend to have more leaf chlorophyll and lower canopy temperatures when stressed
<cit> .
in contrast, genotypes that are susceptible to water stress often have higher canopy temperatures
<cit> , and when such plants are well-watered, with their stomata fully open, water evaporation causes leaf cooling
<cit> .
the ability of a genotype to remain green under water stress conditions may therefore enhance photoassimilation, thus increasing productivity and grain filling
<cit> .
in contrast to these general statements, a low level of leaf chlorophyll can limit the interception of light to avoid oxidative damage caused by excess radiation
<cit>  and could be considered a desirable trait under water stress conditions
<cit> .
the optimization of photosynthesis in crops under water stress therefore requires a productive balance between assimilation under favorable conditions and the avoidance of excess radiation under stress
<cit> .
chlorophylls and carotenoids play an important role in photoassimilation and the synthesis of both molecules is coupled to chloroplast development
<cit> .
thylakoid organization is coordinated with the expression of nuclear genes, encoding pigment-binding proteins that are imported into the chloroplast to assemble the photosynthetic complexes and the expression of chloroplast encoded genes
<cit> .
this process is tightly regulated, so mutants deficient in chlorophyll content can arise not only through the disruption of chlorophyll biosynthesis but also via processes related to photosystem assembly and light-harvesting activity
<cit> .
such chlorophyll mutants have been used to study the function of photosynthetic components as described in rice e.g.
<cit> , barley e.g.
<cit>  and wheat e.g.
<cit> .
carotenoids are involved in photoprotection and in light harvesting, and different carotenoids play different roles in the photosynthetic complexes.
for example, only β-carotene is present in the reaction centers whereas xanthophylls are present in the antennal light harvesting complex  where each protein has a specific carotenoid complement that ensures maximum photosystem efficiency
<cit> .
lutein is involved in light harvesting and the quenching of 3chl states
<cit>  and violaxanthin can be replaced by zeaxanthin if the de-epoxidation cycle is activated by photosystem saturation following the absorption of excess light.
zeaxanthin helps to prevent photo-inhibition by dissipating the excess of energy as heat, either by direct quenching or by inducing conformational changes in the lhc proteins
<cit> .
photo-inhibition is an early consequence of excess light and other environmental stresses, reducing photosynthetic activity and leading to pigment photo-oxidation, the production of reactive oxygen species  and ultimately cell death
<cit> .
chloroplasts and mitochondria are the primary sources of ros, which damage cells by oxidizing nucleic acids, proteins and lipids.
however, low concentrations of ros act as potent signaling molecules that coordinate responses to abiotic stress, pests and pathogens
<cit> .
the balance between ros production and scavenging by antioxidants can be disrupted by different forms of stress
<cit> .
wheat is a staple crop that plays a major role in global food security.
conventional breeding strategies have been used to increase grain yields and stress tolerance, but in order to feed a growing population it will be essential to include molecular breeding as well as conventional approaches to achieve further substantial gains
<cit> .
gene expression profiling with microarrays has been used to characterize the response of wheat to heat and drought stress
<cit> , cold
<cit> , fusarium infection
<cit>  and phase-transition
<cit> , and to investigate the molecular basis of grain quality traits
<cit> .
in the context of drought stress, krugman et al.
<cit>  compared resistant and susceptible wild emmer genotypes to investigate the overlapping regulatory and signaling processes overrepresented in the drought-resistant genotype and to identify candidate genes for drought tolerance.
proteomics has also been used to investigate the response of wheat to nitrogen
<cit> , salinity stress
<cit> , low temperatures
<cit>  and drought
<cit> .
poor correlation between transcriptomic and proteomic data has been reported in some stress experiments although such complementary information highlights the multilevel regulation of responses to complex environments
<cit> .
most of the experiments discussed above involved genotypes with contrasting phenotypes and different genetic backgrounds, an approach that allows many genes and stress tolerance strategies to be identified, but makes it difficult to characterize specific genotype-phenotype relationships.
this can be achieved in experiments that involve genotypes differing at a single locus e.g.
<cit> .
in order to control for genetic background effects, we compared a pale-green mutant  and its wild-type mother variety  to determine the impact of leaf pigment content on gene and protein expression.
we studied the chlorophyll and carotenoid content as well as the transcriptomic and proteomic profiles of the mutant and wild-type plants under well-irrigated  conditions and terminal water stress.
we also tested the performance of the mutant and wild-type plants under irrigated  and rain-fed field conditions in order to assess the effect of the mutation on biomass accumulation and grain yield.
RESULTS
chloroplast ultrastructure
wild-type and mutant leaf sections at the tillering stage were compared by transmission electron microscopy .
as shown in figure
<dig>  lens-shaped chloroplasts were present in the wild-type mesophyll cells whereas those in the mutant plants were typically cylindrical.
the thylakoid grana of the wild-type chloroplasts appeared to be connected by continuous intergranal lamellae parallel to the long axis of the chloroplast, whereas the mutant grana appeared to be disconnected and disorganized within the chloroplast stroma.
osmiophilic lipid globules  were more abundant in the mutant chloroplasts .
physiological responses and field measures
water stress was applied to plants in the glasshouse by reducing the soil water content at anthesis.
this increased the negative value of the midday flag leaf water potential , reduced the leaf conductance to water vapor values  and reduced the mean net photosynthetic rate  but differences between genotypes were not statistically significant .
in the field, differences in aboveground biomass were significant only under rain-fed conditions, but the grain yield of the mutant plants was reduced by  <dig> % under irrigated conditions and by  <dig> % under rain-fed conditions, compared to wild-type plants in the same environment .
w
s
w
s
a) f-values of the analysis of variance and, b) means of soil water content , flag leaf water potential , stomatal conductance  and net photosynthetic rate  for each water treatment and for the wild-type and the mutant plants under control and water stress conditions.
water stress started at booting.
***p <  <dig> , n.s.
p >  <dig> .
for each variable, water treatment means with different letters are significantly different for a tukey test.
average values of grain yield and aboveground biomass were determined at anthesis in the wild-type  and the pale-green mutant.
data represent means of 3 years of field experiments.
genotype means with different letters within a water regime differ at p =  <dig> .
pigment content
the total carotenoid and chlorophyll levels in the flag leaves at anthesis under well-watered  conditions were significantly lower in the mutant than the wild-type, but differences in antheraxanthin and zeaxanthin levels were not statistically significant .
under water stress conditions, only the antheraxanthin content differed significantly between wild-type and mutant leaves, whereas the total carotenoid and chlorophyll contents were similar in both genotypes.
the xanthophyll de-epoxidation cycle appeared to be induced in the mutant plants under control conditions.
whereas under terminal water stress, wild-type plants increased the de-epoxidation rate, mutant plants did not.
therefore, there was no statistically significant difference between the genotypes under water stress conditions .
vs
p
vs
p
pigment quantification  of the flag leaf of wild-type  and pale-green mutant  plants at anthesis under control and water stress conditions was performed by hplc.
values are means of four samples.
xanthophylls de-epoxidation rate .
transcriptomic profiling
microarray quality analysis
microarray data were collected using the affymetrix wheat genome array and normalized using the rma algorithm.
the average background was  <dig> , well within the recommended levels.
the percentage of "present" calls ranged from  <dig> % to  <dig> % among the 61 k probe sets.
the quality of the biological replicates was determined by calculating r <dig> values among replicates of the same sample and the values ranged between  <dig>  and  <dig>  with an average of  <dig> .
principal component analysis highlighted the main sources of total variance.
the two components explaining  <dig> % and  <dig> % of the variance represented the differences between the treatments  and between the genotypes  as shown in figure
<dig>
the mutation effect on gene expression was studied comparing the expression of the mutant to wild-type for each water treatment and indicated along the manuscript with the terms induced or repressed genes.
in addition, the stress effect was evaluated comparing the gene expression of the water stress to control conditions for each genotype and indicated along the manuscript as up/downregulated genes.
gene expression under control conditions
transcriptional profiling at anthesis revealed differences in gene expression between the two genotypes under control conditions.
we identified  <dig> induced and  <dig> repressed probe sets in the mutant .
the array data for selected probe sets were validated by quantitative real-time rt-pcr, showing strong agreement between the fold-change values observed with each method .
mips funcatdb was used to distribute the genes into functional categories, revealing that the induced probe sets in the mutant represented chitin catabolism, defense  and cell wall metabolism .
the most strongly induced probe sets represented peroxidase  <dig> , pathogenesis-related  proteins including beta-glucanases and chitinases, and a tawrky45-like transcription factor .
the repressed probe sets represented functions including systemic interaction with the environment, abscisic acid  responses, hormonal regulation, stress responses and dna degradation , e.g.
sequences representing rab  proteins, late embryogenesis abundant protein lea <dig> and endonucleases .
probe sets related to cell rescue, defense and virulence  were induced or repressed in the mutant when compared with the wild-type.
however, none the subcategories was statistically significant at p ≤  <dig> .
p
significant functional categories represented in the induced and repressed probe sets in the mutant compared to wild-type plants at anthesis under control conditions according to the mips functional catalogue database
<cit> .
only functional categories with a cut-off of p ≤  <dig>  were considered.
a
aspecies: 1: triticum aestivum, 2: hordeum vulgare, 3: zea mays, 4: sorghum vulgare, 5: triticum monococcum, 6: triticum turgidum ssp.
durum, 7: oryza sativa.
-: not determined.
gene expression under water stress
we initially compared each genotype under stress and control conditions to identify both general and genotype-specific stress-modulated genes, which were defined as upregulated  or downregulated , as mentioned above.
we identified a total of  <dig> probe sets that were modulated by water stress in at least one of the genotypes , including  <dig> genes in the wild-type plants,  <dig> genes in the mutant plants and  <dig> in both.
we also compared the wild-type and mutant plants under water stress conditions, to identify genes that were differently-expressed under stress.
we identified  <dig> probe sets that were defined either as induced or repressed in the mutant plants, according to the level of expression under water stress conditions.
genotype-independent stress-response genes
from the  <dig> probe sets modulated in both genotypes,  <dig> were upregulated and  <dig> were downregulated under water stress .
the upregulated genes predominantly represented the following functional categories: stress response, carbon compound and carbohydrate metabolism, metabolite transport, amino acid metabolism, aba response and proline biosynthesis .
genes already known to be associated with water stress are shown in the additional file
3: table s <dig>  these include genes encoding sucrose synthase , glucose-6-phosphate dehydrogenase , branched-chain-amino-acid aminotransferase  and branched-chain alpha-keto acid dehydrogenase e <dig> α subunit , delta 1-pyrroline-5-carboxylate synthetase  <dig> , pyrroline-5-carboxylate reductase  and rab <dig>  the downregulated genes predominantly represented the following functional categories: phosphate and secondary metabolism, e.g.
1-aminocyclopropane-1-carboxylate synthase  and 12-oxophytodienoate reductase ; interaction with the environment, e.g.
chitinases, β-glucanases and pathogenesis-related proteins; immune response, e.g.
transcription factor wrky; and phenylpropanoid metabolism, e.g.
phenylalanine ammonia-lyase.
these genotype-independent stress-modulated genes correspond to the well-characterized drought stress response of wheat, confirming that the mutant plants preserve much of the stress-response signaling network that allows wheat plants to tolerate water stress
<cit> .
genotype-dependent stress-response genes in wild-type plants
we identified  <dig> probe sets that were modulated solely in the wild-type plants or to a greater extent  than in the mutant plants.
this included  <dig> upregulated and  <dig> downregulated genes .
the upregulated genes predominantly represented carbohydrate and pyruvate metabolism and the glyoxylate cycle, whereas the downregulated represented metabolism, interaction with the environment, protein modification and signaling pathways .
selected genes specifically modulated by the wild-type plants under stress are listed in table
<dig>  including carbon fixation enzymes such as malate dehydrogenase  and phosphoenolpyruvate caboxykinase , and glyoxylate cycle enzymes such as isocitrate lyase  and malate synthase .
p
significant functional categories represented in the specifically upregulated and downregulated probe sets in wild-type plants at anthesis under water stress conditions compared to well-watered  conditions according to the mips functional catalogue database
<cit> .
only functional categories with a cut-off of p ≤  <dig>  were considered.
<dig>
<dig>
<dig>
selected genes were specifically significant regulated by the wild-type  and the mutant  under water stress compared with the same genotype under well-watered  conditions.
last column  compares mutant versus wt expression under water stress.
positive and negative values indicate upregulation and downregulation respectively, whereas no statistically significant differences are indicated with a dash .
auniprot id from triticum turgidum subsp.
durum.
bblast executed against tagi  wheat gene index: tc <dig>  homologue to uniref100_p <dig>
genotype-dependent stress-response genes in mutant plants
we identified  <dig> probe sets that were modulated solely in the mutant plants or to a greater extent  than in the wild-type plants.
this included  <dig> upregulated and  <dig> downregulated genes representing diverse functional categories .
the most significant categories in the upregulated group were associated with chloroplast transcription, interactions with the environment , stress responses and metabolism, whereas the downregulated group was dominated by functional categories such as interaction with the environment , stress responses and plant defense .
the downregulated genes also included those induced in the mutant under control conditions.
p
significant functional categories represented in the specifically upregulated and downregulated probe sets lists in the mutant at anthesis under water stress conditions compared to well-watered  conditions according to the mips functional catalogue database
<cit> .
only functional categories with a cut-off of p ≤  <dig>  were considered.
the genes that were upregulated specifically in the mutant included those involved in the synthesis of carotenoid precursors, e.g.
hydroxymethylglutaryl-coa synthase , 1-deoxy-d-xylulose-5-phosphate synthase  and phytoene synthase , and those involved in chloroplast gene expression, photosynthesis, chlorophyll binding and detoxification, e.g.
glutamyl-trna reductase  <dig> , light harvesting complex ii components such as lhb1b <dig> and atpase subunit delta, and the rubisco small subunit.
we observed the downregulation of several genes representing glutamate metabolism such as glutamate decarboxylase , glutamate dehydrogenase , γ-glutamyltranspeptidase  and l-aspartate oxidase ; and several signaling pathway components, including calcineurin b-like protein  <dig> , inositol phosphatase  and mpk <dig> .
differentially-expressed stress-response genes under stress conditions
we compared the transcriptional profiles of wild-type and mutant plants under water stress and identified  <dig> differently-expressed probe sets .
the only functional category significantly represented among the  <dig> induced probe sets was the channel/pore transport class , corresponding to the transcripts for two plasma membrane intrinsic proteins  that were downregulated in both genotypes but to a lesser extent in the mutant .
other genes that were induced in the mutant under water stress included those encoding ascorbate peroxidase , sucrose: fructan 6-fructosyltransferase , myo-inositol oxygenase  and dihydrodipicolinate reductase  <dig>  .
the genes that were repressed in the mutant plants under stress were predominantly related to carbohydrate metabolism, peroxidase/oxidative stress responses, the glyoxylate cycle and chromosome subcellular localization .
representative repressed genes included those encoding rap <dig> , gdh, gad, cbl-interacting protein kinase  <dig>  and glyoxalase .
we also found genes encoding icl, mls and trehalose phosphate-synthase  which were not affected by stress in mutant plants but were upregulated in wild-type plants, thus appearing in the repressed transcript category .
proteomic profiling
the effect of the mutation was also investigated by carrying out a comparative proteomic analysis between wild-type and mutant plants at anthesis under the same conditions as described above.
when wild-type and mutant plants were compared under well-watered  conditions, we matched  <dig> protein spots  among which two showed a significant  difference in signal intensity between genotypes, although neither spot could be identified because of the low abundance of the corresponding protein.
even so, this experiment supported the transcriptomic and suggested that there were only minor differences between the genotypes under normal conditions.
when we compared each genotype under stress and control conditions, we matched  <dig> protein spots among both genotypes.
from the  <dig>  we found that  <dig> spots were modulated by water stress in the wild-type plants and  <dig> were modulated in the mutant plants, including  <dig> spots that were common to both genotypes.
this left seven modulated spots specific to the wild-type plants and  <dig> specific to the mutant.
when the two genotypes were compared under water stress, we identified  <dig> spots that were more abundant in the mutant and  <dig> that were less abundant in the mutant.
we selected  <dig> of the induced spots and all  <dig> of the repressed spots based on the likelihood of extracting useful amounts of protein, and we identified  <dig> proteins from the induced set and  <dig> from the repressed set .
the proteins were identified by peptide mass fingerprinting  and ms/ms analysis .
the induced proteins predominantly represented carbohydrate and amino acid metabolism, photosynthesis, detoxification and phytoalexin biosynthesis, whereas the repressed proteins predominantly represented photosynthesis .
two proteins that were specifically upregulated in the mutant were the oxygen-evolving enhancer protein  <dig> corresponding to the psbp subunit of photosystem ii , and catalase- <dig> .
two proteins that were specifically downregulated in the mutant were the rubisco small subunit chain pw <dig>  and the photosystem i reaction center subunit iv .
b
c
aaccession number from ncbi.
bspecies: 1: hordeum vulgare; 2: zea mays; 3:triticum aestivum; 4: nicotiana tabacum; 5: brachypodium distachyon; 6: arabidopsis thaliana.
cmethod: pmf: peptide mass fingerprinting; ms/ms: maldi tof/tof.
ratios are only indicated for differences statistically significant.
discussion
chloroplast ultrastructure and pigment content
chlorotic mutants with affected chloroplast ultrastructure have been described in several reports
<cit> .
the chloroplasts in these plants often contain fewer grana structures than wild-type plants, and thinner thylakoid membranes that are not stacked in the normal manner
<cit> .
in contrast, the chloroplasts in our pale-green mutant contained abundant grana with normally-stacked thylakoids, but their distribution within the stroma was disorganized, potentially reflecting a lack of membrane continuity or the degradation of inter-grana lamella membranes.
the stacking adhesion of the thylakoids in grana structures is regulated by the phosphorylation of photosystem ii proteins, which also ensures photosynthetic efficiency
<cit> .
we found no significant difference in the photosynthetic rate of the wild-type and mutant and no quantitative differences in transcripts or proteins representing the photosynthetic apparatus at anthesis under control conditions.
the mutant chloroplasts also contained more stromal plastoglobules than wild-type chloroplasts.
these lipoprotein bodies are structurally continuous with the thylakoid membranes and facilitate content exchange with the thylakoid lumen
<cit> .
they may also be involved in thylakoid development and disassembly, increasing in abundance in response to oxidative stress and delivering the antioxidant molecules stored on them to the thylakoid lumen, where they scavenge ros
<cit> .
the structure of the inter-grana lamella in the mutant chloroplasts could therefore reflect thylakoid reorganization or the impact of oxidative stress already at tillering stage under field capacity conditions.
although the pigment content of the mutant plants was ~41% lower than in wild-type plants under control conditions, the photosynthetic rate was similar .
this seems illogical given that carotenoids and chlorophylls are required for the proper folding, assembly and stability of lhc apoproteins
<cit> .
however, we detected a greater xanthophyll de-epoxidation rate in the mutant as indicated by the relationship /.
the zeaxanthin in the mutant may compensate for the lack of other xanthophylls in the antenna proteins
<cit> , and may also preserve the structure of the photosystem and its membranes by dissipating photochemical energy as heat to prevent photo-oxidation
<cit> .
this may explain the abnormal chloroplast ultrastructure in the mutant but the near-normal photosynthetic rate.
our data suggest that, under control conditions, the mutant compensates for the lower pigment content and that minor oxidative stress has no detrimental effect on photosynthetic capacity or biomass accumulation.
however, under water stress conditions the level of de-epoxidation increases in wild-type plants, and both genotypes show similar values for the relationship / .
this suggests that the mutant has a lower capacity for increasing the de-epoxidation rate.
an alternative explanation is that the pool of convertible violaxanthin is rapidly depleted and only non-convertible violaxanthin remains to maintain the structural integrity of the antenna
<cit> .
because xanthophylls help to prevent photo-oxidation, the mutant plants without additional de-epoxidated carotenoids to cope for the water stress damage may sense higher oxidative stress in the chloroplast under water stress.
thus, the pale-green mutant may be more susceptible since the ability of plants to withstand oxidative stress determines the level of overall stress tolerance
<cit> .
differences between the wild-type and mutant transcriptomes at anthesis
there were only minor differences in gene expression between the wild-type and mutant plants under control conditions at anthesis, mirroring the similar net photosynthetic rates .
the few repressed genes in the mutant were predominantly related to aba responses and the induced genes mostly represented pathogenesis-related  proteins and peroxidases involved in plant defense .
apart from their role in defense, peroxidases may be modulated by cellular redox disturbances
<cit> .
in such cases, nuclear gene expression can be regulated by ros-dependent signal transduction or low-molecular-weight antioxidants produced in the chloroplast.
for example, the expression of pr proteins and peroxidases increases in ascorbate-deficient mutants
<cit> .
since no lesion or pathogen attack was detected in our investigation, it is most likely that low-level of oxidative stress triggers defense gene expression in the pale-green mutant.
proteins involved in plant defense are also sensitive to hormones such as salicylic acid , jasmonate  and ethylene, but we did not find any differently-expressed genes related to these pathways in the mutant.
plant defense signaling pathways are also activated through wrky transcription factors that bind to pathogen-response and wound-inducible promoter elements .
the tawrky <dig> gene was induced in the mutant, and may help to activate some of the pr genes we also identified.
this transcription factor confers resistance to fusarium head blight  in wheat
<cit>  and increases the expression of pr- <dig> and pr- <dig> genes in rice to confer resistance to pseudomonas syringae, salinity and drought tolerance, as well as reducing aba sensitivity
<cit> .
genes encoding defense enzymes that synthesize secondary metabolites such as phenylpropanoids and flavonoids were also induced in the mutant, including naringerin, 2-oxoglutarate 3-dioxygenase and agmatine coumaroyltransferase , the latter representing the final step in the hydroxycinnamic acid amide  synthesis pathway.
peroxidases can also couple agmatine conjugates to form antifungal compounds known as hordatines in barley and  in wheat
<cit> .
finally, the blufensin- <dig> gene was also induced in the mutant.
this is a negative regulator conferring sensitivity to powdery mildew in barley by modulating penetration resistance
<cit> .
the repressed probe sets were related to systemic interactions with the environment, especially the aba response, e.g.
genes encoding dehydrins such as rab proteins and other lea proteins that accumulate during seed maturation and in vegetative tissues in response to abiotic stress
<cit> .
rab is a lea family dehydrin that is induced by aba, and the arabidopsis ortholog rab <dig> is a useful marker for the loss of aba sensitivity e.g.
in plants that are deficient for the swi/snf chromatin-remodeling complex
<cit> .
the gene is upregulated when a relevant repressor is mutated, e.g.
sumo e <dig> ligase siz1
<cit> .
we hypothesize that the mutant induces a repressor that acts against the aba pathway or downregulates an inducer of the same pathway.
indeed, two transcription factors were repressed in the mutant under control conditions, the q-type c2h <dig> zinc finger transcription factor tazp <dig> and the homeobox-leucine zipper protein athb- <dig>  tazp <dig> is upregulated in leaves and roots during drought stress and also following aba treatment, suggesting a role in aba-dependent gene regulation
<cit> .
athb- <dig> may act as a growth regulator in response to water deficit, and is induced by aba, salinity and dehydration stress
<cit> .
it also regulates the aba signaling pathway, acting downstream of abi1
<cit> .
the repressed probe sets also included two endonuclease genes related to the barley genes bnuc and ben <dig>  which are expressed during apoptosis
<cit> .
bnuc is also induced by salinity stress
<cit> .
the papain-like cysteine proteinase  and tds <dig> were also represented in the repressed probe sets, and these also facilitate senescence and apoptosis
<cit> .
hvpap <dig> also generates mature forms of storage proteins in seeds, provides free amino acids during germination
<cit> , and confers stress tolerance and defense against pathogens
<cit> .
tds <dig> is induced during post-flowering senescence in wheat
<cit> , and also by ja, sa and in chlorotic/necrotic leaf tissue following infection with pyrenophora teres <cit> .
there were few differences between the mutant and wild-type plants in the absence of water stress at anthesis and no statistically significant differentially-expressed genes could be identified also at the tillering stage .
therefore despite a clear difference in chlorophyll content and chloroplast structure, the mutation appears to have a minimal impact on metabolism and gene regulation, a finding that does not provide sufficient explanation for the yield loss detected under field conditions.
water stress enhances the differences between mutant and wild-type plants
we investigated the molecular basis of the difference in yield between the two genotypes by exposing them to water stress, which is the most common environmental constraint for durum wheat
<cit> .
most of the genes modulated by water stress were similarly regulated in both genotypes, suggesting a common response .
genes involved in the synthesis of osmoprotectants were upregulated in both genotypes, whereas genes involved in aba-dependent signaling, ethylene and ja synthesis were downregulated in both genotypes.
genes related to primary metabolism  were also similarly modulated, indicating that both genotypes use metabolic strategies to counter the effects of drought.
in contrast, genes encoding photosystem components and enzymes representing carbohydrate metabolism and the tricarboxylic acid  cycle showed significant differences that may explain the difference in performance between wild-type and mutant plants in the field.
photosynthesis and pigment synthesis
both transcriptomic and proteomic analysis indicated that the components of photosystems i and ii  and atpase were differentially expressed in the wild-type and mutant plants under water stress.
several psi proteins were underrepresented in the mutant, including reaction center  protein subunit iv , which is required for optimal electron transport to ferredoxin and flavodoxin
<cit> , and light harvesting complex lhca <dig>  .
psi takes longer than psii to recover from photo-inhibition because rc proteins are degraded instead of being repaired
<cit> .
therefore, the loss of psi rc proteins in the mutant is likely to exacerbate the damage caused by stress.
psbo and psbp maintain the structure of psii
<cit>  and were specifically overrepresented in the mutant.
cp <dig> was upregulated in both genotypes, but this was underrepresented in the mutant under stress conditions .
this protein binds lhcb proteins to the reaction center and confers photoprotection
<cit> .
the lhcb proteins were downregulated in both genotypes and underrepresented in the mutant.
in contrast, the genes encoding lhcb proteins were upregulated in the mutant .
the beta and delta subunits of atpase synthase were also overrepresented in the mutant, suggesting that the h+ flux across the thylakoid membrane is greater in the mutant, with a knock-on effect on atp production.
three genes responsible for the synthesis of carotenoid precursors were upregulated by stress specifically in the mutant: 1-deoxy-d-xylulose-5-phosphate synthase , hydroxymethylglutaryl-coa synthase  and phytoene synthase .
one gene encoding glutamyl-trna reductase  <dig> , which is responsible for the synthesis of chlorophyll precursors, was also upregulated by stress specifically in the mutant.
the pigment content of the mutant plants did not increase relative to the control but the activation of these genes could help to prevent a steeper decline in pigment content under water stress conditions .
redox changes in the chloroplast result in the stoichiometric adjustment of photosystem proteins and regulate the expression of both nuclear and plastid photosynthesis-related genes
<cit> .
because the mutant showed evidence of oxidative stress even under control conditions, the re-organization of the photosynthetic apparatus under water stress conditions could reflect the additional oxidative stress that occurs during the early stages of drought.
the loss of photosynthetic proteins  by the mutant in response to drought stress reduces the amount of captured energy and thus avoids the over-excitation of psii which would generate additional ros and cause oxidative damage
<cit> .
oxidative stress and ros signaling
the balance between ros synthesis and scavenging determines the level of oxidative stress in plants
<cit> .
ros scavenging and detoxifying proteins such as peroxidases, glutathione transferases and glyoxalase were generally modulated in a similar manner in both genotypes, but more probe sets of this group were downregulated specifically in the mutant under stress.
in the same direction, a glutaredoxin gene was upregulated in the wild-type plants under stress but was unaffected in the mutant .
thylakoid ascorbate peroxidase and catalase were upregulated specifically in the mutant, suggesting a greater need for protection against ros derived from chloroplast and peroxisome activity .
h2o <dig> can spread from the chloroplast to other organelles by diffusion through aquaporin-like pip <dig> proteins
<cit>  which were downregulated in the mutant to a lesser extent than in wild-type plants .
these results indicate that the mutant experiences greater oxidative stress than wild-type plants under water stress conditions.
two probe sets related to ros and stomatal closure were specifically modulated in the mutant under water stress.
the specific downregulation of glyoxalase gene suggests that the mutant is less able than wild-type plants to detoxify methylglyoxal using reduced glutathione, thus allowing the production of ros and stomatal closure
<cit> .
similarly, the specific upregulation of respiratory burst oxidase homologue d in the mutant  suggests that the aba-induced production of ros in guard cells is promoted to encourage stomatal closure
<cit> .
however, the differential expression of genes that regulate the stomatal aperture did not result in any differences between the genotypes in terms of stomatal conductance under water stress.
ros can induce ca2+-dependent signaling that triggers interactions between different calcineurin b-like  sensors and their cbl-interacting protein kinase  targets
<cit> .
genotype-dependent differences were found in expression of these genes: whereas cipk <dig> and cipk <dig> were upregulated by stress in both genotypes, cipk <dig> and cipk <dig> were upregulated specifically in wild-type plants and cbl <dig> and cipk <dig> were downregulated specifically in the mutant.
these two proteins cooperate with cbl <dig> to activate k+-channels and regulate stomatal responses
<cit> .
cipk <dig> and cipk <dig> belong to the snrk3-type family and mediate nutrient sensing, stress responses and aba signaling
<cit> .
as a result of altered expression of cbls and cipks different ca2+-dependent downstream responses under water stress are expected between mutant and wild-type plants.
mpk <dig>  which was specifically downregulated in the mutant, is also involved in ros signaling
<cit> .
once activated, it phosphorylates other stress-related proteins such as the transcription factors zat <dig> and azf <dig>  and enzymes in the ethylene synthesis pathway such as acs
<cit> , all of which were downregulated by stress in both genotypes.
zat <dig>  is a transcriptional repressor responsive to chitin, which mediates the response to photo-oxidative stress and ros
<cit> .
despite not showing damaged leaves or altered stomatal response, the data suggest that the pale-green mutant differs significantly from wild-type plants in terms of ros sensing and signaling under water stress.
ros generated in the chloroplast could move more easily inside the cells and elicit a signal triggering specific responses.
carbohydrate metabolism
transcriptomic profiling revealed differences between the genotypes in the regulation of carbohydrate metabolism under water stress conditions .
the synthesis of osmoprotectants such as raffinose family oligosaccharides  and trehalose was specifically upregulated in the wild-type plants, including the genes encoding galactokinase and raffinose synthases.
rfos protect membranes and scavenge ros
<cit> .
trehalose-phosphate synthase  was also specifically induced in wild-type plants and thus repressed in the mutant under stress.
trehalose and trehalose-6-phosphate act not only as osmolytes to stabilize proteins and membranes, but also as signaling molecules to regulate metabolism.
two of the probe sets upregulated in the mutant plants encoded sucrose: sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase , which are responsible for synthesizing fructans from sucrose in the vacuole
<cit> .
the mutant plants therefore appeared to promote fructan synthesis for remobilization as free carbohydrates, which are directly correlated with the water-soluble carbohydrate content in wheat
<cit> .
conversely, probe sets encoding cellulose synthase and cellulose synthase-like enzymes were upregulated specifically in the wild-type plants and xylosyltransferases were repressed in the mutant plants under stress .
these enzymes divert carbon to the synthesis of cell wall polysaccharides  and their loss reduces growth and also inhibits photosystem activity and carbohydrate metabolism genes
<cit> .
this shows the importance of cell wall structure not only for growth and turgor changes under stress but also for carbohydrate metabolism.
furthermore, sucrose, rfos and fructans can directly or indirectly scavenge ros, as reviewed by keunen et al.
<cit> .
the general picture that emerges from the modulation of carbohydrate metabolism suggests that the pale-green mutant is less able to deal with water stress than wild-type plants.
cell wall synthesis genes are activated in the wild-type plants whereas fructans are produced in the mutant.
this correlates with the lower biomass of the mutant plants at anthesis and the lower yield under rain-fed conditions .
tca cycle and related pathways
the tricarboxylic acid  cycle generates energy and reducing power  but consumes h2o and releases co <dig>  the substrates feeding the cycle are acetyl-coa and other intermediates from lipid or protein catabolism.
acetyl-coa is also produced from pyruvate  by the action of the pyruvate dehydrogenase complex, and the subunit e <dig> gene representing this complex was upregulated in both genotypes under water stress.
however, under conditions of carbon starvation or demand, additional strategies are used to supply tca substrates
<cit> .
the wild-type plants appear to be more effective at these strategies given the specific upregulation of the following genes :
i) genes encoding products that convert amino acids into substrates suitable for the tca cycle
<cit>  were upregulated in both genotypes , but three were upregulated specifically in wild-type plants: methylcrotonoyl-coa carboxylase, α and β subunits , and 2-oxoisovalerate dehydrogenase e <dig> .
ii) genes encoding the nadp-malic enzyme , which converts malate into pyruvate, and phosphoenolpyruvate carboxykinase , which converts oxaloacetate into phosphoenolpyruvate, were upregulated specifically in the wild-type plants.
iii) genes encoding isocitrate lyase  and malate synthase  from the glyoxylate cycle were upregulated specifically in wild-type plants and thus, repressed in the mutant.
this cycle produces succinate and malate acting as a bypass to avoid the loss of co <dig> from the normal tca cycle.
it also metabolizes glyoxylate generated by photorespiration and prevents photo-inhibition
<cit> .
iv) genes encoding glutamate dehydrogenase and glutamate decarboxylase  were specifically downregulated and consequently repressed in the mutant under stress.
they participate in glutamate metabolism, which balances nitrogen and carbon use in the cell
<cit>  either by storing nitrogen as gaba, glutamate or glutamine, or by supplying carbon back to the tca cycle.
a functional gaba shunt is required for stress tolerance
<cit> .
in the pale-green mutant, carbon stored as glutamate and gaba may not be introduced efficiently into the tca cycle, disrupting the nitrogen/carbon balance and reducing growth under carbon demand or starvation imposed by stress
<cit> .
gdh can also be inhibited by excess light or sucrose
<cit> .
the feed-in to the tca cycle and the ability to derive energy from it appear to be more limited in the mutant under stress, potentially explaining the differences between genotypes in terms of biomass and yield in the field experiments.
the mutant mobilizes carbon less efficiently, with a significant impact on grain filling and yield as observed in our field experiments.
regulators of carbon metabolism and signaling
genes that regulate carbohydrate and nitrogen metabolism were also differentially expressed between the wild-type and mutant plants under water stress, helping to explain the basis of the metabolic differences between the genotypes .
the bzip <dig> gene was specifically downregulated and repressed in the mutant plants under stress.
the transcription factor encoded by this gene is an important node in the glucose-aba interaction network and is repressed by both aba and glucose
<cit> .
it may promote starvation tolerance by regulating transcription of genes such as the raffinose synthase .
ap2/erebp proteins mediate hormone, sugar and redox signaling in the context of cold and drought stress
<cit> .
each member has a unique expression profile and the individual proteins are differentially regulated by glucose
<cit> .
we found that rap <dig>  and rap <dig>  were specifically upregulated in wild-type plants and as a result appeared repressed in the mutant, and that rap <dig>  was specifically downregulated and also appeared repressed in the mutant, suggesting that the mutant and wild-type plants respond differently to the presence of sugars.
pi3p5k was upregulated specifically in the mutant plants.
this enzyme increases the levels of phosphatidylinositol-5-phosphate, which functions as a signaling molecule during osmotic stress and can repress the expression of wrky70
<cit> .
sal <dig>  which was downregulated specifically in the mutant, acts as negative regulator of drought tolerance by modulating the levels of different sugars and controlling the corresponding signaling pathways
<cit> .
since the results highlighted some modifications of signaling molecules, the enzymes of the metabolism could be fine-tuned specifically in each genotype to adjust the concentration of metabolites.
CONCLUSIONS
under well-watered glasshouse conditions the reduced chlorophyll and carotenoids content of our pale-green mutant resulted on mild oxidative stress symptoms that could be compensated without altering photosynthesis and physiological performance by the induction of stress-response genes.
when the availability of water was limited, we observed more extreme modulation of antioxidant enzymes and photosynthetic proteins in the mutant than in wild-type plants, suggesting that the latter were better protected against oxidative stress.
concomitant molecular changes in the mutant revealed that the regulation of carbohydrate metabolism differed from wild-type plants, particularly the energy-producing pathways and pathways feeding the tca cycle.
the modification of carbohydrate metabolism in the mutant resulted in lower biomass accumulation and hence reduced yield under field conditions.
this yield reduction was significant  under irrigation, but was exacerbated in rain-fed conditions  in agreement with the gene expression study, thus indicating that the mutant plants were more sensitive to water scarcity.
our results support the conclusion that the pale-green mutant was less able to adapt to terminal water stress, sustaining that reduced pigment content may be disadvantageous in durum wheat under water limited conditions, although a case by case study would be required when working with other pale-green mutants.

 the leaf chlorophyll content of plants determines their capacity to absorb energy from sunlight and is therefore considered a measure of photosynthetic potential. plants typically respond to water stress by reducing the leaf chlorophyll content, and hence their photosynthetic activity. genotypes that tolerate water stress tend to have more leaf chlorophyll and lower canopy temperatures when stressed. in contrast, genotypes that are susceptible to water stress often have higher canopy temperatures, and when such plants are well-watered, with their stomata fully open, water evaporation causes leaf cooling. the ability of a genotype to remain green under water stress conditions may therefore enhance photoassimilation, thus increasing productivity and grain filling. in contrast to these general statements, a low level of leaf chlorophyll can limit the interception of light to avoid oxidative damage caused by excess radiation and could be considered a desirable trait under water stress conditions. the optimization of photosynthesis in crops under water stress therefore requires a productive balance between assimilation under favorable conditions and the avoidance of excess radiation under stress. chlorophylls and carotenoids play an important role in photoassimilation and the synthesis of both molecules is coupled to chloroplast development. thylakoid organization is coordinated with the expression of nuclear genes, encoding pigment-binding proteins that are imported into the chloroplast to assemble the photosynthetic complexes and the expression of chloroplast encoded genes. this process is tightly regulated, so mutants deficient in chlorophyll content can arise not only through the disruption of chlorophyll biosynthesis but also via processes related to photosystem assembly and light-harvesting activity. such chlorophyll mutants have been used to study the function of photosynthetic components as described in rice e.g., barley e.g. and wheat e.g.. carotenoids are involved in photoprotection and in light harvesting, and different carotenoids play different roles in the photosynthetic complexes. for example, only β-carotene is present in the reaction centers whereas xanthophylls are present in the antennal light harvesting complex where each protein has a specific carotenoid complement that ensures maximum photosystem efficiency. lutein is involved in light harvesting and the quenching of 3chl states and violaxanthin can be replaced by zeaxanthin if the de-epoxidation cycle is activated by photosystem saturation following the absorption of excess light. zeaxanthin helps to prevent photo-inhibition by dissipating the excess of energy as heat, either by direct quenching or by inducing conformational changes in the lhc proteins. photo-inhibition is an early consequence of excess light and other environmental stresses, reducing photosynthetic activity and leading to pigment photo-oxidation, the production of reactive oxygen species and ultimately cell death. chloroplasts and mitochondria are the primary sources of ros, which damage cells by oxidizing nucleic acids, proteins and lipids. however, low concentrations of ros act as potent signaling molecules that coordinate responses to abiotic stress, pests and pathogens. the balance between ros production and scavenging by antioxidants can be disrupted by different forms of stress. wheat is a staple crop that plays a major role in global food security. conventional breeding strategies have been used to increase grain yields and stress tolerance, but in order to feed a growing population it will be essential to include molecular breeding as well as conventional approaches to achieve further substantial gains. gene expression profiling with microarrays has been used to characterize the response of wheat to heat and drought stress, cold, fusarium infection and phase-transition, and to investigate the molecular basis of grain quality traits. in the context of drought stress, krugman et al. compared resistant and susceptible wild emmer genotypes to investigate the overlapping regulatory and signaling processes overrepresented in the drought-resistant genotype and to identify candidate genes for drought tolerance. proteomics has also been used to investigate the response of wheat to nitrogen, salinity stress, low temperatures and drought. poor correlation between transcriptomic and proteomic data has been reported in some stress experiments although such complementary information highlights the multilevel regulation of responses to complex environments. most of the experiments discussed above involved genotypes with contrasting phenotypes and different genetic backgrounds, an approach that allows many genes and stress tolerance strategies to be identified, but makes it difficult to characterize specific genotype-phenotype relationships. this can be achieved in experiments that involve genotypes differing at a single locus e.g.. in order to control for genetic background effects, we compared a pale-green mutant and its wild-type mother variety to determine the impact of leaf pigment content on gene and protein expression. we studied the chlorophyll and carotenoid content as well as the transcriptomic and proteomic profiles of the mutant and wild-type plants under well-irrigated conditions and terminal water stress. we also tested the performance of the mutant and wild-type plants under irrigated and rain-fed field conditions in order to assess the effect of the mutation on biomass accumulation and grain yield.  chloroplast ultrastructure wild-type and mutant leaf sections at the tillering stage were compared by transmission electron microscopy. as shown in figure  lens-shaped chloroplasts were present in the wild-type mesophyll cells whereas those in the mutant plants were typically cylindrical. the thylakoid grana of the wild-type chloroplasts appeared to be connected by continuous intergranal lamellae parallel to the long axis of the chloroplast, whereas the mutant grana appeared to be disconnected and disorganized within the chloroplast stroma. osmiophilic lipid globules were more abundant in the mutant chloroplasts. physiological responses and field measures water stress was applied to plants in the glasshouse by reducing the soil water content at anthesis. this increased the negative value of the midday flag leaf water potential, reduced the leaf conductance to water vapor values and reduced the mean net photosynthetic rate but differences between genotypes were not statistically significant. in the field, differences in aboveground biomass were significant only under rain-fed conditions, but the grain yield of the mutant plants was reduced by % under irrigated conditions and by % under rain-fed conditions, compared to wild-type plants in the same environment. w s w s a) f-values of the analysis of variance and, b) means of soil water content, flag leaf water potential, stomatal conductance and net photosynthetic rate for each water treatment and for the wild-type and the mutant plants under control and water stress conditions. water stress started at booting. ***p <, n.s. p >. for each variable, water treatment means with different letters are significantly different for a tukey test. average values of grain yield and aboveground biomass were determined at anthesis in the wild-type and the pale-green mutant. data represent means of 3 years of field experiments. genotype means with different letters within a water regime differ at p = . pigment content the total carotenoid and chlorophyll levels in the flag leaves at anthesis under well-watered conditions were significantly lower in the mutant than the wild-type, but differences in antheraxanthin and zeaxanthin levels were not statistically significant. under water stress conditions, only the antheraxanthin content differed significantly between wild-type and mutant leaves, whereas the total carotenoid and chlorophyll contents were similar in both genotypes. the xanthophyll de-epoxidation cycle appeared to be induced in the mutant plants under control conditions. whereas under terminal water stress, wild-type plants increased the de-epoxidation rate, mutant plants did not. therefore, there was no statistically significant difference between the genotypes under water stress conditions. vs p vs p pigment quantification of the flag leaf of wild-type and pale-green mutant plants at anthesis under control and water stress conditions was performed by hplc. values are means of four samples. xanthophylls de-epoxidation rate. transcriptomic profiling microarray quality analysis microarray data were collected using the affymetrix wheat genome array and normalized using the rma algorithm. the average background was, well within the recommended levels. the percentage of "present" calls ranged from % to % among the 61 k probe sets. the quality of the biological replicates was determined by calculating r values among replicates of the same sample and the values ranged between and with an average of. principal component analysis highlighted the main sources of total variance. the two components explaining % and % of the variance represented the differences between the treatments and between the genotypes as shown in figure  the mutation effect on gene expression was studied comparing the expression of the mutant to wild-type for each water treatment and indicated along the manuscript with the terms induced or repressed genes. in addition, the stress effect was evaluated comparing the gene expression of the water stress to control conditions for each genotype and indicated along the manuscript as up/downregulated genes. gene expression under control conditions transcriptional profiling at anthesis revealed differences in gene expression between the two genotypes under control conditions. we identified induced and repressed probe sets in the mutant. the array data for selected probe sets were validated by quantitative real-time rt-pcr, showing strong agreement between the fold-change values observed with each method. mips funcatdb was used to distribute the genes into functional categories, revealing that the induced probe sets in the mutant represented chitin catabolism, defense and cell wall metabolism. the most strongly induced probe sets represented peroxidase, pathogenesis-related proteins including beta-glucanases and chitinases, and a tawrky45-like transcription factor. the repressed probe sets represented functions including systemic interaction with the environment, abscisic acid responses, hormonal regulation, stress responses and dna degradation, e.g. sequences representing rab proteins, late embryogenesis abundant protein lea and endonucleases. probe sets related to cell rescue, defense and virulence were induced or repressed in the mutant when compared with the wild-type. however, none the subcategories was statistically significant at p ≤ . p significant functional categories represented in the induced and repressed probe sets in the mutant compared to wild-type plants at anthesis under control conditions according to the mips functional catalogue database. only functional categories with a cut-off of p ≤  were considered. a aspecies: 1: triticum aestivum, 2: hordeum vulgare, 3: zea mays, 4: sorghum vulgare, 5: triticum monococcum, 6: triticum turgidum ssp. durum, 7: oryza sativa. -: not determined. gene expression under water stress we initially compared each genotype under stress and control conditions to identify both general and genotype-specific stress-modulated genes, which were defined as upregulated or downregulated, as mentioned above. we identified a total of probe sets that were modulated by water stress in at least one of the genotypes, including genes in the wild-type plants, genes in the mutant plants and in both. we also compared the wild-type and mutant plants under water stress conditions, to identify genes that were differently-expressed under stress. we identified probe sets that were defined either as induced or repressed in the mutant plants, according to the level of expression under water stress conditions. genotype-independent stress-response genes from the probe sets modulated in both genotypes, were upregulated and were downregulated under water stress. the upregulated genes predominantly represented the following functional categories: stress response, carbon compound and carbohydrate metabolism, metabolite transport, amino acid metabolism, aba response and proline biosynthesis. genes already known to be associated with water stress are shown in the additional file 3: table s these include genes encoding sucrose synthase, glucose-6-phosphate dehydrogenase, branched-chain-amino-acid aminotransferase and branched-chain alpha-keto acid dehydrogenase e α subunit, delta 1-pyrroline-5-carboxylate synthetase, pyrroline-5-carboxylate reductase and rab the downregulated genes predominantly represented the following functional categories: phosphate and secondary metabolism, e.g. 1-aminocyclopropane-1-carboxylate synthase and 12-oxophytodienoate reductase; interaction with the environment, e.g. chitinases, β-glucanases and pathogenesis-related proteins; immune response, e.g. transcription factor wrky; and phenylpropanoid metabolism, e.g. phenylalanine ammonia-lyase. these genotype-independent stress-modulated genes correspond to the well-characterized drought stress response of wheat, confirming that the mutant plants preserve much of the stress-response signaling network that allows wheat plants to tolerate water stress. genotype-dependent stress-response genes in wild-type plants we identified probe sets that were modulated solely in the wild-type plants or to a greater extent than in the mutant plants. this included upregulated and downregulated genes. the upregulated genes predominantly represented carbohydrate and pyruvate metabolism and the glyoxylate cycle, whereas the downregulated represented metabolism, interaction with the environment, protein modification and signaling pathways. selected genes specifically modulated by the wild-type plants under stress are listed in table  including carbon fixation enzymes such as malate dehydrogenase and phosphoenolpyruvate caboxykinase, and glyoxylate cycle enzymes such as isocitrate lyase and malate synthase. p significant functional categories represented in the specifically upregulated and downregulated probe sets in wild-type plants at anthesis under water stress conditions compared to well-watered conditions according to the mips functional catalogue database. only functional categories with a cut-off of p ≤  were considered. selected genes were specifically significant regulated by the wild-type and the mutant under water stress compared with the same genotype under well-watered conditions. last column compares mutant versus wt expression under water stress. positive and negative values indicate upregulation and downregulation respectively, whereas no statistically significant differences are indicated with a dash. auniprot id from triticum turgidum subsp. durum. bblast executed against tagi wheat gene index: tc homologue to uniref100_p genotype-dependent stress-response genes in mutant plants we identified probe sets that were modulated solely in the mutant plants or to a greater extent than in the wild-type plants. this included upregulated and downregulated genes representing diverse functional categories. the most significant categories in the upregulated group were associated with chloroplast transcription, interactions with the environment, stress responses and metabolism, whereas the downregulated group was dominated by functional categories such as interaction with the environment, stress responses and plant defense. the downregulated genes also included those induced in the mutant under control conditions. p significant functional categories represented in the specifically upregulated and downregulated probe sets lists in the mutant at anthesis under water stress conditions compared to well-watered conditions according to the mips functional catalogue database. only functional categories with a cut-off of p ≤  were considered. the genes that were upregulated specifically in the mutant included those involved in the synthesis of carotenoid precursors, e.g. hydroxymethylglutaryl-coa synthase, 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase, and those involved in chloroplast gene expression, photosynthesis, chlorophyll binding and detoxification, e.g. glutamyl-trna reductase, light harvesting complex ii components such as lhb1b and atpase subunit delta, and the rubisco small subunit. we observed the downregulation of several genes representing glutamate metabolism such as glutamate decarboxylase, glutamate dehydrogenase, γ-glutamyltranspeptidase and l-aspartate oxidase; and several signaling pathway components, including calcineurin b-like protein, inositol phosphatase and mpk. differentially-expressed stress-response genes under stress conditions we compared the transcriptional profiles of wild-type and mutant plants under water stress and identified differently-expressed probe sets. the only functional category significantly represented among the induced probe sets was the channel/pore transport class, corresponding to the transcripts for two plasma membrane intrinsic proteins that were downregulated in both genotypes but to a lesser extent in the mutant. other genes that were induced in the mutant under water stress included those encoding ascorbate peroxidase, sucrose: fructan 6-fructosyltransferase, myo-inositol oxygenase and dihydrodipicolinate reductase. the genes that were repressed in the mutant plants under stress were predominantly related to carbohydrate metabolism, peroxidase/oxidative stress responses, the glyoxylate cycle and chromosome subcellular localization. representative repressed genes included those encoding rap, gdh, gad, cbl-interacting protein kinase and glyoxalase. we also found genes encoding icl, mls and trehalose phosphate-synthase which were not affected by stress in mutant plants but were upregulated in wild-type plants, thus appearing in the repressed transcript category. proteomic profiling the effect of the mutation was also investigated by carrying out a comparative proteomic analysis between wild-type and mutant plants at anthesis under the same conditions as described above. when wild-type and mutant plants were compared under well-watered conditions, we matched protein spots among which two showed a significant difference in signal intensity between genotypes, although neither spot could be identified because of the low abundance of the corresponding protein. even so, this experiment supported the transcriptomic and suggested that there were only minor differences between the genotypes under normal conditions. when we compared each genotype under stress and control conditions, we matched protein spots among both genotypes. from the we found that spots were modulated by water stress in the wild-type plants and were modulated in the mutant plants, including spots that were common to both genotypes. this left seven modulated spots specific to the wild-type plants and specific to the mutant. when the two genotypes were compared under water stress, we identified spots that were more abundant in the mutant and that were less abundant in the mutant. we selected of the induced spots and all of the repressed spots based on the likelihood of extracting useful amounts of protein, and we identified proteins from the induced set and from the repressed set. the proteins were identified by peptide mass fingerprinting and ms/ms analysis. the induced proteins predominantly represented carbohydrate and amino acid metabolism, photosynthesis, detoxification and phytoalexin biosynthesis, whereas the repressed proteins predominantly represented photosynthesis. two proteins that were specifically upregulated in the mutant were the oxygen-evolving enhancer protein corresponding to the psbp subunit of photosystem ii, and catalase-. two proteins that were specifically downregulated in the mutant were the rubisco small subunit chain pw and the photosystem i reaction center subunit iv. b c aaccession number from ncbi. bspecies: 1: hordeum vulgare; 2: zea mays; 3:triticum aestivum; 4: nicotiana tabacum; 5: brachypodium distachyon; 6: arabidopsis thaliana. cmethod: pmf: peptide mass fingerprinting; ms/ms: maldi tof/tof. ratios are only indicated for differences statistically significant. discussion chloroplast ultrastructure and pigment content chlorotic mutants with affected chloroplast ultrastructure have been described in several reports. the chloroplasts in these plants often contain fewer grana structures than wild-type plants, and thinner thylakoid membranes that are not stacked in the normal manner. in contrast, the chloroplasts in our pale-green mutant contained abundant grana with normally-stacked thylakoids, but their distribution within the stroma was disorganized, potentially reflecting a lack of membrane continuity or the degradation of inter-grana lamella membranes. the stacking adhesion of the thylakoids in grana structures is regulated by the phosphorylation of photosystem ii proteins, which also ensures photosynthetic efficiency. we found no significant difference in the photosynthetic rate of the wild-type and mutant and no quantitative differences in transcripts or proteins representing the photosynthetic apparatus at anthesis under control conditions. the mutant chloroplasts also contained more stromal plastoglobules than wild-type chloroplasts. these lipoprotein bodies are structurally continuous with the thylakoid membranes and facilitate content exchange with the thylakoid lumen. they may also be involved in thylakoid development and disassembly, increasing in abundance in response to oxidative stress and delivering the antioxidant molecules stored on them to the thylakoid lumen, where they scavenge ros. the structure of the inter-grana lamella in the mutant chloroplasts could therefore reflect thylakoid reorganization or the impact of oxidative stress already at tillering stage under field capacity conditions. although the pigment content of the mutant plants was ~41% lower than in wild-type plants under control conditions, the photosynthetic rate was similar. this seems illogical given that carotenoids and chlorophylls are required for the proper folding, assembly and stability of lhc apoproteins. however, we detected a greater xanthophyll de-epoxidation rate in the mutant as indicated by the relationship /. the zeaxanthin in the mutant may compensate for the lack of other xanthophylls in the antenna proteins, and may also preserve the structure of the photosystem and its membranes by dissipating photochemical energy as heat to prevent photo-oxidation. this may explain the abnormal chloroplast ultrastructure in the mutant but the near-normal photosynthetic rate. our data suggest that, under control conditions, the mutant compensates for the lower pigment content and that minor oxidative stress has no detrimental effect on photosynthetic capacity or biomass accumulation. however, under water stress conditions the level of de-epoxidation increases in wild-type plants, and both genotypes show similar values for the relationship /. this suggests that the mutant has a lower capacity for increasing the de-epoxidation rate. an alternative explanation is that the pool of convertible violaxanthin is rapidly depleted and only non-convertible violaxanthin remains to maintain the structural integrity of the antenna. because xanthophylls help to prevent photo-oxidation, the mutant plants without additional de-epoxidated carotenoids to cope for the water stress damage may sense higher oxidative stress in the chloroplast under water stress. thus, the pale-green mutant may be more susceptible since the ability of plants to withstand oxidative stress determines the level of overall stress tolerance. differences between the wild-type and mutant transcriptomes at anthesis there were only minor differences in gene expression between the wild-type and mutant plants under control conditions at anthesis, mirroring the similar net photosynthetic rates. the few repressed genes in the mutant were predominantly related to aba responses and the induced genes mostly represented pathogenesis-related proteins and peroxidases involved in plant defense. apart from their role in defense, peroxidases may be modulated by cellular redox disturbances. in such cases, nuclear gene expression can be regulated by ros-dependent signal transduction or low-molecular-weight antioxidants produced in the chloroplast. for example, the expression of pr proteins and peroxidases increases in ascorbate-deficient mutants. since no lesion or pathogen attack was detected in our investigation, it is most likely that low-level of oxidative stress triggers defense gene expression in the pale-green mutant. proteins involved in plant defense are also sensitive to hormones such as salicylic acid, jasmonate and ethylene, but we did not find any differently-expressed genes related to these pathways in the mutant. plant defense signaling pathways are also activated through wrky transcription factors that bind to pathogen-response and wound-inducible promoter elements. the tawrky gene was induced in the mutant, and may help to activate some of the pr genes we also identified. this transcription factor confers resistance to fusarium head blight in wheat and increases the expression of pr- and pr- genes in rice to confer resistance to pseudomonas syringae, salinity and drought tolerance, as well as reducing aba sensitivity. genes encoding defense enzymes that synthesize secondary metabolites such as phenylpropanoids and flavonoids were also induced in the mutant, including naringerin, 2-oxoglutarate 3-dioxygenase and agmatine coumaroyltransferase, the latter representing the final step in the hydroxycinnamic acid amide synthesis pathway. peroxidases can also couple agmatine conjugates to form antifungal compounds known as hordatines in barley and in wheat. finally, the blufensin- gene was also induced in the mutant. this is a negative regulator conferring sensitivity to powdery mildew in barley by modulating penetration resistance. the repressed probe sets were related to systemic interactions with the environment, especially the aba response, e.g. genes encoding dehydrins such as rab proteins and other lea proteins that accumulate during seed maturation and in vegetative tissues in response to abiotic stress. rab is a lea family dehydrin that is induced by aba, and the arabidopsis ortholog rab is a useful marker for the loss of aba sensitivity e.g. in plants that are deficient for the swi/snf chromatin-remodeling complex. the gene is upregulated when a relevant repressor is mutated, e.g. sumo e ligase siz1. we hypothesize that the mutant induces a repressor that acts against the aba pathway or downregulates an inducer of the same pathway. indeed, two transcription factors were repressed in the mutant under control conditions, the q-type c2h zinc finger transcription factor tazp and the homeobox-leucine zipper protein athb- tazp is upregulated in leaves and roots during drought stress and also following aba treatment, suggesting a role in aba-dependent gene regulation. athb- may act as a growth regulator in response to water deficit, and is induced by aba, salinity and dehydration stress. it also regulates the aba signaling pathway, acting downstream of abi1. the repressed probe sets also included two endonuclease genes related to the barley genes bnuc and ben which are expressed during apoptosis. bnuc is also induced by salinity stress. the papain-like cysteine proteinase and tds were also represented in the repressed probe sets, and these also facilitate senescence and apoptosis. hvpap also generates mature forms of storage proteins in seeds, provides free amino acids during germination, and confers stress tolerance and defense against pathogens. tds is induced during post-flowering senescence in wheat, and also by ja, sa and in chlorotic/necrotic leaf tissue following infection with pyrenophora teres. there were few differences between the mutant and wild-type plants in the absence of water stress at anthesis and no statistically significant differentially-expressed genes could be identified also at the tillering stage. therefore despite a clear difference in chlorophyll content and chloroplast structure, the mutation appears to have a minimal impact on metabolism and gene regulation, a finding that does not provide sufficient explanation for the yield loss detected under field conditions. water stress enhances the differences between mutant and wild-type plants we investigated the molecular basis of the difference in yield between the two genotypes by exposing them to water stress, which is the most common environmental constraint for durum wheat. most of the genes modulated by water stress were similarly regulated in both genotypes, suggesting a common response. genes involved in the synthesis of osmoprotectants were upregulated in both genotypes, whereas genes involved in aba-dependent signaling, ethylene and ja synthesis were downregulated in both genotypes. genes related to primary metabolism were also similarly modulated, indicating that both genotypes use metabolic strategies to counter the effects of drought. in contrast, genes encoding photosystem components and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle showed significant differences that may explain the difference in performance between wild-type and mutant plants in the field. photosynthesis and pigment synthesis both transcriptomic and proteomic analysis indicated that the components of photosystems i and ii and atpase were differentially expressed in the wild-type and mutant plants under water stress. several psi proteins were underrepresented in the mutant, including reaction center protein subunit iv, which is required for optimal electron transport to ferredoxin and flavodoxin, and light harvesting complex lhca. psi takes longer than psii to recover from photo-inhibition because rc proteins are degraded instead of being repaired. therefore, the loss of psi rc proteins in the mutant is likely to exacerbate the damage caused by stress. psbo and psbp maintain the structure of psii and were specifically overrepresented in the mutant. cp was upregulated in both genotypes, but this was underrepresented in the mutant under stress conditions. this protein binds lhcb proteins to the reaction center and confers photoprotection. the lhcb proteins were downregulated in both genotypes and underrepresented in the mutant. in contrast, the genes encoding lhcb proteins were upregulated in the mutant. the beta and delta subunits of atpase synthase were also overrepresented in the mutant, suggesting that the h+ flux across the thylakoid membrane is greater in the mutant, with a knock-on effect on atp production. three genes responsible for the synthesis of carotenoid precursors were upregulated by stress specifically in the mutant: 1-deoxy-d-xylulose-5-phosphate synthase, hydroxymethylglutaryl-coa synthase and phytoene synthase. one gene encoding glutamyl-trna reductase, which is responsible for the synthesis of chlorophyll precursors, was also upregulated by stress specifically in the mutant. the pigment content of the mutant plants did not increase relative to the control but the activation of these genes could help to prevent a steeper decline in pigment content under water stress conditions. redox changes in the chloroplast result in the stoichiometric adjustment of photosystem proteins and regulate the expression of both nuclear and plastid photosynthesis-related genes. because the mutant showed evidence of oxidative stress even under control conditions, the re-organization of the photosynthetic apparatus under water stress conditions could reflect the additional oxidative stress that occurs during the early stages of drought. the loss of photosynthetic proteins by the mutant in response to drought stress reduces the amount of captured energy and thus avoids the over-excitation of psii which would generate additional ros and cause oxidative damage. oxidative stress and ros signaling the balance between ros synthesis and scavenging determines the level of oxidative stress in plants. ros scavenging and detoxifying proteins such as peroxidases, glutathione transferases and glyoxalase were generally modulated in a similar manner in both genotypes, but more probe sets of this group were downregulated specifically in the mutant under stress. in the same direction, a glutaredoxin gene was upregulated in the wild-type plants under stress but was unaffected in the mutant. thylakoid ascorbate peroxidase and catalase were upregulated specifically in the mutant, suggesting a greater need for protection against ros derived from chloroplast and peroxisome activity. h2o can spread from the chloroplast to other organelles by diffusion through aquaporin-like pip proteins which were downregulated in the mutant to a lesser extent than in wild-type plants. these results indicate that the mutant experiences greater oxidative stress than wild-type plants under water stress conditions. two probe sets related to ros and stomatal closure were specifically modulated in the mutant under water stress. the specific downregulation of glyoxalase gene suggests that the mutant is less able than wild-type plants to detoxify methylglyoxal using reduced glutathione, thus allowing the production of ros and stomatal closure. similarly, the specific upregulation of respiratory burst oxidase homologue d in the mutant suggests that the aba-induced production of ros in guard cells is promoted to encourage stomatal closure. however, the differential expression of genes that regulate the stomatal aperture did not result in any differences between the genotypes in terms of stomatal conductance under water stress. ros can induce ca2+-dependent signaling that triggers interactions between different calcineurin b-like sensors and their cbl-interacting protein kinase targets. genotype-dependent differences were found in expression of these genes: whereas cipk and cipk were upregulated by stress in both genotypes, cipk and cipk were upregulated specifically in wild-type plants and cbl and cipk were downregulated specifically in the mutant. these two proteins cooperate with cbl to activate k+-channels and regulate stomatal responses. cipk and cipk belong to the snrk3-type family and mediate nutrient sensing, stress responses and aba signaling. as a result of altered expression of cbls and cipks different ca2+-dependent downstream responses under water stress are expected between mutant and wild-type plants. mpk which was specifically downregulated in the mutant, is also involved in ros signaling. once activated, it phosphorylates other stress-related proteins such as the transcription factors zat and azf and enzymes in the ethylene synthesis pathway such as acs, all of which were downregulated by stress in both genotypes. zat is a transcriptional repressor responsive to chitin, which mediates the response to photo-oxidative stress and ros. despite not showing damaged leaves or altered stomatal response, the data suggest that the pale-green mutant differs significantly from wild-type plants in terms of ros sensing and signaling under water stress. ros generated in the chloroplast could move more easily inside the cells and elicit a signal triggering specific responses. carbohydrate metabolism transcriptomic profiling revealed differences between the genotypes in the regulation of carbohydrate metabolism under water stress conditions. the synthesis of osmoprotectants such as raffinose family oligosaccharides and trehalose was specifically upregulated in the wild-type plants, including the genes encoding galactokinase and raffinose synthases. rfos protect membranes and scavenge ros. trehalose-phosphate synthase was also specifically induced in wild-type plants and thus repressed in the mutant under stress. trehalose and trehalose-6-phosphate act not only as osmolytes to stabilize proteins and membranes, but also as signaling molecules to regulate metabolism. two of the probe sets upregulated in the mutant plants encoded sucrose: sucrose 1-fructosyltransferase and sucrose:fructan 6-fructosyltransferase, which are responsible for synthesizing fructans from sucrose in the vacuole. the mutant plants therefore appeared to promote fructan synthesis for remobilization as free carbohydrates, which are directly correlated with the water-soluble carbohydrate content in wheat. conversely, probe sets encoding cellulose synthase and cellulose synthase-like enzymes were upregulated specifically in the wild-type plants and xylosyltransferases were repressed in the mutant plants under stress. these enzymes divert carbon to the synthesis of cell wall polysaccharides and their loss reduces growth and also inhibits photosystem activity and carbohydrate metabolism genes. this shows the importance of cell wall structure not only for growth and turgor changes under stress but also for carbohydrate metabolism. furthermore, sucrose, rfos and fructans can directly or indirectly scavenge ros, as reviewed by keunen et al.. the general picture that emerges from the modulation of carbohydrate metabolism suggests that the pale-green mutant is less able to deal with water stress than wild-type plants. cell wall synthesis genes are activated in the wild-type plants whereas fructans are produced in the mutant. this correlates with the lower biomass of the mutant plants at anthesis and the lower yield under rain-fed conditions. tca cycle and related pathways the tricarboxylic acid cycle generates energy and reducing power but consumes h2o and releases co the substrates feeding the cycle are acetyl-coa and other intermediates from lipid or protein catabolism. acetyl-coa is also produced from pyruvate by the action of the pyruvate dehydrogenase complex, and the subunit e gene representing this complex was upregulated in both genotypes under water stress. however, under conditions of carbon starvation or demand, additional strategies are used to supply tca substrates. the wild-type plants appear to be more effective at these strategies given the specific upregulation of the following genes: i) genes encoding products that convert amino acids into substrates suitable for the tca cycle were upregulated in both genotypes, but three were upregulated specifically in wild-type plants: methylcrotonoyl-coa carboxylase, α and β subunits, and 2-oxoisovalerate dehydrogenase e. ii) genes encoding the nadp-malic enzyme, which converts malate into pyruvate, and phosphoenolpyruvate carboxykinase, which converts oxaloacetate into phosphoenolpyruvate, were upregulated specifically in the wild-type plants. iii) genes encoding isocitrate lyase and malate synthase from the glyoxylate cycle were upregulated specifically in wild-type plants and thus, repressed in the mutant. this cycle produces succinate and malate acting as a bypass to avoid the loss of co from the normal tca cycle. it also metabolizes glyoxylate generated by photorespiration and prevents photo-inhibition. iv) genes encoding glutamate dehydrogenase and glutamate decarboxylase were specifically downregulated and consequently repressed in the mutant under stress. they participate in glutamate metabolism, which balances nitrogen and carbon use in the cell either by storing nitrogen as gaba, glutamate or glutamine, or by supplying carbon back to the tca cycle. a functional gaba shunt is required for stress tolerance. in the pale-green mutant, carbon stored as glutamate and gaba may not be introduced efficiently into the tca cycle, disrupting the nitrogen/carbon balance and reducing growth under carbon demand or starvation imposed by stress. gdh can also be inhibited by excess light or sucrose. the feed-in to the tca cycle and the ability to derive energy from it appear to be more limited in the mutant under stress, potentially explaining the differences between genotypes in terms of biomass and yield in the field experiments. the mutant mobilizes carbon less efficiently, with a significant impact on grain filling and yield as observed in our field experiments. regulators of carbon metabolism and signaling genes that regulate carbohydrate and nitrogen metabolism were also differentially expressed between the wild-type and mutant plants under water stress, helping to explain the basis of the metabolic differences between the genotypes. the bzip gene was specifically downregulated and repressed in the mutant plants under stress. the transcription factor encoded by this gene is an important node in the glucose-aba interaction network and is repressed by both aba and glucose. it may promote starvation tolerance by regulating transcription of genes such as the raffinose synthase. ap2/erebp proteins mediate hormone, sugar and redox signaling in the context of cold and drought stress. each member has a unique expression profile and the individual proteins are differentially regulated by glucose. we found that rap and rap were specifically upregulated in wild-type plants and as a result appeared repressed in the mutant, and that rap was specifically downregulated and also appeared repressed in the mutant, suggesting that the mutant and wild-type plants respond differently to the presence of sugars. pi3p5k was upregulated specifically in the mutant plants. this enzyme increases the levels of phosphatidylinositol-5-phosphate, which functions as a signaling molecule during osmotic stress and can repress the expression of wrky70. sal which was downregulated specifically in the mutant, acts as negative regulator of drought tolerance by modulating the levels of different sugars and controlling the corresponding signaling pathways. since the results highlighted some modifications of signaling molecules, the enzymes of the metabolism could be fine-tuned specifically in each genotype to adjust the concentration of metabolites.  under well-watered glasshouse conditions the reduced chlorophyll and carotenoids content of our pale-green mutant resulted on mild oxidative stress symptoms that could be compensated without altering photosynthesis and physiological performance by the induction of stress-response genes. when the availability of water was limited, we observed more extreme modulation of antioxidant enzymes and photosynthetic proteins in the mutant than in wild-type plants, suggesting that the latter were better protected against oxidative stress. concomitant molecular changes in the mutant revealed that the regulation of carbohydrate metabolism differed from wild-type plants, particularly the energy-producing pathways and pathways feeding the tca cycle. the modification of carbohydrate metabolism in the mutant resulted in lower biomass accumulation and hence reduced yield under field conditions. this yield reduction was significant under irrigation, but was exacerbated in rain-fed conditions in agreement with the gene expression study, thus indicating that the mutant plants were more sensitive to water scarcity. our results support the conclusion that the pale-green mutant was less able to adapt to terminal water stress, sustaining that reduced pigment content may be disadvantageous in durum wheat under water limited conditions, although a case by case study would be required when working with other pale-green mutants. 
leaf pigment content is an important trait involved in environmental interactions. the pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. this study provides insight on the effect of pigment content in the molecular response to drought. we also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction. chlorophyll contentoxidative stresspale-green mutantproteomicstranscriptomicstriticum turgidum l var. transcriptomic analysis under control conditions showed that defense genes were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat. in order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant under contrasting water availability conditions.
 leaf pigment content is an important trait involved in environmental interactions. in order to determine its impact on drought tolerance in wheat, we characterized a pale-green durum wheat mutant under contrasting water availability conditions.  the pale-green mutant was investigated by comparing pigment content and gene/protein expression profiles to wild-type plants at anthesis. under well-watered conditions the mutant had lower levels of chlorophylls and carotenoids, but higher levels of xanthophyll de-epoxidation compared to wild-type. transcriptomic analysis under control conditions showed that defense genes were upregulated in the mutant, suggesting the presence of mild oxidative stress that was compensated without altering the net rate of photosynthesis. transcriptomic analysis under terminal water stress conditions, revealed the modulation of antioxidant enzymes, photosystem components, and enzymes representing carbohydrate metabolism and the tricarboxylic acid cycle, indicating that the mutant was exposed to greater oxidative stress than the wild-type plants, but had a limited capacity to respond. we also compared the two genotypes under irrigated and rain-fed field conditions over three years, finding that the greater oxidative stress and corresponding molecular changes in the pale-green mutant were associated to a yield reduction.  this study provides insight on the effect of pigment content in the molecular response to drought. identified genes differentially expressed under terminal water stress may be valuable for further studies addressing drought resistance in wheat. chlorophyll contentoxidative stresspale-green mutantproteomicstranscriptomicstriticum turgidum l var. durumwater stress 