BACKGROUND
grapevine  is a fruit crop traditionally subjected to moderate or severe water stress, as this is an efficient strategy to improve fruit and wine quality .
vitis species adapt well to drought conditions due to good osmotic adjustment, large and deep root systems, efficient control of stomatal aperture and xylem embolism  <cit> .
the strength and timing of these responses varies between different cultivars and major differences in water stress tolerance can be found when compared to other species or hybrids from the vitis genus  <cit> .
although these genotype-related variations involve different aspects of the physiology of the plant, they are largely linked to differences in stomatal conductance   <cit> .
stomata are microscopic pores distributed on the surface of leaves and stems, surrounded by two highly specialized guard cells.
the opening and closure of the pore, in response to internal signals and environmental cues, allows the plant to cope with the conflicting needs of ensuring adequate uptake of co <dig> for photosynthesis and preventing water loss by transpiration  <cit> .
under drought, abscisic acid  is accumulated, inducing rapid stomatal closure to limit water loss.
increasing evidence indicates a role for transcription factors belonging to the r2r <dig> myb subfamily as key modulators of physiological responses in stomata  <cit> .
in particular, atmyb <dig> has been shown to be differentially expressed in guard cells in response to aba, and the loss-of function atmyb60- <dig> mutant displays constitutive reduction of light-induced stomatal opening and enhanced tolerance to dehydration  <cit> .
guard cell-specific myb genes are thus focal points in understanding stomatal regulation in plants and represent molecular targets to modulate guard cell activity to improve crop survival and productivity during drought.
the grapevine genome has been estimated to contain a total of  <dig> myb genes  <cit> , of which  <dig> belong to the r2r <dig> subfamily  <cit> .
a phylogenetic tree, constructed with the complete grape, arabidopsis and rice r2r3myb subfamilies, showed that many genes sharing similar functions were clustered in the same phylogenetic groups.
some of these clades were conserved in gene copy number  while in those controlling flavonoid synthesis several expansions events may have occurred  <cit> .
in this work, we report the identification of two close homologues of the guard cell-related atmyb <dig> gene in the grape genome, namely vvmyb <dig> and vvmyb <dig>  analysis of gene expression in grape tissues revealed that both vvmyb <dig> and vvmyb <dig> were expressed in green tissues and developing seeds.
as opposite to vvmyb <dig>  vvmyb <dig> transcript abundance was greatly reduced by aba and osmotic stress.
a gus reporter gene approach in arabidopsis showed that activity of the vvmyb <dig> promoter was restricted to stomatal guard cells and was down-regulated by aba.
comparative analysis of regulatory regions revealed the presence of common guard cell-specific motifs in the promoters of the grape and arabidopsis myb <dig> genes.
finally, vvmyb <dig>  unlike vvmyb <dig>  fully complemented the stomatal defects of the atmyb60- <dig> mutant, thus indicating that vvmyb <dig> is a functional ortholog of the arabidopsis atmyb <dig> stomatal regulator.
RESULTS
phylogenetic relationships of myb <dig> homologues
as a first approach to identify grape homologues of the atmyb <dig> transcription factor, we searched the  <dig> r2r <dig> myb proteins identified in the vitis vinifera pn <dig> genome  <cit> , for the presence of a distinctive c-terminal motif , found in members of subgroup  <dig> of the arabidopsis myb family  <cit> .
this subgroup includes: atmyb <dig>  regulating light-induced stomatal aperture  <cit> ; atmyb <dig>  related to the regulation of brassinosteroid-induced gene expression  <cit>  and to the biosynthesis of very-long-chain fatty acids involved in hypersensitive cell death  <cit> ; atmyb <dig>  an aba/auxin cross-talker, mediating aba signaling during drought stress and involved in promoting pathogen resistance  <cit>  and atmyb <dig>  whose function is still unknown.
our search yielded two grape close homologues in the grape genome version 12x: the annotated gene models gsvivt <dig>  and gsvivt <dig> .
a parsimony consensus tree was constructed to investigate the phylogenetic relationships within these grape proteins and members of arabidopsis r2r <dig> myb subgroup  <dig>  subgroup  <dig> was also included as some of its members are involved in drought responses and aba signaling  <cit> .
from this subgroup, a grape myb <dig> homologue had also been previously isolated  <cit> .
atmyb <dig>  regulating stomatal activity  <cit> , but not belonging to any of these subgroups, was included as an out-group.
as shown in figure 1a, the two grape proteins acf <dig> and abk <dig> clustered with members of the arabidopsis subgroup  <dig>  interestingly, atmyb <dig>  the most distant member of subgroup  <dig>  was more closely related to the grape protein accession acf <dig> than to the other members of the subgroup .
on the other hand, the grape accession protein abk <dig> was closely related to atmyb <dig> and atmyb <dig> and to a lesser extent to atmyb <dig> and atmyb <dig> .
hereafter, we will refer to acf <dig> and abk <dig> as vvmyb <dig> and vvmyb <dig>  respectively.
based on these results, we further divided subgroup <dig> into subgroup  <dig> ,  and subgroup  <dig>   .
as expected, all the proteins included in the tree disclosed a highly conserved r2r <dig> dna binding domain .
the identity between the r2r <dig> domain of atmyb <dig> and vvmyb <dig> and vvmyb <dig> was 99%, and 90%, respectively.
in addition, atmyb <dig> and vvmyb <dig> disclosed a distinctive pheeg signature, encompassing the two highly conserved glutamic acid residues, located in the loop connecting the r <dig> and r <dig> repeats .
the complete protein sequence of atmyb <dig> showed 51% amino acid identity to vvmyb <dig>  and 48% identity to vvmyb <dig>  all of these proteins share two c-terminal motifs  which are only found in subgroup  <dig>  in addition, atmyb <dig>   <dig>   <dig> and  <dig> possess a third myb domain , which is absent in atmyb <dig> and vvmyb <dig> .
the function of these c-terminal domains is still unknown although they might reflect the functional differences between subgroups  <dig>  and  <dig> .
we determined the precise gene structure of both vvmyb <dig> and vvmyb <dig>  by comparing the complete coding sequence with the full length cdna sequence, amplified from pinot noir pn <dig> genomic dna and leaf cdna, respectively .
it was interesting to note that in the 12x version of the grape genome, gsvivp <dig>  representing the vvmyb <dig> gene model, was misannotated in terms of exon number.
indeed, our results indicate the presence of three exons, as opposed to the five exons predicted by the gene model, thus revealing a conserved exon/intron organization for vvmyb <dig>  vvmyb <dig> and atmyb <dig> .
based on gene structure, myb genes have been classified in four different groups  <cit> .
vvmyb <dig>  vvmyb <dig> and atmyb <dig> all belong to group i, which contains genes with a characteristic r <dig> domain split between exons  <dig> and  <dig>  and a r <dig> domain split between exons  <dig> and  <dig> .
the biggest differences in lengths were found in the first intron and the third exon, which were longer in the grape genes compared to atmyb <dig>
expression of vvmyb <dig> and vvmyb <dig> in grape tissues and in response to hormonal and stress factors
qpcr analysis of gene expression in different grape organs indicated that vvmyb <dig> and vvmyb <dig> transcripts were most abundant in leaves, seeds and ripened berry skins .
interestingly, vvmyb <dig> and vvmyb <dig> revealed completely opposite expression patterns during seed development; while vvmyb <dig> expression was gradually down-regulated towards the onset of ripening , vvmyb <dig> expression was rapidly activated .
during berry skin development, vvmyb <dig> expression also showed a dramatic decrease to full repression at veraison, followed by a slight increase towards ripening .
in this tissue, vvmyb <dig> was mostly constantly expressed throughout the green and ripening stages .
in arabidopsis, it has been shown that the expression of the atmyb <dig> gene is rapidly down-regulated following treatment with aba  <cit> .
we thus analysed the expression of the grape genes in leaves treated with  <dig> or  <dig> μm aba .
as reported in figure 2d, vvmyb <dig> showed a significant decrease in expression levels in both  <dig> and  <dig> μm aba treated samples, when compared to the mock treated leaves.
conversely, vvmyb <dig> did not show any change in expression after exposure to the hormone under these conditions .
to further investigate the expression of vvmyb <dig> and vvmyb <dig> in response to osmotic stresses, we designed an in vitro long-term salt stress experiment.
nodal explants were placed vertically on sterile ms media supplemented with  <dig> ,  <dig> or  <dig> mm nacl.
explants were maintained for a month in a growth chamber until roots and/or leaves were visible and fully expanded.
at the end of the experiment, plantlets from the  <dig> mm nacl treatment had a small radicule and high leaf anthocyanin accumulation, as a clear sign of stress in the plant, while plants at  <dig> mm showed more severe symptoms, including systemic wilting and brown pigmentation .
under these conditions, vvmyb <dig> and vvmyb <dig> showed opposite responses to the increasing salt concentrations; while vvmyb <dig> expression was reduced five-fold at both concentrations when compared to the control treatment, vvmyb <dig> expression increased three-fold on addition of  <dig> mm nacl .
activity of the vvmyb <dig> and vvmyb <dig> promoters in arabidopsis transgenic lines
we employed a reporter gene approach in the heterologous model system arabidopsis thaliana to investigate the activity of both vvmyb <dig> and vvmyb <dig> promoters.
a region of approximately  <dig> kb located upstream of the atg codon of vvmyb <dig> and vvmyb <dig> was fused to the β-glucuronidase  reporter gene and the resulting pvvmyb30:gus and pvvmyb60:gus constructs were introduced in arabidopsis by agrobacterium-mediated transformation  <cit> .
we assessed the cell and tissue specificity of reporter gene expression in ten independent t <dig> transgenic lines for each promoter:gus combination.
fifteen-day-old pvvmyb30:gus seedlings displayed expression of the reporter at the shoot apex, at the base of trichomes located on leaf primordia, and in the emerging lateral roots .
at the same developmental stage, pvvmyb60:gus seedlings showed gus expression exclusively in guard cells distributed on cotyledons, hypocotyls and developing leaves .
no expression of the reporter gene was detected in rosette leaves from mature pvvmyb30:gus plants, even after prolonged incubation of plant tissues in the gus solution .
on the other hand, we observed guard cell-specific signals in mature leaves of pvvmyb60:gus plants, consistent with the gus profile observed in young seedlings .
next, we investigated expression of the reporter in flowers and siliques from adult plants.
prior to pollination, pvvmyb30:gus flowers revealed a diffuse staining of carpels and stigmatic tissues .
we did not observe gus expression in pre- and post-fertilization flowers from most pvvmyb60:gus lines.
in two transgenic lines, a weak staining was occasionally detected in stamens, at the interface of filaments and anthers .
finally, we did not detect expression of the reporter in developing seeds from either pvvmyb30:gus or pvvmyb60:gus transgenic lines .
expression of both the endogenous arabidopsis and grape myb <dig> genes is rapidly down-regulated following treatment with aba  <cit>  .
we thus investigated changes in gus expression in the pvvmyb60:gus lines in response to exogenous applications of this hormone, using both qpcr and histochemical analyses.
a previously described transgenic line carrying a transcriptional fusion between the arabidopsis atmyb <dig> promoter and the reporter gus  was used as a control for the experiment  <cit> .
as expected, qpcr analysis of gus expression revealed a significant and rapid decrease in the accumulation of gus transcripts in the control patmyb60:gus plants following exposure to aba  .
we observed a comparable reduction in gus expression in two independent pvvmyb60:gus lines, that were randomly selected for the qpcr experiment  .
staining of rosette leaves excised from all the ten pvvmyb60:gus lines, before and after treatment with aba, confirmed the negative effect of the hormone on the activity of the vvmyb <dig> promoter .
conversely, treatment of pvvmyb30:gus plants with aba did not significantly affect the expression of the reporter .
occurrence of guard cell-specific motifs in the vvmyb <dig> promoter
the conserved activity of the arabidopsis and grape myb <dig> promoters, as emphasized by the analysis of the corresponding promoter:gus transgenic lines, suggests that these two regulatory regions might share common cis-elements responsible for the guard cell-specific expression of the reporter.
previous evidence indicates a role for dna consensus sequences for dof-type transcription factors  as guard cell-specific cis-active enhancers  <cit> .
specifically, clusters of at least three aaag motifs located on the same strand within a region of at most  <dig> bp were identified as putative guard cell-specific cis-regulatory elements  <cit> .
the arabidopsis atmyb <dig> promoter contains multiple aaag clusters, of which the most proximal to the translation start codon , is necessary and sufficient to drive expression in guard cells  .
we thus searched the grape vvmyb <dig> and vvmyb <dig> promoters for the occurrence of aaag oligonucleotides, in a region of  <dig> bp upstream of the translation start site.
we identified a cluster of three aaag motifs in the vvmyb <dig> promoter, located at - <dig> bp from the atg codon of the endogenous gene, a distance comparable to the position of the guard cell regulatory element found in the promoter of atmyb <dig>  consistent with the lack of activity in arabidopsis guard cells no aaag clusters were identified in the promoter of the grape vvmyb <dig> gene .
the cellular specificity of gene expression has been investigated for a very limited number of grape genes.
among these, vvsirk, encoding a k+ channel, has been reported to be specifically expressed in guard cells  <cit> .
interestingly, we discovered an aaag cluster upstream of the translation start codon  of vvsirk, in the opposite orientation relative to the direction of transcription .
functional complementation of the arabidopsis atmyb60- <dig> mutant by vvmyb60
a null allele of the arabidopsis atmyb <dig> gene  displays constitutive reduction of the opening of the stomatal pores and reduced water loss during drought  <cit> .
interestingly, despite its increased tolerance to dehydration relative to the wild type, the atmyb60- <dig> mutant does not show obvious alterations in the sensitivity of guard cells to aba  <cit> .
we used the atmyb60- <dig> allele to investigate the role of vvmyb <dig> in the regulation of stomatal activity and to explore the conservation of the myb <dig> gene function between grape and arabidopsis.
to this end, we introduced the full length vvmyb <dig> cdna in transgenic mutant plants  to assess the ability of the grape gene to rescue the stomatal defects of the atmyb60- <dig> allele.
as a control for the complementation, we generated a second series of transgenic plants, in which we transformed the full length vvmyb <dig> cdna in the atmyb60- <dig> background .
it is important to note that the two vvmyb <dig> and vvmyb <dig> promoters displayed very different patterns of activity in arabidopsis .
hence, for a more robust and reliable comparison of the two grape genes in the atmyb60- <dig> background we used the  <dig>  kb atmyb <dig> promoter  <cit>  to drive the expression of vvmyb <dig> and vvmyb <dig> in guard cells.
three independent transgenic mutant lines with a single insertion locus and comparable levels of expression of the transgene were selected for further analysis of each grape gene .
we performed an in-vitro assay to evaluate the aperture of the stomatal pore in epidermal strips excised from mutant and transgenic lines.
in agreement with a previous report  <cit> , light-induced stomatal opening was reduced in the atmyb60- <dig> mutant compared to the wild-type .
mutant lines expressing the vvmyb <dig> gene did not display significant differences in the aperture of the stomatal pores compared to atmyb60- <dig> .
conversely, all the mutant lines transformed with the vvmyb <dig> gene displayed a wild-type response in terms of light-induced stomatal opening, indicating full complementation of the atmyb60- <dig> mutation .
to substantiate the results obtained in vitro, we investigated the effect of both vvmyb <dig> and vvmyb <dig> in vivo, by estimating whole-plant transpiration under stress conditions.
wild-type, atmyb60- <dig>  atmyb60-c <dig> and atmyb60-c <dig> plants were grown in soil and pots were covered with tin foil to prevent evaporation, so that water loss occurring through stomatal transpiration could be quantified.
pots were regularly watered for  <dig> days, and subsequently drought stress was imposed by terminating irrigation.
as expected, transpirational water loss, as determined by soil water content measurements, was significantly reduced in atmyb60- <dig> compared to the wild-type  .
consistently with results from the in vitro assay, mutant lines expressing the vvmyb <dig> gene did not show any difference in term of water loss compared to the atmyb60- <dig> mutant .
conversely, under the same conditions, the lines expressing the vvmyb <dig> gene displayed a rate of water loss indistinguishable from the one observed in the wild-type, thus demonstrating complete rescue of the stomatal defects of the atmyb60- <dig> mutant .
discussion
identification of a grape ortholog of the atmyb <dig> transcription factor
the myb superfamily constitutes the most abundant group of transcription factors found in plants, with at least  <dig> members in arabidopsis and  <dig> in rice  <cit> .
in grape,  <dig> putative r2r3- myb family genes were found in the first genome version   <cit> , whereas more than  <dig> r2r <dig> myb genes can be found using the 12x version .
plant r2r3-type myb transcription factors are implicated in several processes related to cell fate, plant development, hormonal responses, pathogen-disease resistance, drought and cold tolerance, light sensing and flavonoid biosynthesis, among many other functions  <cit> .
myb genes have been intensively investigated in grape, yet most studies have focused on members of the r2r <dig> clade involved in the regulation of the anthocyanin and pro-anthocyanidin biosynthetic pathway, as the accumulation of these flavonoid compounds in fruit tissues is a key determinant of berry and wine quality  <cit> .
conversely, myb genes clustered outside the flavonoid biosynthesis functional group received little attention.
this work shows the identification of the grape vvmyb <dig> gene, as a functional ortholog of the arabidopsis atmyb <dig> gene, involved in the regulation of light-induced stomatal aperture  <cit> .
four lines of evidence support this conclusion: i) the aminoacidic sequence of the vvmyb <dig> and atmyb <dig> proteins is highly conserved, ii) the vvmyb <dig> and atmyb <dig> genes show very similar expression profiles, both in terms of tissue- and cell-specificity and response to aba, iii) the vvmyb <dig> and atmyb <dig> promoters drive expression of reporter genes exclusively in guard cells and share common cis-regulatory elements, iv) the expression of vvmyb <dig> in the atmyb60- <dig> mutant background completely rescues the loss of the atmyb <dig> function.
the arabidopsis and grape myb <dig> proteins resulted more similar to each other than to any other myb in grape or arabidopsis, even inside subgroup  <dig>  reason why we denoted subgroups  <dig>  and  <dig>  for further classification.
two main features discriminate between the arabidopsis and grape myb <dig> proteins and other closely related proteins from subgroup 1: a distinctive pheeg signature in the myb domain, located in the loop connecting the r <dig> and r <dig> repeats, and the lack of the first  of three c-terminal motifs present in all the other myb proteins assigned to subgroup  <dig> .
notably, both characteristics are conserved in putative myb <dig> orthologs that we identified in other plant genomes, including oilseed rape, tomato, cucumber and poplar .
even though a role for the pheeg and ctm <dig> motifs has not yet been described, it is intriguing to speculate that the presence of the former and the absence of the latter, might contribute to the specificity of the myb <dig> function in guard cells.
expression features of vvmyb <dig> in grape organs
it has been previously shown that the arabidopsis atmyb <dig> gene is expressed in seedlings, rosette leaves, stems and flowers and its level of expression is rapidly down-regulated by the stress hormone aba  <cit> .
in addition, publicly available repositories of microarray-based gene profiling experiments indicate that atmyb <dig> is transiently expressed during seed development, peaking in stage  <dig> seeds  and rapidly declining in mature seeds .
our survey of vvmyb <dig> and vvmyb <dig> expression in grape tissues revealed that both genes are preferentially expressed in leaves, berry skin and seeds .
similarly to atmyb <dig>  and opposite to vvmyb <dig>  expression of vvmyb <dig> in seeds was down-regulated during seed development .
in berry skin vvmyb <dig> expression was higher before veraison, when the grape berry is photosynthetically active and stomata are functional, and was reduced after veraison, when stomata evolve into non-functional lenticels  <cit>  .
interestingly, at this stage, the onset of ripening and the accumulation of sugars are correlated to increasing levels of aba in the berry  <cit> , suggesting a possible negative effect of the hormone on the expression of vvmyb <dig> in grape tissues.
indeed, treatment of leaves with exogenous aba resulted in the rapid down-regulation of vvmyb <dig> expression .
in contrast, the hormone did not have any effect on the accumulation of the vvmyb <dig> transcripts .
additionally, osmotic stresses which trigger aba-mediated responses, as high concentrations of nacl, caused the rapid down-regulation of vvmyb <dig> expression in grape tissues .
interestingly, it has been recently shown that applications of low concentrations of aba can trigger a transient up-regulation of myb <dig> expression in arabidopsis seedlings  <cit> .
this suggests that the pattern of atmyb <dig> expression in response to osmotic stress might be rather complex and dose-dependent.
even though the detailed analysis of the mechanisms that regulate the expression of the vvmyb <dig> gene extends beyond the scope of this work, it will be intriguing to further investigate the expression profile of vvmyb <dig> in different grape tissues in response to a wider range of aba concentrations.
the vvmyb <dig> promoter specifically drives reporter gene expression in arabidopsis guard cells
reporter gene analysis and rt-pcr experiments performed on purified arabidopsis stomata, clearly demonstrated that in green tissues, atmyb <dig> is exclusively expressed in guard cells  <cit> .
we produced arabidopsis lines harboring the gus marker gene under the control of the vvmyb <dig> and vvmyb <dig> promoters to establish the cellular localization of gene expression.
histochemical analysis of gus expression in several independent lines indicated that the activity of the vvmyb <dig> promoter is restricted to guard cells .
this result is consistent with the expression of the endogenous vvmyb <dig> gene in leaves and berry skin, which both contain stomata, and with the lack of expression in roots .
reporter gene approaches in arabidopsis provide efficient and reliable tools to investigate the expression of grape genes and to identify gene regulatory elements  <cit> .
yet, we did not observe reporter activity in developing seeds of the pvvmyb30:gus and pvvmyb60:gus lines .
this finding is in contrast with data from qpcr experiments, which showed that both genes are highly expressed in grape seeds .
this discrepancy could simply be artefactual, because of the heterologous genetic background.
however, it is important to note that we did not detect gus activity in developing seeds of patmyb60:gus plants, used as a positive control in this study, despite the high expression of the endogenous arabidopsis gene in these organs  <cit> .
different hypotheses can be formulated to explain the lack of activity of the atmyb <dig> and vvmyb <dig> promoters in seeds.
first, cis-elements responsible for the seed expression of the endogenous genes could be located outside the regulatory genomic regions considered in this work.
however, cominelli and colleagues reported that the complete 5' and 3' atmyb <dig> intergenic regions, cloned upstream and downstream of the gus gene, do not drive expression of the reporter in seeds  <cit> .
alternatively, expression of atmyb <dig> and vvmyb <dig> in seeds could be mediated by intragenic regulatory elements.
cis-acting motifs, located in introns, have been demonstrated to be required to establish the correct expression domain of transcription factors, such as the mads-box agamous gene  <cit> .
most interestingly, seed-specific enhancers have been mapped in the intronic regions of seed-expressed genes in different plant species  <cit> .
finally, the finding that the atmyb <dig> mrna is associated with polyribosomes purified from guard cells but not from other plant tissues  <cit> , opens the possibility for a translational level of regulation for myb <dig> expression in seeds.
clearly, more work is needed to unravel the nature of the cis-regulatory elements that modulate myb <dig> expression in seeds, together with revealing the function of this gene in these organs.
nevertheless, it is reasonable to conclude that the stomata-specific activity of the vvmyb <dig> promoter in arabidopsis mirrors the expression of the endogenous gene in grape guard cells.
while the identity of the cis-acting elements required for the expression of atmyb <dig> and vvmyb <dig> in seeds remains elusive, their expression in stomata is most likely regulated, in cis, by dof recognition dna motifs.
we identified a cluster of aaag dof target sites in close proximity to the vvmyb <dig> translational start codon .
such a cluster has been described as a guard cell-specific cis-regulatory element in different plant species, including arabidopsis and potato  <cit> .
the occurrence of aaag motifs in the guard cell-specific vvmyb <dig> and vvsirk grape promoters lends further support to the conservation of the cis- and, possibly, trans-mechanisms that direct expression in guard cells in distantly related plant species.
interestingly, strong conservation across a wide range of flowering plant species has also been reported for other cell-specific cis-motifs, such as the root hair-specific cis-elements   <cit> .
vvmyb <dig> is a functional ortholog of atmyb60
the ability of vvmyb <dig> to fully complement, both in vitro and in vivo, the stomatal defects exhibited by the atmy60- <dig> mutant unequivocally demonstrates that vvmyb <dig> is a true ortholog of the arabidopsis atmyb <dig> transcription factor.
importantly, the vvmyb <dig> gene product, which shares 47% identity to vvmyb <dig>  did not complement the atmyb60- <dig> mutation.
this result is in agreement with functional studies which indicate that, despite the high degree of homology between the atmyb <dig> and atmyb <dig> aminoacidic sequences, these two proteins play two distinct functional roles.
in arabidopsis, atmyb <dig> mediates brassinosteroid-induced gene expression  <cit>  and pathogen-induced hypersensitive response  <cit> , whereas atmyb <dig> positively regulates light-induced stomatal opening and modulates water loss under drought  <cit> .
as a whole, our findings indicate a role for vvmyb <dig> in the regulation of guard cell activity and transpiration rate in grapevine.
stomatal conductance is a key trait in grapevine, as it directly determines the isohydric/anisohydric behavior displayed by different genotypes.
these differences are due to stomatal control over evaporative demand rather than stomatal density in vegetative tissues  <cit> .
cultivar-specific differences have also been described for the effects of water deficit on aba metabolism and signaling  <cit> .
anisohydric cultivars such as pinot noir possess insufficient stomatal regulation and show high transpiration rates and stomatal conductance, whereas isohydric cultivars as shiraz, display much lower values  <cit> .
in this perspective, it will be interesting to survey variations in naturally occurring vvmyb <dig> alleles and to establish their contribution to differences in stomatal activity in different vitis species and cultivars.
CONCLUSIONS
vvmyb <dig> could represent a valuable target for downstream biotechnological applications.
although grapevine is a highly productive water stress-adapted plant, the availability of molecular targets for engineering or breeding of new cultivars with enhanced stomatal responses represents an attractive approach to increase water use efficiency and, possibly, to reduce pathogen penetration through the stomatal pore  <cit> .

 grapevine is a fruit crop traditionally subjected to moderate or severe water stress, as this is an efficient strategy to improve fruit and wine quality. vitis species adapt well to drought conditions due to good osmotic adjustment, large and deep root systems, efficient control of stomatal aperture and xylem embolism. the strength and timing of these responses varies between different cultivars and major differences in water stress tolerance can be found when compared to other species or hybrids from the vitis genus. although these genotype-related variations involve different aspects of the physiology of the plant, they are largely linked to differences in stomatal conductance. stomata are microscopic pores distributed on the surface of leaves and stems, surrounded by two highly specialized guard cells. the opening and closure of the pore, in response to internal signals and environmental cues, allows the plant to cope with the conflicting needs of ensuring adequate uptake of co for photosynthesis and preventing water loss by transpiration. under drought, abscisic acid is accumulated, inducing rapid stomatal closure to limit water loss. increasing evidence indicates a role for transcription factors belonging to the r2r myb subfamily as key modulators of physiological responses in stomata. in particular, atmyb has been shown to be differentially expressed in guard cells in response to aba, and the loss-of function atmyb60- mutant displays constitutive reduction of light-induced stomatal opening and enhanced tolerance to dehydration. guard cell-specific myb genes are thus focal points in understanding stomatal regulation in plants and represent molecular targets to modulate guard cell activity to improve crop survival and productivity during drought. the grapevine genome has been estimated to contain a total of myb genes, of which belong to the r2r subfamily. a phylogenetic tree, constructed with the complete grape, arabidopsis and rice r2r3myb subfamilies, showed that many genes sharing similar functions were clustered in the same phylogenetic groups. some of these clades were conserved in gene copy number while in those controlling flavonoid synthesis several expansions events may have occurred. in this work, we report the identification of two close homologues of the guard cell-related atmyb gene in the grape genome, namely vvmyb and vvmyb analysis of gene expression in grape tissues revealed that both vvmyb and vvmyb were expressed in green tissues and developing seeds. as opposite to vvmyb vvmyb transcript abundance was greatly reduced by aba and osmotic stress. a gus reporter gene approach in arabidopsis showed that activity of the vvmyb promoter was restricted to stomatal guard cells and was down-regulated by aba. comparative analysis of regulatory regions revealed the presence of common guard cell-specific motifs in the promoters of the grape and arabidopsis myb genes. finally, vvmyb unlike vvmyb fully complemented the stomatal defects of the atmyb60- mutant, thus indicating that vvmyb is a functional ortholog of the arabidopsis atmyb stomatal regulator.  phylogenetic relationships of myb homologues as a first approach to identify grape homologues of the atmyb transcription factor, we searched the r2r myb proteins identified in the vitis vinifera pn genome, for the presence of a distinctive c-terminal motif, found in members of subgroup of the arabidopsis myb family. this subgroup includes: atmyb regulating light-induced stomatal aperture; atmyb related to the regulation of brassinosteroid-induced gene expression and to the biosynthesis of very-long-chain fatty acids involved in hypersensitive cell death; atmyb an aba/auxin cross-talker, mediating aba signaling during drought stress and involved in promoting pathogen resistance and atmyb whose function is still unknown. our search yielded two grape close homologues in the grape genome version 12x: the annotated gene models gsvivt and gsvivt. a parsimony consensus tree was constructed to investigate the phylogenetic relationships within these grape proteins and members of arabidopsis r2r myb subgroup subgroup was also included as some of its members are involved in drought responses and aba signaling. from this subgroup, a grape myb homologue had also been previously isolated. atmyb regulating stomatal activity, but not belonging to any of these subgroups, was included as an out-group. as shown in figure 1a, the two grape proteins acf and abk clustered with members of the arabidopsis subgroup interestingly, atmyb the most distant member of subgroup was more closely related to the grape protein accession acf than to the other members of the subgroup. on the other hand, the grape accession protein abk was closely related to atmyb and atmyb and to a lesser extent to atmyb and atmyb. hereafter, we will refer to acf and abk as vvmyb and vvmyb respectively. based on these results, we further divided subgroup into subgroup, and subgroup. as expected, all the proteins included in the tree disclosed a highly conserved r2r dna binding domain. the identity between the r2r domain of atmyb and vvmyb and vvmyb was 99%, and 90%, respectively. in addition, atmyb and vvmyb disclosed a distinctive pheeg signature, encompassing the two highly conserved glutamic acid residues, located in the loop connecting the r and r repeats. the complete protein sequence of atmyb showed 51% amino acid identity to vvmyb and 48% identity to vvmyb all of these proteins share two c-terminal motifs which are only found in subgroup in addition, atmyb and possess a third myb domain, which is absent in atmyb and vvmyb. the function of these c-terminal domains is still unknown although they might reflect the functional differences between subgroups and. we determined the precise gene structure of both vvmyb and vvmyb by comparing the complete coding sequence with the full length cdna sequence, amplified from pinot noir pn genomic dna and leaf cdna, respectively. it was interesting to note that in the 12x version of the grape genome, gsvivp representing the vvmyb gene model, was misannotated in terms of exon number. indeed, our results indicate the presence of three exons, as opposed to the five exons predicted by the gene model, thus revealing a conserved exon/intron organization for vvmyb vvmyb and atmyb. based on gene structure, myb genes have been classified in four different groups. vvmyb vvmyb and atmyb all belong to group i, which contains genes with a characteristic r domain split between exons and and a r domain split between exons and. the biggest differences in lengths were found in the first intron and the third exon, which were longer in the grape genes compared to atmyb expression of vvmyb and vvmyb in grape tissues and in response to hormonal and stress factors qpcr analysis of gene expression in different grape organs indicated that vvmyb and vvmyb transcripts were most abundant in leaves, seeds and ripened berry skins. interestingly, vvmyb and vvmyb revealed completely opposite expression patterns during seed development; while vvmyb expression was gradually down-regulated towards the onset of ripening, vvmyb expression was rapidly activated. during berry skin development, vvmyb expression also showed a dramatic decrease to full repression at veraison, followed by a slight increase towards ripening. in this tissue, vvmyb was mostly constantly expressed throughout the green and ripening stages. in arabidopsis, it has been shown that the expression of the atmyb gene is rapidly down-regulated following treatment with aba. we thus analysed the expression of the grape genes in leaves treated with or μm aba. as reported in figure 2d, vvmyb showed a significant decrease in expression levels in both and μm aba treated samples, when compared to the mock treated leaves. conversely, vvmyb did not show any change in expression after exposure to the hormone under these conditions. to further investigate the expression of vvmyb and vvmyb in response to osmotic stresses, we designed an in vitro long-term salt stress experiment. nodal explants were placed vertically on sterile ms media supplemented with, or mm nacl. explants were maintained for a month in a growth chamber until roots and/or leaves were visible and fully expanded. at the end of the experiment, plantlets from the mm nacl treatment had a small radicule and high leaf anthocyanin accumulation, as a clear sign of stress in the plant, while plants at mm showed more severe symptoms, including systemic wilting and brown pigmentation. under these conditions, vvmyb and vvmyb showed opposite responses to the increasing salt concentrations; while vvmyb expression was reduced five-fold at both concentrations when compared to the control treatment, vvmyb expression increased three-fold on addition of mm nacl. activity of the vvmyb and vvmyb promoters in arabidopsis transgenic lines we employed a reporter gene approach in the heterologous model system arabidopsis thaliana to investigate the activity of both vvmyb and vvmyb promoters. a region of approximately kb located upstream of the atg codon of vvmyb and vvmyb was fused to the β-glucuronidase reporter gene and the resulting pvvmyb30:gus and pvvmyb60:gus constructs were introduced in arabidopsis by agrobacterium-mediated transformation. we assessed the cell and tissue specificity of reporter gene expression in ten independent t transgenic lines for each promoter:gus combination. fifteen-day-old pvvmyb30:gus seedlings displayed expression of the reporter at the shoot apex, at the base of trichomes located on leaf primordia, and in the emerging lateral roots. at the same developmental stage, pvvmyb60:gus seedlings showed gus expression exclusively in guard cells distributed on cotyledons, hypocotyls and developing leaves. no expression of the reporter gene was detected in rosette leaves from mature pvvmyb30:gus plants, even after prolonged incubation of plant tissues in the gus solution. on the other hand, we observed guard cell-specific signals in mature leaves of pvvmyb60:gus plants, consistent with the gus profile observed in young seedlings. next, we investigated expression of the reporter in flowers and siliques from adult plants. prior to pollination, pvvmyb30:gus flowers revealed a diffuse staining of carpels and stigmatic tissues. we did not observe gus expression in pre- and post-fertilization flowers from most pvvmyb60:gus lines. in two transgenic lines, a weak staining was occasionally detected in stamens, at the interface of filaments and anthers. finally, we did not detect expression of the reporter in developing seeds from either pvvmyb30:gus or pvvmyb60:gus transgenic lines. expression of both the endogenous arabidopsis and grape myb genes is rapidly down-regulated following treatment with aba. we thus investigated changes in gus expression in the pvvmyb60:gus lines in response to exogenous applications of this hormone, using both qpcr and histochemical analyses. a previously described transgenic line carrying a transcriptional fusion between the arabidopsis atmyb promoter and the reporter gus was used as a control for the experiment. as expected, qpcr analysis of gus expression revealed a significant and rapid decrease in the accumulation of gus transcripts in the control patmyb60:gus plants following exposure to aba. we observed a comparable reduction in gus expression in two independent pvvmyb60:gus lines, that were randomly selected for the qpcr experiment. staining of rosette leaves excised from all the ten pvvmyb60:gus lines, before and after treatment with aba, confirmed the negative effect of the hormone on the activity of the vvmyb promoter. conversely, treatment of pvvmyb30:gus plants with aba did not significantly affect the expression of the reporter. occurrence of guard cell-specific motifs in the vvmyb promoter the conserved activity of the arabidopsis and grape myb promoters, as emphasized by the analysis of the corresponding promoter:gus transgenic lines, suggests that these two regulatory regions might share common cis-elements responsible for the guard cell-specific expression of the reporter. previous evidence indicates a role for dna consensus sequences for dof-type transcription factors as guard cell-specific cis-active enhancers. specifically, clusters of at least three aaag motifs located on the same strand within a region of at most bp were identified as putative guard cell-specific cis-regulatory elements. the arabidopsis atmyb promoter contains multiple aaag clusters, of which the most proximal to the translation start codon, is necessary and sufficient to drive expression in guard cells. we thus searched the grape vvmyb and vvmyb promoters for the occurrence of aaag oligonucleotides, in a region of bp upstream of the translation start site. we identified a cluster of three aaag motifs in the vvmyb promoter, located at - bp from the atg codon of the endogenous gene, a distance comparable to the position of the guard cell regulatory element found in the promoter of atmyb consistent with the lack of activity in arabidopsis guard cells no aaag clusters were identified in the promoter of the grape vvmyb gene. the cellular specificity of gene expression has been investigated for a very limited number of grape genes. among these, vvsirk, encoding a k+ channel, has been reported to be specifically expressed in guard cells. interestingly, we discovered an aaag cluster upstream of the translation start codon of vvsirk, in the opposite orientation relative to the direction of transcription. functional complementation of the arabidopsis atmyb60- mutant by vvmyb60 a null allele of the arabidopsis atmyb gene displays constitutive reduction of the opening of the stomatal pores and reduced water loss during drought. interestingly, despite its increased tolerance to dehydration relative to the wild type, the atmyb60- mutant does not show obvious alterations in the sensitivity of guard cells to aba. we used the atmyb60- allele to investigate the role of vvmyb in the regulation of stomatal activity and to explore the conservation of the myb gene function between grape and arabidopsis. to this end, we introduced the full length vvmyb cdna in transgenic mutant plants to assess the ability of the grape gene to rescue the stomatal defects of the atmyb60- allele. as a control for the complementation, we generated a second series of transgenic plants, in which we transformed the full length vvmyb cdna in the atmyb60- background. it is important to note that the two vvmyb and vvmyb promoters displayed very different patterns of activity in arabidopsis. hence, for a more robust and reliable comparison of the two grape genes in the atmyb60- background we used the kb atmyb promoter to drive the expression of vvmyb and vvmyb in guard cells. three independent transgenic mutant lines with a single insertion locus and comparable levels of expression of the transgene were selected for further analysis of each grape gene. we performed an in-vitro assay to evaluate the aperture of the stomatal pore in epidermal strips excised from mutant and transgenic lines. in agreement with a previous report, light-induced stomatal opening was reduced in the atmyb60- mutant compared to the wild-type. mutant lines expressing the vvmyb gene did not display significant differences in the aperture of the stomatal pores compared to atmyb60-. conversely, all the mutant lines transformed with the vvmyb gene displayed a wild-type response in terms of light-induced stomatal opening, indicating full complementation of the atmyb60- mutation. to substantiate the results obtained in vitro, we investigated the effect of both vvmyb and vvmyb in vivo, by estimating whole-plant transpiration under stress conditions. wild-type, atmyb60- atmyb60-c and atmyb60-c plants were grown in soil and pots were covered with tin foil to prevent evaporation, so that water loss occurring through stomatal transpiration could be quantified. pots were regularly watered for days, and subsequently drought stress was imposed by terminating irrigation. as expected, transpirational water loss, as determined by soil water content measurements, was significantly reduced in atmyb60- compared to the wild-type. consistently with results from the in vitro assay, mutant lines expressing the vvmyb gene did not show any difference in term of water loss compared to the atmyb60- mutant. conversely, under the same conditions, the lines expressing the vvmyb gene displayed a rate of water loss indistinguishable from the one observed in the wild-type, thus demonstrating complete rescue of the stomatal defects of the atmyb60- mutant. discussion identification of a grape ortholog of the atmyb transcription factor the myb superfamily constitutes the most abundant group of transcription factors found in plants, with at least members in arabidopsis and in rice. in grape, putative r2r3- myb family genes were found in the first genome version, whereas more than r2r myb genes can be found using the 12x version. plant r2r3-type myb transcription factors are implicated in several processes related to cell fate, plant development, hormonal responses, pathogen-disease resistance, drought and cold tolerance, light sensing and flavonoid biosynthesis, among many other functions. myb genes have been intensively investigated in grape, yet most studies have focused on members of the r2r clade involved in the regulation of the anthocyanin and pro-anthocyanidin biosynthetic pathway, as the accumulation of these flavonoid compounds in fruit tissues is a key determinant of berry and wine quality. conversely, myb genes clustered outside the flavonoid biosynthesis functional group received little attention. this work shows the identification of the grape vvmyb gene, as a functional ortholog of the arabidopsis atmyb gene, involved in the regulation of light-induced stomatal aperture. four lines of evidence support this conclusion: i) the aminoacidic sequence of the vvmyb and atmyb proteins is highly conserved, ii) the vvmyb and atmyb genes show very similar expression profiles, both in terms of tissue- and cell-specificity and response to aba, iii) the vvmyb and atmyb promoters drive expression of reporter genes exclusively in guard cells and share common cis-regulatory elements, iv) the expression of vvmyb in the atmyb60- mutant background completely rescues the loss of the atmyb function. the arabidopsis and grape myb proteins resulted more similar to each other than to any other myb in grape or arabidopsis, even inside subgroup reason why we denoted subgroups and for further classification. two main features discriminate between the arabidopsis and grape myb proteins and other closely related proteins from subgroup 1: a distinctive pheeg signature in the myb domain, located in the loop connecting the r and r repeats, and the lack of the first of three c-terminal motifs present in all the other myb proteins assigned to subgroup. notably, both characteristics are conserved in putative myb orthologs that we identified in other plant genomes, including oilseed rape, tomato, cucumber and poplar. even though a role for the pheeg and ctm motifs has not yet been described, it is intriguing to speculate that the presence of the former and the absence of the latter, might contribute to the specificity of the myb function in guard cells. expression features of vvmyb in grape organs it has been previously shown that the arabidopsis atmyb gene is expressed in seedlings, rosette leaves, stems and flowers and its level of expression is rapidly down-regulated by the stress hormone aba. in addition, publicly available repositories of microarray-based gene profiling experiments indicate that atmyb is transiently expressed during seed development, peaking in stage seeds and rapidly declining in mature seeds. our survey of vvmyb and vvmyb expression in grape tissues revealed that both genes are preferentially expressed in leaves, berry skin and seeds. similarly to atmyb and opposite to vvmyb expression of vvmyb in seeds was down-regulated during seed development. in berry skin vvmyb expression was higher before veraison, when the grape berry is photosynthetically active and stomata are functional, and was reduced after veraison, when stomata evolve into non-functional lenticels. interestingly, at this stage, the onset of ripening and the accumulation of sugars are correlated to increasing levels of aba in the berry, suggesting a possible negative effect of the hormone on the expression of vvmyb in grape tissues. indeed, treatment of leaves with exogenous aba resulted in the rapid down-regulation of vvmyb expression. in contrast, the hormone did not have any effect on the accumulation of the vvmyb transcripts. additionally, osmotic stresses which trigger aba-mediated responses, as high concentrations of nacl, caused the rapid down-regulation of vvmyb expression in grape tissues. interestingly, it has been recently shown that applications of low concentrations of aba can trigger a transient up-regulation of myb expression in arabidopsis seedlings. this suggests that the pattern of atmyb expression in response to osmotic stress might be rather complex and dose-dependent. even though the detailed analysis of the mechanisms that regulate the expression of the vvmyb gene extends beyond the scope of this work, it will be intriguing to further investigate the expression profile of vvmyb in different grape tissues in response to a wider range of aba concentrations. the vvmyb promoter specifically drives reporter gene expression in arabidopsis guard cells reporter gene analysis and rt-pcr experiments performed on purified arabidopsis stomata, clearly demonstrated that in green tissues, atmyb is exclusively expressed in guard cells. we produced arabidopsis lines harboring the gus marker gene under the control of the vvmyb and vvmyb promoters to establish the cellular localization of gene expression. histochemical analysis of gus expression in several independent lines indicated that the activity of the vvmyb promoter is restricted to guard cells. this result is consistent with the expression of the endogenous vvmyb gene in leaves and berry skin, which both contain stomata, and with the lack of expression in roots. reporter gene approaches in arabidopsis provide efficient and reliable tools to investigate the expression of grape genes and to identify gene regulatory elements. yet, we did not observe reporter activity in developing seeds of the pvvmyb30:gus and pvvmyb60:gus lines. this finding is in contrast with data from qpcr experiments, which showed that both genes are highly expressed in grape seeds. this discrepancy could simply be artefactual, because of the heterologous genetic background. however, it is important to note that we did not detect gus activity in developing seeds of patmyb60:gus plants, used as a positive control in this study, despite the high expression of the endogenous arabidopsis gene in these organs. different hypotheses can be formulated to explain the lack of activity of the atmyb and vvmyb promoters in seeds. first, cis-elements responsible for the seed expression of the endogenous genes could be located outside the regulatory genomic regions considered in this work. however, cominelli and colleagues reported that the complete 5' and 3' atmyb intergenic regions, cloned upstream and downstream of the gus gene, do not drive expression of the reporter in seeds. alternatively, expression of atmyb and vvmyb in seeds could be mediated by intragenic regulatory elements. cis-acting motifs, located in introns, have been demonstrated to be required to establish the correct expression domain of transcription factors, such as the mads-box agamous gene. most interestingly, seed-specific enhancers have been mapped in the intronic regions of seed-expressed genes in different plant species. finally, the finding that the atmyb mrna is associated with polyribosomes purified from guard cells but not from other plant tissues, opens the possibility for a translational level of regulation for myb expression in seeds. clearly, more work is needed to unravel the nature of the cis-regulatory elements that modulate myb expression in seeds, together with revealing the function of this gene in these organs. nevertheless, it is reasonable to conclude that the stomata-specific activity of the vvmyb promoter in arabidopsis mirrors the expression of the endogenous gene in grape guard cells. while the identity of the cis-acting elements required for the expression of atmyb and vvmyb in seeds remains elusive, their expression in stomata is most likely regulated, in cis, by dof recognition dna motifs. we identified a cluster of aaag dof target sites in close proximity to the vvmyb translational start codon. such a cluster has been described as a guard cell-specific cis-regulatory element in different plant species, including arabidopsis and potato. the occurrence of aaag motifs in the guard cell-specific vvmyb and vvsirk grape promoters lends further support to the conservation of the cis- and, possibly, trans-mechanisms that direct expression in guard cells in distantly related plant species. interestingly, strong conservation across a wide range of flowering plant species has also been reported for other cell-specific cis-motifs, such as the root hair-specific cis-elements. vvmyb is a functional ortholog of atmyb60 the ability of vvmyb to fully complement, both in vitro and in vivo, the stomatal defects exhibited by the atmy60- mutant unequivocally demonstrates that vvmyb is a true ortholog of the arabidopsis atmyb transcription factor. importantly, the vvmyb gene product, which shares 47% identity to vvmyb did not complement the atmyb60- mutation. this result is in agreement with functional studies which indicate that, despite the high degree of homology between the atmyb and atmyb aminoacidic sequences, these two proteins play two distinct functional roles. in arabidopsis, atmyb mediates brassinosteroid-induced gene expression and pathogen-induced hypersensitive response, whereas atmyb positively regulates light-induced stomatal opening and modulates water loss under drought. as a whole, our findings indicate a role for vvmyb in the regulation of guard cell activity and transpiration rate in grapevine. stomatal conductance is a key trait in grapevine, as it directly determines the isohydric/anisohydric behavior displayed by different genotypes. these differences are due to stomatal control over evaporative demand rather than stomatal density in vegetative tissues. cultivar-specific differences have also been described for the effects of water deficit on aba metabolism and signaling. anisohydric cultivars such as pinot noir possess insufficient stomatal regulation and show high transpiration rates and stomatal conductance, whereas isohydric cultivars as shiraz, display much lower values. in this perspective, it will be interesting to survey variations in naturally occurring vvmyb alleles and to establish their contribution to differences in stomatal activity in different vitis species and cultivars.  vvmyb could represent a valuable target for downstream biotechnological applications. although grapevine is a highly productive water stress-adapted plant, the availability of molecular targets for engineering or breeding of new cultivars with enhanced stomatal responses represents an attractive approach to increase water use efficiency and, possibly, to reduce pathogen penetration through the stomatal pore. 
unlike vvmyb vvmyb was able to complement the loss-of-function atmyb60- mutant, indicating that vvmyb is the only true ortholog of atmyb in the grape genome. this event is trigged by a series of signals produced inside guard cells which finally reduce their turgor. promoter-gus transcriptional fusion assays showed that expression of vvmyb was restricted to stomatal guard cells and was attenuated in response to aba. these results show that vvmyb modulates physiological responses in guard cells, leading to the possibility of engineering stomatal conductance in grapevine, reducing water loss and helping this species to tolerate drought under extreme climatic conditions. under drought, plants accumulate the signaling hormone abscisic acid, which induces the rapid closure of stomatal pores to prevent water loss. in addition, vvmyb was differentially regulated during development of grape organs and in response to aba and drought-related stress conditions. many of these events are tightly regulated at the transcriptional level, including the control exerted by myb proteins.
 under drought, plants accumulate the signaling hormone abscisic acid, which induces the rapid closure of stomatal pores to prevent water loss. this event is trigged by a series of signals produced inside guard cells which finally reduce their turgor. many of these events are tightly regulated at the transcriptional level, including the control exerted by myb proteins. in a previous study, while identifying the grapevine r2r myb family, two closely related genes, vvmyb and vvmyb were found with high similarity to atmyb an arabidopsis guard cell-related drought responsive gene.  promoter-gus transcriptional fusion assays showed that expression of vvmyb was restricted to stomatal guard cells and was attenuated in response to aba. unlike vvmyb vvmyb was able to complement the loss-of-function atmyb60- mutant, indicating that vvmyb is the only true ortholog of atmyb in the grape genome. in addition, vvmyb was differentially regulated during development of grape organs and in response to aba and drought-related stress conditions.  these results show that vvmyb modulates physiological responses in guard cells, leading to the possibility of engineering stomatal conductance in grapevine, reducing water loss and helping this species to tolerate drought under extreme climatic conditions. 