BACKGROUND
decreasing water resources and increasing global warming have the potential to reduce food production in the future  <cit> .
abiotic stresses such as drought, cold and salinity have large impacts on plant growth and development leading to a loss of production and reduced crop quality, which results in the loss of hundreds of millions of dollars each year.
changes in climate that lead to an increase in the frequency and magnitude of drought stress will increase a crop’s dependence on irrigation to maintain productivity.
grapevine  is one of the most economically important fruit crops affected by abiotic stresses.
grapes have a multi-billion dollar impact on the economy, as well as having health benefits, such as providing nutrients and antioxidants  <cit> .
cabernet sauvignon is one of the most well known red wine grapes in the world and is widely cultivated in water-limited areas of the world  where production is highly dependent on irrigation.
grape yields are influenced by plant water status and water stress can lead to decreases in grape production and affect wine quality  <cit> .
the plant hormone abscisic acid  plays a crucial role in responding to a variety of environmental stresses such as drought, salinity and chilling stress  <cit>  and has essential functions involved in plant growth and development, including seed germination, seed dormancy and bud dormancy .
aba has an important role in vegetative tissues in conserving water loss by closing stomata and reducing the leaf surface area.
aba is increased in response to water deficit in grapevine leaves, xylem sap, and berries  <cit>  and water deficit affects a large number of transcripts involved in aba metabolism .
in the past years, aba signal transduction has been extensively studied at the molecular level .
as a result, numerous secondary messengers associated with aba signaling such as calcium , reactive oxygen species , and nitric oxide  were identified.
an aba model of action utilizing pyr/pyl/rcar receptors, type 2c protein phosphatases  and sucrose non-fermenting- <dig> -related protein kinase  <dig>  was proposed and validated .
the soluble pyr/pyl/rcar receptors function at the apex of a negative regulatory pathway to directly regulate pp2c, which in turn negatively regulates snrk <dig>  snrk <dig> is auto-phosphorylated and then phosphorylates other transcription factors , such as members of the bzip/abre, nac, myc/myb, and ap2/erf tf families.
however, their interactions in the framework of an aba signaling network remain to be clarified.
the first step of de novo aba biosynthesis in response to stimuli occurs in the plastid and in the final step, aba-aldehyde is converted to aba in the cytosol  <cit> .
aba is catabolized by aba 8′-hydroxylases and conjugated by aba glucosyltransferases.
aba-glucose ester  levels in leaves were shown to be relatively constant under normal conditions and substantially increase during drought stress  <cit> .
aba-ge is a transport and storage form of aba, which is critical for aba homeostasis  <cit> .
there are at least two different plasma membrane-localized aba transporters; atp-binding cassette g <dig>  is a transporter for aba efflux from vascular tissue  <cit>  and abcg <dig> is responsible for aba transport into guard cells in arabidopsis  <cit> .
plants contain multiple organs that have specific physiological functions with unique gene expression patterns during different developmental stages and stress responses.
for example, leaves specialize in photosynthesis and roots specialize in ion and water transport.
aba affects gene expression differently in seeds and seedlings of arabidopsis  <cit> ; however little is known about other organs, particularly in a woody perennial fruit crop like grapevine.
physiological responses to aba differ in different organs and cells types such as roots, shoots and guard cells.
we hypothesize that aba signaling will differ in different organs as well.
investigations of aba signaling in different organs will improve our understanding of plant responses to osmotic stress and plant development.
this study focuses on aba signaling, investigating the down-stream transcriptional gene expression in different organs: roots shoot tips, mature leaves, berries and cell culture .
the work presented here elucidates the effect of aba on the transcription of genes involved in aba biosynthesis, degradation, conjugation, transport, signaling pathways and metabolic pathways in different organs of grapevine.
RESULTS
transcriptomic analysis of grapevine shows organ-specific change in response to aba
five different organs  of cabernet sauvignon were directly exposed to 10 μm aba for 2 h, except the leaf samples, which were harvested from vines that had their roots treated aeroponically.
although anatomically incorrect, we refer to cell cultures as artificial organs representing meristem-like functions in this paper.
the nimblegen vitis whole genome microarray was used to test our hypothesis that different organs had different aba signaling responses.
a two-way anova identified  <dig> genes with a statistically significant treatment effect, and  <dig> genes with a statistically significant aba treatment and organ interaction  .
the term “significant” will mean “statistically significant” at a p-value at or below  <dig>  throughout this paper.
a post-hoc tukey test was performed to identify significant treatment effects among each organ; there were  <dig> significant  differentially expressed genes  based upon the tukey post-hoc test between control and aba treatment in at least one organ .
from the post-hoc test, the root had  <dig> degs, which is the largest number of genes in response to aba, while the shoot tip had the lowest number with  <dig> genes .
to show the distribution of the degs between control and aba treatment among berries, shoot tips, leaves, roots, and cell cultures, a five-way venn diagram was created .
roots and cell cultures had the most overlap with  <dig> genes responding in common to aba.
our hypothesis was confirmed, there was not a single gene whose transcript abundance changed in common with all organs; the response to aba was organ-specific.
further supporting our hypothesis, principal component analysis  revealed distinct differences among the different organs and treatments within the transcriptomic data .
principal component  <dig> and  <dig>  show the overall variance of transcription expression values.
grape organs were clearly separated from each other on the first principal component; however, on the second principal component, berries and shoot tips were separated from leaves as well as cell cultures and roots.fig.
<dig> gene expression of control and aba treated-plants.
a numbers of significantly differentially expressed genes induced by aba.
b venn diagram showing the overlap of significantly differentially expressed genes between control and aba treatment in berries, shoots, roots, leaves and cell cultures.
c principal component analysis of berries , shoot tips , leaves , roots , and cell cultures .
c = control; a = aba treated.
the following numbers refers to an experimental replicate sample
aba affects aba biosynthesis, degradation, conjugation, transport, signaling and metabolic pathways
aba had significant effects on the transcript abundance of genes involved in aba metabolism .
carotenoids are the precursors for aba biosynthesis.
aba biosynthesis is often controlled by the rate-limiting step, nine-cis epoxycarotenoid dioxygenase   <cit> .
the transcript abundance of vvinced <dig>  significantly increased in cell cultures.
note this gene was originally named vvnced <dig>  <cit> , but following the recommendations of the international grape genome program supernomenclature committee  <cit> , we have named this gene vvinced <dig>  because its closest ortholog in arabidopsis is atnced <dig> fig.
<dig> aba biosynthesis, degradation, and conjugation.
the profiles of the log <dig> ratio  of the transcript abundance of the genes  matched to each enzyme in the aba metabolic pathway are shown as heat maps in the boxes.
boxes from left to right are berries , shoot tips , leaves , roots , and cell cultures , respectively.
ec numbers and all abbreviations are:  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  , aba-ge: aba-glucose ester, abcg: atp-binding cassette subfamily g. all current v <dig> ids for a-r found in additional file  <dig> are: a = ensemblplants:vit_19s0093g <dig>  b = ensemblplants:vit_10s0003g <dig>  c = ensemblplants:vit_05s0051g <dig>  d = ensemblplants:vit_13s0019g <dig>  e = ensemblplants:vit_06s0009g <dig>  f = ensemblplants:vit_06s0004g <dig>  g = ensemblplants:vit_03s0063g <dig>  h = ensemblplants:vit_02s0087g <dig>  i = ensemblplants:vit_06s0004g <dig>  j = ensemblplants:vit_01s0011g <dig>  k = ensemblplants:vit_17s0000g <dig>  l = ensemblplants:vit_12s0034g <dig>  m = ensemblplants:vit_12s0055g <dig>  n = ensemblplants:vit_12s0055g <dig>  o = ensemblplants:vit_03s0063g <dig>  p = ensemblplants:vit_18s0072g <dig>  q = ensemblplants:vit_09s0002g <dig>  r = ensemblplants:vit_18s0166g <dig>  * indicate the significant genes from the tukey post-hoc test
the transcript abundances of genes encoding enzymes in the aba biosynthesis pathway before the rate-limiting step did not change significantly in response to exogenous aba in most organs; the exception was a significant increase in transcript abundance for β-carotene hydroxylase  in cell culture .
the transcript abundance of xanthoxin dehydrogenase and abscisic aldehyde oxidase genes, two steps involved in aba biosynthesis after nced, was not significantly affected by aba.
the aba level in cells is highly regulated by degradation and conjugation processes.
aba is metabolized to 8′-hydroxyabscisic acid by aba 8’-hydroxylase  and conjugated to aba-ge by aba udp-glucosyltransferase .
the relative expression of genes involved in aba catabolism, cyp707as   <cit>  were significantly increased in roots and cell cultures in response to aba.
for aba conjugation, the transcript abundance of the ugt genes was decreased in the shoot tips and roots but increased in cell cultures.
the transcript abundance of an aba transporter, vviabcg <dig> , significantly decreased in roots.
another transcript annotated as an abc transporter g member 22-like  may also be involved in aba transport as it is a paralog of the vitis ortholog  of atabcg <dig> ; its transcript abundance increased significantly in berries in response to aba.
heat maps displaying the gene expression profile of core components of the classic aba signaling pathway  are shown in fig.
<dig> and additional file  <dig>  the transcript abundance of the genes encoding pyl12/rcar <dig>  and pyl4/rcar <dig>  decreased significantly in roots and cell cultures in the presence of aba.
in contrast, the relative gene expression of pp2cs increased significantly in roots  and cell cultures  in response to aba.
there were no significant changes in gene expression of snrk2s following exposure to aba.
a significant increase in transcript abundance of vviabf <dig>  was found in roots.fig.
<dig> aba signaling pathway.
the profiles of the log <dig> ratio  of the transcript abundance of the genes  matched to each enzyme in the aba signaling pathway are shown as heat maps in the boxes.
boxes from left to right are berries , shoot tips , leaves , roots , and cell cultures , respectively.
all abbreviations are: pyr/pyl/rcar: pyrabactin resistance/pyr-like/regulatory components of aba receptor, pp2c: protein phosphatase 2c, snrk2: sucrose non-fermenting 1-related protein kinase  <dig>  areb/abf: aba-responsive element binding protein/abre binding factor, abre: aba-responsive element.
all current v <dig> ids for a-m found in additional file  <dig> are: a = ensemblplants:vit_08s0058g <dig>  b = ensemblplants:vit_13s0067g <dig>  c = ensemblplants:vit_04s0008g <dig>  d = ensemblplants:vit_11s0016g <dig>  e = ensemblplants:vit_16s0050g <dig>  f = ensemblplants:vit_06s0004g <dig>  g = ensemblplants:vit_16s0050g <dig>  h = ensemblplants:vit_00s0179g <dig>  i = ensemblplants:vit_13s0019g <dig>  j = ensemblplants:vit_06s0004g <dig>  k = ensemblplants:vit_07s0197g <dig>  l = ensemblplants:vit_03s0063g <dig>  m = ensemblplants:vit_18s0001g <dig>  n = ensemblplants:vit_07s0005g <dig>  * indicate the significant genes from the tukey post-hoc test
both aba-dependent and -independent transcription factors such as members of the ap2/erf, nac, bzip/abre, and myc/myb families were affected by aba .
interestingly, the greatest change in transcript abundance of transcription factors was found in the ap2/erf family, with  <dig> members changing in their transcript abundance with statistical significance.
the highest increase in transcript abundance induced by aba of the ap2/erf tf superfamily was vvidreb2h ; the response of vvidreb2h was root specific.
in the nac tf family, there were  <dig> degs.
a transcript encoding a gene ortholog to vvinac <dig>  underwent a significant increase in transcript abundance in leaves.
there was a slight change in the transcript abundances of seven members of the bzip/abre tf family.
one deg of the bzip/abre tf family was vvigbf <dig> , which was found to be significantly upregulated in roots and cell cultures.
there is no ortholog of this gene in arabidopsis; its function is unknown.
for the myc/myb tf family, there were  <dig> degs.
most notably, vvimyb <dig>  exhibited the greatest significant increase in transcript abundance in roots.fig.
<dig> transcription factors affected by aba.
the profiles of the log <dig> ratio  of the transcript abundance of the genes  matched to each transcription factors in the aba signaling pathway are shown as heat maps in the boxes.
boxes from left to right are berries , shoot tips , leaves , roots , and cell cultures , respectively.
* indicate the significant genes from the tukey post-hoc test
the transcript abundance of genes in several metabolic pathways was affected by exogenous aba, especially the flavonoid biosynthesis pathway .
genes encoding stilbene synthase  significantly increased in transcript abundance in response to aba treatment in berries but decreased in roots and cell cultures.
on the contrary, genes encoding flavonol synthase  and flavonoid 3’-hydroxylase  were significantly increased in roots in response to aba.
interestingly, the transcript abundance of many genes encoding anthocyanidin 3-o-glucosyltransferase was significantly increased in response to aba only in roots, while other organs showed decreasing or unchanging mrna abundance.fig.
<dig> flavonoid biosynthesis pathway.
the profiles of the log <dig> ratio  of the transcript abundance of the genes  matched to each enzyme in the flavonoid biosynthesis pathway are shown as heat maps in the boxes.
boxes from left to right are berries , shoot tips , leaves , roots , and cell cultures , respectively.
ec numbers are:  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  ,  <dig> .
<dig>  .
all current v <dig> ids for a-r found in additional file  <dig> are: a = , b = , c = , d = , e = , f = , g = , h = , i = , j = , k = , l = , m = , n = , o = , p = , q = , r = [ensemblplants:vit_17s0000g <dig>  * indicate the significant genes from the tukey post-hoc test
differential gene expression responses of organs to aba
aba affected many biological processes of the gene ontology  functional categories; statistically significant degs were involved in protein folding and response to heat, hydrogen peroxide, high light, and temperature .
however, each plant organ had a different response to aba.
the individual responses of each organ are summarized below:
berry
there were  <dig> genes that showed organ-specific gene expression responses to aba in berries .
the gene with the highest increase in transcript abundance to aba was vviabcg22-like ; while the largest decrease in transcript abundance was a gene encoding a glycine-rich cell wall structural protein .
the gene encoding the transcription factor  with the highest transcript abundance in response to aba in berries was abscisic acid insensitive 3-like .
abi <dig> is a b3-domain transcription factor that is a part of the core aba signaling network  <cit> .
the transcript abundance of a peroxidase gene  involved in porphyrin and chlorophyll metabolism significantly decreased in response to aba.
another transporter that significantly increased in transcript abundance was a vacuolar amino acid transporter .
the transcript abundance of a gabat  gene  increased significantly, while the expression of ast  decreased significantly, which may cause an increase of succinate followed by an increase of malate.
transcript abundances of genes encoding enzymes involving flavonoid biosynthesis such as sts  significantly increased only in the berry.
shoot tip
genes with significantly decreased transcript abundance in the aba treatment of shoot tips that had significantly overrepresented go categories were involved in protein amino acid phosphorylation  such as a clavata1-like receptor kinase , receptor kinase homolog lrk14-like , and malectin/receptor-like protein kinase-like .
there were  <dig> genes that showed organ-specific gene expression responses to aba .
the most highly expressed genes in response to aba were the genes in the ap2/erf tf family.
a vviddf <dig>  was the gene with the greatest increase in transcript abundance and was significantly changed in the shoot tips.
a vviora <dig>  was also significantly increased in response to aba.
the genes with the largest decrease in transcript abundance in response to aba were involved in secondary cell wall biosynthesis .
another gene that is also involved in cell wall biosynthesis  was significantly decreased in transcript abundance in response to aba.
this result indicates that there may be an inhibition of shoot growth in response to aba.
leaf
there were  <dig> genes that showed leaf organ-specific gene expression in response to aba .
the greatest increase of transcript abundance of tfs was vvinac <dig> .
the gene with the largest decrease in transcript abundance in response to aba was a gene encoding ent-copalyl diphosphate synthase , which is involved in gibberellic acid  biosynthesis.
the transcript abundance of ent-copalyl diphosphate synthase  in arabidopsis also decreased in response to aba  <cit> .
most genes with significantly increased transcript abundance in leaves were involved in protein folding.
there were also genes encoding -alpha-farnesene synthases  that significantly increased in transcript abundance in response to aba in leaves but significantly decreased in shoot tips.
-alpha-farnesene synthases are involved in terpenoid biosynthesis.
some genes with statistically significant decreased transcript abundance, such as epidermal patterning factor-like protein  <dig>  and tapetum  <dig>  have roles in leaf morphogenesis and bract formation.
furthermore, there were genes with significantly decreased transcript abundance related to transport such as an aquaporin gamma-tip <dig>  and an ortholog of abc transporter member  <dig>  atmrp <dig> , which is an inositol hexakisphosphate transporter involved in aba signaling and regulation of guard cells  <cit> .
root
roots have the largest number of genes responding to aba and there were a number of go categories that were significantly overrepresented .
the main biological process go categories for genes with a significant increase in transcript abundance in root in response to aba was response to stimuli such as light, heat, and reactive oxygen species.
the molecular function of -abscisic acid 8′-hydroxylase activity was significantly overrepresented for genes with a significant increase in response to aba.
for genes with a significant decrease in transcript abundance to aba, many of them were involved in protein amino acid phosphorylation.
the transcript abundance of vvimyb <dig>  and vvidreb2h  was significantly upregulated only in the root .
these two transcription factors have been found to respond to abiotic stress.
qpcr results also confirmed that the transcript abundance of vvimyb <dig> and vvidreb2h increased in response to aba in roots .
the genes with the largest decrease in transcript abundance in response to aba in the root were two pectinesterases  , pectin-degrading enzymes that are involved in cell wall metabolism.
the transcript abundance of a number of heat shock protein genes increased in the roots.
the transcript abundance of genes encoding glucosyltransferase enzymes  was significantly downregulated by aba in the root.
interestingly, there were auxin-, anthocyanin-, and cytokinin- glucosyltransferases degs whose transcript abundance was decreased by aba only in roots.
several genes involved in transport were significantly increased in roots such as vviabcg <dig>  an ortholog of atpdr <dig>  the aquaporin gamma-tip <dig> , and a sulfate transporter .
note that the same aquaporin was downregulated in the leaf by aba .
cell culture
the go categories that were significantly overrepresented in cell cultures of up-regulated genes in response to aba were involved in hormone stimulus .
in addition, there were a large number of genes with increased transcript abundance that were associated with xyloglucosyltransferase activity such as ensemblplants:vit_11s0052g <dig>  ensemblplants:vit_11s0052g <dig>  ensemblplants:vit_11s0052g <dig>  and ensemblplants:vit_11s0052g <dig>  the tf that had the highest increase in transcript abundance in response to aba in cell cultures was an ortholog  .
this result is consistent with the high expression of a pp2c , which is part of a negative feedback loop in the aba signaling pathway.
lignin-forming anionic peroxidase genes were also decreased in transcript abundance by aba .
down-regulation of an anionic peroxidase alters both lignin content and composition  <cit> .
the gene with the highest increase in transcript abundance was a u-box domain-containing protein  <dig>  that may have a u-box type e <dig> ubiquitin ligase function .
discussion
water deficit alters the metabolic homeostasis of plants .
plants reduce water loss by closing stomata and decreasing photosynthesis, which can be triggered by aba  <cit> .
aba can be synthesized in all cells and organs  <cit> .
a root organ senses the soil water availability, synthesizes de novo aba and transports aba via the xylem under mild water deficits  <cit> .
however, it is still uncertain the degree to which aba is transported from the root to different parts of the plant via the xylem sap or how much aba is directly synthesized in leaves or guard cells, subsequently causing stomatal closure  <cit> .
aba signaling varies with the organ
our microarray data clearly support our hypothesis that different organs had different aba signaling responses.
no single deg was affected in the same manner in all organs.
in our study, equal concentrations of aba were applied to the organs ; the larger change in gene expression found in the root  may be due to a higher sensitivity for aba in the root, however, we cannot rule out the possibility that there were differences in aba uptake into the organs.
furthermore, with time or higher aba concentrations, more genes may have had similar expression patterns.
nevertheless, the results support differences in aba sensitivity and signaling.
another contributing factor to the variation in aba response could be the presence of other interacting signals or epigenetic regulation  <cit> .
aba is known to interact or have crosstalk with other plant hormones and nutrients such as sugars  <cit> .
critical processes including seed germination, fruit development, root and shoot growth have been used to understand the role of hormone crosstalk in protein activities and gene expression.
it is a major challenge to decipher the intricate complexity of these multiple signals in the regulation of gene expression.
our data provide direct transcriptional evidence of aba action that differs from one organ to another.
this indicates the involvement of other “signaling partners” along with aba that are essential to coordinate gene expression.
future experiments will be needed to identify what are the major factors that may interact with aba in order to explain the different responses of aba in the organs studied here.
aba effects on aba metabolism
our data indicate that aba triggered feedback loops on aba metabolism and signaling and these responses differed among the organs.
multiple studies showed that nced genes  were induced strongly by drought-stressed conditions in grape, arabidopsis, maize, tomato, bean, cowpea and avocado .
in our study, the relative expression of vvinced <dig> was significantly increased in cell culture, but not other organs.
the transcript abundance of genes involved in aba degradation and conjugation was highly increased .
this is consistent with evidence from several studies that aba negatively regulates its own accumulation, in part through activation of catabolic enzymes  <cit> .
in the aba signaling pathway, the transcript abundance of pyr/pyl/rcar significantly decreased and the transcript abundance of pp2cs significantly increased in roots and cell culture .
the high level of aba from stress conditions can induce the expression of pp2cs  <cit> .
for aba transport, an abc transporter was found to be able to transport aba from the cytoplasm to the vacuole in order to control the level of aba in the cytosol  <cit> .
the relatively high expression of vviabcg22-like in berries indicates that there may be aba transport into cells  <cit> .
in contrast, the significant decrease in transcript abundance of vviabcg <dig> in cell cultures may lessen aba levels in cells, which corresponds to the negative-feedback response found in cell cultures.
overall, these results indicate that there is a negative-feedback loop from the increasing aba concentration to balance aba action  <cit> .
biochemical effects of aba on berry ripening
in plant growth and development, the aba level increases during grape berry ripening; possibly independent of osmotic stress  <cit> .
processes involved during grape berry ripening include: fruit softening, sugar accumulation, organic acid reduction, and increases in potassium level and phenolic compounds  <cit> .
our study is consistent with these studies that aba is involved in grape berry ripening processes.
the significant increase in transcript abundance of vviabi <dig> in berries was found during the lag phase of berry ripening  <cit>  indicating a relationship between aba and grape ripening.
a glycine-rich protein was found in our microarray analyses whose ortholog is involved in cell wall biogenesis and degradation in arabidopsis .
this result is consistent with the hypothesis that aba regulates genes involved in cell wall modification during the ripening processes contributing to fruit softening.
the relative expression of genes involved in malate biosynthesis such as gabat and ast increased.
gaba metabolism is able to contribute to the activity of both the tca cycle and the respiratory electron transfer chain by generating succinate and nadh through ssadh activity.
it is consistent with a low expression of an aspartate aminotransferase gene, which catalyzes oxaloacetate to aspartate leading to an increase of malate level.
moreover, aba enhances the production of phenolic compounds such as stilbenes and anthocyanins  <cit> , which agrees well with the up-regulation of stilbene synthases and anthocyanin glucosyltransferases genes in berries in this study .
aba effects on energy conservation and antioxidant defense during water deficit
aba is a defense hormone that helps protect plants from water deficits.
plants are very sensitive to water deficits and maintaining a sufficient level of energy is difficult when stomata are closed and photosynthesis is inhibited.
under energy-limited conditions, it has been suggested that plants prioritize metabolic pathways that support energy conservation, by reducing growth and protein synthesis, and utilizing the conserved energy for defense, such as antioxidant defenses  <cit> .
as a consequence of energy conservation during stress, metabolic flux can be diverted from cell wall production, an energy intensive process.
here, the decreasing transcript abundance of vvifla <dig> found only in the shoot tips by the aba treatment may be associated with the inhibition of shoot growth.
a previous study showed that the fla <dig> mutant has a reduced ability to regenerate shoots in an in vitro shoot-induction assay in arabidopsis  <cit> .
atfla <dig> and atfla <dig> in arabidopsis are involved in cell wall composition, resulting in changes in cell-wall architecture  <cit> .
this case also occurs in leaves, where a gene involved in ga biosynthesis was decreased in transcript abundance.
ga regulates the transition from cell division to expansion that controls organ growth and size.
therefore, ga levels may be decreased under stress to limit the leaf area surface, which aids the plant by reducing water loss  <cit> .
additionally, the transcript abundance of other cell wall enzymes were affected such as a pectinesterase in the root and cell wall peroxidases in cell culture.
overexpressing genes involved in flavonol and anthocyanin biosynthesis increase the stress tolerance of plants  <cit> .
furthermore it appears that different organs  of maize produce different forms of terpenoid antioxidants in response to water deficit, with the root-specific terpenoids conferring drought tolerance to the plants  <cit> .
anthocyanin biosynthesis has been found to be affected by aba, which plays a role in antioxidant and uv-b protection  <cit> .
in addition, terpenes in the leaves increase in response to abiotic stress and can act as defense or protective responses  <cit> .
consistent with these observations, aba increased the transcript abundance of genes involved in antioxidant defense in our study.
there was a differential expression of genes involved in both flavonoid and terpenoid metabolism.
different steps in flavonoid biosynthesis were affected in the different organs .
the transcript abundance of -alpha-farnesene synthase genes  was increased in the leaf by aba.
-alpha-farnesene synthase is categorized in the tps-b family, which can produce monoterpenes in cabernet sauvignon  <cit> .
terpenes can act as antioxidants during abiotic stress, for example, by transfer of hydrogen or electron and quenching of singlet oxygen  <cit> .
aba effects on transcription
multiple tfs are triggered by aba to initiate or inhibit signaling downstream.
in our study, tfs that had the largest change in transcript abundance belonged to the ap2/erf superfamily .
dreb is a subfamily of ap2/erf tfs that regulate the response of plants to abiotic stress conditions  <cit> .
ap2/erf tfs can crosstalk with other hormones such as ethylene, gibberellin, and salicylic acid.
little is known about which particular drebs act as a sensor in each case.
our results showed a unique and high expression of the vviddf <dig> gene in shoot tips, vvidreb2h specifically in roots, and vvierf <dig> in cell culture in response to aba.
overexpression of the atddf <dig> in arabidopsis results in a dwarf phenotype, on account of a reduction of ga, which is consistent with our results in leaves where genes involved in ga biosynthesis were down regulated in response to aba  <cit> .
other tfs families such as bzip/abre, nac, and myc/myb also have a differential change in gene expression.
transcript abundance of the vvimyb <dig> uniquely increased in roots in response to aba.
this result is consistent with an arabidopsis study where expression of atmyb <dig> increased in response to aba treatment and only in roots; it also showed the greatest change in transcript abundance in response to osmotic stress  <cit> .
in leaves, transcript abundance of a vvinac <dig> had the highest increase in transcript abundance of any tf in response to aba.
the ortholog of this gene in arabidopsis only shows a high expression in senescent leaves  <cit> , and some nac tfs have been found to be part of the aba induction of leaf senescence  <cit> .
vvinac <dig> responds to aba and other defense-related hormones; it confers stress tolerance when overexpressed in arabidopsis  <cit> .
interestingly, the tf that had the highest transcript abundance in berries was vviabi <dig>  the close ortholog of this gene in arabidopsis is found to be essential for seed maturation and is part of the aba signaling network  <cit> .
these tfs are good candidate genes to study the regulation of organ-specific responses to aba in grapevine.
aba effects on protein modification
genes involved in protein modification and metabolism were affected by aba in our study.
posttranslational modifications such as phosphorylation, ubiquitination, and nitrosylation have been found to be involved in aba signaling pathways  <cit> .
phosphorylation is a key process for aba to trigger downstream targets.
pp2c is a key protein that negatively regulates snrk2s via dephosphorylation.
when snrk2s are phosphorylated they are active, resulting in the phosphorylation of downstream activator and repressor proteins.
pp2cs had increased transcript abundance to slowdown the activation of the aba signaling pathway that occurs from a rapid increase in the amount of exogenously applied aba.
these transcripts fall within the go category for protein amino acid phosphorylation process that was found to decrease in shoot tips, roots and cell cultures .
ubiquitination is a rapid posttranslational modification that responds to various environmental stresses leading to protein degradation.
in our study, we found a large increase in transcript abundance of a plant u-box domain-containing protein  <dig>  in cell culture and roots in response to aba.
a previous study found that atpub <dig>  gene expression was induced by aba and drought and it has e <dig> ligase activity  <cit> .
pub <dig> is a negative regulator in aba-mediated drought stress response; the atpub <dig> mutant showed more sensitivity to aba with enhanced tolerance to drought stress  <cit> .
CONCLUSIONS
our transcriptomic analysis has revealed unique effects of aba in different grapevine organs.
supporting our hypothesis that the response to a mild level of aba was complex and dependent on the organ involved.
while there was not a common response to aba for any gene in all organs, there were common pathways or gene ontologies that were affected by applied aba, including transcription factor activities, aba metabolism and signaling, and flavonoid metabolism.
this study provides aba-responsive candidate genes in each grapevine organ.
identifying the differences in gene expression that regulate grapevine aba responses in individual organs is crucial for fully understanding hormone action and the physiological responses to water deficit in the whole plant.
ultimately this knowledge can be utilized to manipulate the effects of aba in different organs to reach desirable outcomes such as enhanced drought tolerance and grape quality.

 decreasing water resources and increasing global warming have the potential to reduce food production in the future. abiotic stresses such as drought, cold and salinity have large impacts on plant growth and development leading to a loss of production and reduced crop quality, which results in the loss of hundreds of millions of dollars each year. changes in climate that lead to an increase in the frequency and magnitude of drought stress will increase a crop’s dependence on irrigation to maintain productivity. grapevine is one of the most economically important fruit crops affected by abiotic stresses. grapes have a multi-billion dollar impact on the economy, as well as having health benefits, such as providing nutrients and antioxidants. cabernet sauvignon is one of the most well known red wine grapes in the world and is widely cultivated in water-limited areas of the world where production is highly dependent on irrigation. grape yields are influenced by plant water status and water stress can lead to decreases in grape production and affect wine quality. the plant hormone abscisic acid plays a crucial role in responding to a variety of environmental stresses such as drought, salinity and chilling stress and has essential functions involved in plant growth and development, including seed germination, seed dormancy and bud dormancy. aba has an important role in vegetative tissues in conserving water loss by closing stomata and reducing the leaf surface area. aba is increased in response to water deficit in grapevine leaves, xylem sap, and berries and water deficit affects a large number of transcripts involved in aba metabolism. in the past years, aba signal transduction has been extensively studied at the molecular level. as a result, numerous secondary messengers associated with aba signaling such as calcium, reactive oxygen species, and nitric oxide were identified. an aba model of action utilizing pyr/pyl/rcar receptors, type 2c protein phosphatases and sucrose non-fermenting- -related protein kinase was proposed and validated. the soluble pyr/pyl/rcar receptors function at the apex of a negative regulatory pathway to directly regulate pp2c, which in turn negatively regulates snrk snrk is auto-phosphorylated and then phosphorylates other transcription factors, such as members of the bzip/abre, nac, myc/myb, and ap2/erf tf families. however, their interactions in the framework of an aba signaling network remain to be clarified. the first step of de novo aba biosynthesis in response to stimuli occurs in the plastid and in the final step, aba-aldehyde is converted to aba in the cytosol. aba is catabolized by aba 8′-hydroxylases and conjugated by aba glucosyltransferases. aba-glucose ester levels in leaves were shown to be relatively constant under normal conditions and substantially increase during drought stress. aba-ge is a transport and storage form of aba, which is critical for aba homeostasis. there are at least two different plasma membrane-localized aba transporters; atp-binding cassette g is a transporter for aba efflux from vascular tissue and abcg is responsible for aba transport into guard cells in arabidopsis. plants contain multiple organs that have specific physiological functions with unique gene expression patterns during different developmental stages and stress responses. for example, leaves specialize in photosynthesis and roots specialize in ion and water transport. aba affects gene expression differently in seeds and seedlings of arabidopsis; however little is known about other organs, particularly in a woody perennial fruit crop like grapevine. physiological responses to aba differ in different organs and cells types such as roots, shoots and guard cells. we hypothesize that aba signaling will differ in different organs as well. investigations of aba signaling in different organs will improve our understanding of plant responses to osmotic stress and plant development. this study focuses on aba signaling, investigating the down-stream transcriptional gene expression in different organs: roots shoot tips, mature leaves, berries and cell culture. the work presented here elucidates the effect of aba on the transcription of genes involved in aba biosynthesis, degradation, conjugation, transport, signaling pathways and metabolic pathways in different organs of grapevine.  transcriptomic analysis of grapevine shows organ-specific change in response to aba five different organs of cabernet sauvignon were directly exposed to 10 μm aba for 2 h, except the leaf samples, which were harvested from vines that had their roots treated aeroponically. although anatomically incorrect, we refer to cell cultures as artificial organs representing meristem-like functions in this paper. the nimblegen vitis whole genome microarray was used to test our hypothesis that different organs had different aba signaling responses. a two-way anova identified genes with a statistically significant treatment effect, and genes with a statistically significant aba treatment and organ interaction. the term “significant” will mean “statistically significant” at a p-value at or below throughout this paper. a post-hoc tukey test was performed to identify significant treatment effects among each organ; there were significant differentially expressed genes based upon the tukey post-hoc test between control and aba treatment in at least one organ. from the post-hoc test, the root had degs, which is the largest number of genes in response to aba, while the shoot tip had the lowest number with genes. to show the distribution of the degs between control and aba treatment among berries, shoot tips, leaves, roots, and cell cultures, a five-way venn diagram was created. roots and cell cultures had the most overlap with genes responding in common to aba. our hypothesis was confirmed, there was not a single gene whose transcript abundance changed in common with all organs; the response to aba was organ-specific. further supporting our hypothesis, principal component analysis revealed distinct differences among the different organs and treatments within the transcriptomic data. principal component and show the overall variance of transcription expression values. grape organs were clearly separated from each other on the first principal component; however, on the second principal component, berries and shoot tips were separated from leaves as well as cell cultures and roots.fig. gene expression of control and aba treated-plants. a numbers of significantly differentially expressed genes induced by aba. b venn diagram showing the overlap of significantly differentially expressed genes between control and aba treatment in berries, shoots, roots, leaves and cell cultures. c principal component analysis of berries, shoot tips, leaves, roots, and cell cultures. c = control; a = aba treated. the following numbers refers to an experimental replicate sample aba affects aba biosynthesis, degradation, conjugation, transport, signaling and metabolic pathways aba had significant effects on the transcript abundance of genes involved in aba metabolism. carotenoids are the precursors for aba biosynthesis. aba biosynthesis is often controlled by the rate-limiting step, nine-cis epoxycarotenoid dioxygenase. the transcript abundance of vvinced significantly increased in cell cultures. note this gene was originally named vvnced, but following the recommendations of the international grape genome program supernomenclature committee, we have named this gene vvinced because its closest ortholog in arabidopsis is atnced fig. aba biosynthesis, degradation, and conjugation. the profiles of the log ratio of the transcript abundance of the genes matched to each enzyme in the aba metabolic pathway are shown as heat maps in the boxes. boxes from left to right are berries, shoot tips, leaves, roots, and cell cultures, respectively. ec numbers and all abbreviations are:.,.,.,.,.,., aba-ge: aba-glucose ester, abcg: atp-binding cassette subfamily g. all current v ids for a-r found in additional file are: a = ensemblplants:vit_19s0093g b = ensemblplants:vit_10s0003g c = ensemblplants:vit_05s0051g d = ensemblplants:vit_13s0019g e = ensemblplants:vit_06s0009g f = ensemblplants:vit_06s0004g g = ensemblplants:vit_03s0063g h = ensemblplants:vit_02s0087g i = ensemblplants:vit_06s0004g j = ensemblplants:vit_01s0011g k = ensemblplants:vit_17s0000g l = ensemblplants:vit_12s0034g m = ensemblplants:vit_12s0055g n = ensemblplants:vit_12s0055g o = ensemblplants:vit_03s0063g p = ensemblplants:vit_18s0072g q = ensemblplants:vit_09s0002g r = ensemblplants:vit_18s0166g * indicate the significant genes from the tukey post-hoc test the transcript abundances of genes encoding enzymes in the aba biosynthesis pathway before the rate-limiting step did not change significantly in response to exogenous aba in most organs; the exception was a significant increase in transcript abundance for β-carotene hydroxylase in cell culture. the transcript abundance of xanthoxin dehydrogenase and abscisic aldehyde oxidase genes, two steps involved in aba biosynthesis after nced, was not significantly affected by aba. the aba level in cells is highly regulated by degradation and conjugation processes. aba is metabolized to 8′-hydroxyabscisic acid by aba 8’-hydroxylase and conjugated to aba-ge by aba udp-glucosyltransferase. the relative expression of genes involved in aba catabolism, cyp707as were significantly increased in roots and cell cultures in response to aba. for aba conjugation, the transcript abundance of the ugt genes was decreased in the shoot tips and roots but increased in cell cultures. the transcript abundance of an aba transporter, vviabcg, significantly decreased in roots. another transcript annotated as an abc transporter g member 22-like may also be involved in aba transport as it is a paralog of the vitis ortholog of atabcg; its transcript abundance increased significantly in berries in response to aba. heat maps displaying the gene expression profile of core components of the classic aba signaling pathway are shown in fig.  and additional file the transcript abundance of the genes encoding pyl12/rcar and pyl4/rcar decreased significantly in roots and cell cultures in the presence of aba. in contrast, the relative gene expression of pp2cs increased significantly in roots and cell cultures in response to aba. there were no significant changes in gene expression of snrk2s following exposure to aba. a significant increase in transcript abundance of vviabf was found in roots.fig. aba signaling pathway. the profiles of the log ratio of the transcript abundance of the genes matched to each enzyme in the aba signaling pathway are shown as heat maps in the boxes. boxes from left to right are berries, shoot tips, leaves, roots, and cell cultures, respectively. all abbreviations are: pyr/pyl/rcar: pyrabactin resistance/pyr-like/regulatory components of aba receptor, pp2c: protein phosphatase 2c, snrk2: sucrose non-fermenting 1-related protein kinase areb/abf: aba-responsive element binding protein/abre binding factor, abre: aba-responsive element. all current v ids for a-m found in additional file are: a = ensemblplants:vit_08s0058g b = ensemblplants:vit_13s0067g c = ensemblplants:vit_04s0008g d = ensemblplants:vit_11s0016g e = ensemblplants:vit_16s0050g f = ensemblplants:vit_06s0004g g = ensemblplants:vit_16s0050g h = ensemblplants:vit_00s0179g i = ensemblplants:vit_13s0019g j = ensemblplants:vit_06s0004g k = ensemblplants:vit_07s0197g l = ensemblplants:vit_03s0063g m = ensemblplants:vit_18s0001g n = ensemblplants:vit_07s0005g * indicate the significant genes from the tukey post-hoc test both aba-dependent and -independent transcription factors such as members of the ap2/erf, nac, bzip/abre, and myc/myb families were affected by aba. interestingly, the greatest change in transcript abundance of transcription factors was found in the ap2/erf family, with members changing in their transcript abundance with statistical significance. the highest increase in transcript abundance induced by aba of the ap2/erf tf superfamily was vvidreb2h; the response of vvidreb2h was root specific. in the nac tf family, there were degs. a transcript encoding a gene ortholog to vvinac underwent a significant increase in transcript abundance in leaves. there was a slight change in the transcript abundances of seven members of the bzip/abre tf family. one deg of the bzip/abre tf family was vvigbf, which was found to be significantly upregulated in roots and cell cultures. there is no ortholog of this gene in arabidopsis; its function is unknown. for the myc/myb tf family, there were degs. most notably, vvimyb exhibited the greatest significant increase in transcript abundance in roots.fig. transcription factors affected by aba. the profiles of the log ratio of the transcript abundance of the genes matched to each transcription factors in the aba signaling pathway are shown as heat maps in the boxes. boxes from left to right are berries, shoot tips, leaves, roots, and cell cultures, respectively. * indicate the significant genes from the tukey post-hoc test the transcript abundance of genes in several metabolic pathways was affected by exogenous aba, especially the flavonoid biosynthesis pathway. genes encoding stilbene synthase significantly increased in transcript abundance in response to aba treatment in berries but decreased in roots and cell cultures. on the contrary, genes encoding flavonol synthase and flavonoid 3’-hydroxylase were significantly increased in roots in response to aba. interestingly, the transcript abundance of many genes encoding anthocyanidin 3-o-glucosyltransferase was significantly increased in response to aba only in roots, while other organs showed decreasing or unchanging mrna abundance.fig. flavonoid biosynthesis pathway. the profiles of the log ratio of the transcript abundance of the genes matched to each enzyme in the flavonoid biosynthesis pathway are shown as heat maps in the boxes. boxes from left to right are berries, shoot tips, leaves, roots, and cell cultures, respectively. ec numbers are:.,.,.,.,.,.. all current v ids for a-r found in additional file are: a =, b =, c =, d =, e =, f =, g =, h =, i =, j =, k =, l =, m =, n =, o =, p =, q =, r = [ensemblplants:vit_17s0000g * indicate the significant genes from the tukey post-hoc test differential gene expression responses of organs to aba aba affected many biological processes of the gene ontology functional categories; statistically significant degs were involved in protein folding and response to heat, hydrogen peroxide, high light, and temperature. however, each plant organ had a different response to aba. the individual responses of each organ are summarized below: berry there were genes that showed organ-specific gene expression responses to aba in berries. the gene with the highest increase in transcript abundance to aba was vviabcg22-like; while the largest decrease in transcript abundance was a gene encoding a glycine-rich cell wall structural protein. the gene encoding the transcription factor with the highest transcript abundance in response to aba in berries was abscisic acid insensitive 3-like. abi is a b3-domain transcription factor that is a part of the core aba signaling network. the transcript abundance of a peroxidase gene involved in porphyrin and chlorophyll metabolism significantly decreased in response to aba. another transporter that significantly increased in transcript abundance was a vacuolar amino acid transporter. the transcript abundance of a gabat gene increased significantly, while the expression of ast decreased significantly, which may cause an increase of succinate followed by an increase of malate. transcript abundances of genes encoding enzymes involving flavonoid biosynthesis such as sts significantly increased only in the berry. shoot tip genes with significantly decreased transcript abundance in the aba treatment of shoot tips that had significantly overrepresented go categories were involved in protein amino acid phosphorylation such as a clavata1-like receptor kinase, receptor kinase homolog lrk14-like, and malectin/receptor-like protein kinase-like. there were genes that showed organ-specific gene expression responses to aba. the most highly expressed genes in response to aba were the genes in the ap2/erf tf family. a vviddf was the gene with the greatest increase in transcript abundance and was significantly changed in the shoot tips. a vviora was also significantly increased in response to aba. the genes with the largest decrease in transcript abundance in response to aba were involved in secondary cell wall biosynthesis. another gene that is also involved in cell wall biosynthesis was significantly decreased in transcript abundance in response to aba. this result indicates that there may be an inhibition of shoot growth in response to aba. leaf there were genes that showed leaf organ-specific gene expression in response to aba. the greatest increase of transcript abundance of tfs was vvinac. the gene with the largest decrease in transcript abundance in response to aba was a gene encoding ent-copalyl diphosphate synthase, which is involved in gibberellic acid biosynthesis. the transcript abundance of ent-copalyl diphosphate synthase in arabidopsis also decreased in response to aba. most genes with significantly increased transcript abundance in leaves were involved in protein folding. there were also genes encoding -alpha-farnesene synthases that significantly increased in transcript abundance in response to aba in leaves but significantly decreased in shoot tips. -alpha-farnesene synthases are involved in terpenoid biosynthesis. some genes with statistically significant decreased transcript abundance, such as epidermal patterning factor-like protein and tapetum have roles in leaf morphogenesis and bract formation. furthermore, there were genes with significantly decreased transcript abundance related to transport such as an aquaporin gamma-tip and an ortholog of abc transporter member atmrp, which is an inositol hexakisphosphate transporter involved in aba signaling and regulation of guard cells. root roots have the largest number of genes responding to aba and there were a number of go categories that were significantly overrepresented. the main biological process go categories for genes with a significant increase in transcript abundance in root in response to aba was response to stimuli such as light, heat, and reactive oxygen species. the molecular function of -abscisic acid 8′-hydroxylase activity was significantly overrepresented for genes with a significant increase in response to aba. for genes with a significant decrease in transcript abundance to aba, many of them were involved in protein amino acid phosphorylation. the transcript abundance of vvimyb and vvidreb2h was significantly upregulated only in the root. these two transcription factors have been found to respond to abiotic stress. qpcr results also confirmed that the transcript abundance of vvimyb and vvidreb2h increased in response to aba in roots. the genes with the largest decrease in transcript abundance in response to aba in the root were two pectinesterases, pectin-degrading enzymes that are involved in cell wall metabolism. the transcript abundance of a number of heat shock protein genes increased in the roots. the transcript abundance of genes encoding glucosyltransferase enzymes was significantly downregulated by aba in the root. interestingly, there were auxin-, anthocyanin-, and cytokinin- glucosyltransferases degs whose transcript abundance was decreased by aba only in roots. several genes involved in transport were significantly increased in roots such as vviabcg an ortholog of atpdr the aquaporin gamma-tip, and a sulfate transporter. note that the same aquaporin was downregulated in the leaf by aba. cell culture the go categories that were significantly overrepresented in cell cultures of up-regulated genes in response to aba were involved in hormone stimulus. in addition, there were a large number of genes with increased transcript abundance that were associated with xyloglucosyltransferase activity such as ensemblplants:vit_11s0052g ensemblplants:vit_11s0052g ensemblplants:vit_11s0052g and ensemblplants:vit_11s0052g the tf that had the highest increase in transcript abundance in response to aba in cell cultures was an ortholog. this result is consistent with the high expression of a pp2c, which is part of a negative feedback loop in the aba signaling pathway. lignin-forming anionic peroxidase genes were also decreased in transcript abundance by aba. down-regulation of an anionic peroxidase alters both lignin content and composition. the gene with the highest increase in transcript abundance was a u-box domain-containing protein that may have a u-box type e ubiquitin ligase function. discussion water deficit alters the metabolic homeostasis of plants. plants reduce water loss by closing stomata and decreasing photosynthesis, which can be triggered by aba. aba can be synthesized in all cells and organs. a root organ senses the soil water availability, synthesizes de novo aba and transports aba via the xylem under mild water deficits. however, it is still uncertain the degree to which aba is transported from the root to different parts of the plant via the xylem sap or how much aba is directly synthesized in leaves or guard cells, subsequently causing stomatal closure. aba signaling varies with the organ our microarray data clearly support our hypothesis that different organs had different aba signaling responses. no single deg was affected in the same manner in all organs. in our study, equal concentrations of aba were applied to the organs; the larger change in gene expression found in the root may be due to a higher sensitivity for aba in the root, however, we cannot rule out the possibility that there were differences in aba uptake into the organs. furthermore, with time or higher aba concentrations, more genes may have had similar expression patterns. nevertheless, the results support differences in aba sensitivity and signaling. another contributing factor to the variation in aba response could be the presence of other interacting signals or epigenetic regulation. aba is known to interact or have crosstalk with other plant hormones and nutrients such as sugars. critical processes including seed germination, fruit development, root and shoot growth have been used to understand the role of hormone crosstalk in protein activities and gene expression. it is a major challenge to decipher the intricate complexity of these multiple signals in the regulation of gene expression. our data provide direct transcriptional evidence of aba action that differs from one organ to another. this indicates the involvement of other “signaling partners” along with aba that are essential to coordinate gene expression. future experiments will be needed to identify what are the major factors that may interact with aba in order to explain the different responses of aba in the organs studied here. aba effects on aba metabolism our data indicate that aba triggered feedback loops on aba metabolism and signaling and these responses differed among the organs. multiple studies showed that nced genes were induced strongly by drought-stressed conditions in grape, arabidopsis, maize, tomato, bean, cowpea and avocado. in our study, the relative expression of vvinced was significantly increased in cell culture, but not other organs. the transcript abundance of genes involved in aba degradation and conjugation was highly increased. this is consistent with evidence from several studies that aba negatively regulates its own accumulation, in part through activation of catabolic enzymes. in the aba signaling pathway, the transcript abundance of pyr/pyl/rcar significantly decreased and the transcript abundance of pp2cs significantly increased in roots and cell culture. the high level of aba from stress conditions can induce the expression of pp2cs. for aba transport, an abc transporter was found to be able to transport aba from the cytoplasm to the vacuole in order to control the level of aba in the cytosol. the relatively high expression of vviabcg22-like in berries indicates that there may be aba transport into cells. in contrast, the significant decrease in transcript abundance of vviabcg in cell cultures may lessen aba levels in cells, which corresponds to the negative-feedback response found in cell cultures. overall, these results indicate that there is a negative-feedback loop from the increasing aba concentration to balance aba action. biochemical effects of aba on berry ripening in plant growth and development, the aba level increases during grape berry ripening; possibly independent of osmotic stress. processes involved during grape berry ripening include: fruit softening, sugar accumulation, organic acid reduction, and increases in potassium level and phenolic compounds. our study is consistent with these studies that aba is involved in grape berry ripening processes. the significant increase in transcript abundance of vviabi in berries was found during the lag phase of berry ripening indicating a relationship between aba and grape ripening. a glycine-rich protein was found in our microarray analyses whose ortholog is involved in cell wall biogenesis and degradation in arabidopsis. this result is consistent with the hypothesis that aba regulates genes involved in cell wall modification during the ripening processes contributing to fruit softening. the relative expression of genes involved in malate biosynthesis such as gabat and ast increased. gaba metabolism is able to contribute to the activity of both the tca cycle and the respiratory electron transfer chain by generating succinate and nadh through ssadh activity. it is consistent with a low expression of an aspartate aminotransferase gene, which catalyzes oxaloacetate to aspartate leading to an increase of malate level. moreover, aba enhances the production of phenolic compounds such as stilbenes and anthocyanins, which agrees well with the up-regulation of stilbene synthases and anthocyanin glucosyltransferases genes in berries in this study. aba effects on energy conservation and antioxidant defense during water deficit aba is a defense hormone that helps protect plants from water deficits. plants are very sensitive to water deficits and maintaining a sufficient level of energy is difficult when stomata are closed and photosynthesis is inhibited. under energy-limited conditions, it has been suggested that plants prioritize metabolic pathways that support energy conservation, by reducing growth and protein synthesis, and utilizing the conserved energy for defense, such as antioxidant defenses. as a consequence of energy conservation during stress, metabolic flux can be diverted from cell wall production, an energy intensive process. here, the decreasing transcript abundance of vvifla found only in the shoot tips by the aba treatment may be associated with the inhibition of shoot growth. a previous study showed that the fla mutant has a reduced ability to regenerate shoots in an in vitro shoot-induction assay in arabidopsis. atfla and atfla in arabidopsis are involved in cell wall composition, resulting in changes in cell-wall architecture. this case also occurs in leaves, where a gene involved in ga biosynthesis was decreased in transcript abundance. ga regulates the transition from cell division to expansion that controls organ growth and size. therefore, ga levels may be decreased under stress to limit the leaf area surface, which aids the plant by reducing water loss. additionally, the transcript abundance of other cell wall enzymes were affected such as a pectinesterase in the root and cell wall peroxidases in cell culture. overexpressing genes involved in flavonol and anthocyanin biosynthesis increase the stress tolerance of plants. furthermore it appears that different organs of maize produce different forms of terpenoid antioxidants in response to water deficit, with the root-specific terpenoids conferring drought tolerance to the plants. anthocyanin biosynthesis has been found to be affected by aba, which plays a role in antioxidant and uv-b protection. in addition, terpenes in the leaves increase in response to abiotic stress and can act as defense or protective responses. consistent with these observations, aba increased the transcript abundance of genes involved in antioxidant defense in our study. there was a differential expression of genes involved in both flavonoid and terpenoid metabolism. different steps in flavonoid biosynthesis were affected in the different organs. the transcript abundance of -alpha-farnesene synthase genes was increased in the leaf by aba. -alpha-farnesene synthase is categorized in the tps-b family, which can produce monoterpenes in cabernet sauvignon. terpenes can act as antioxidants during abiotic stress, for example, by transfer of hydrogen or electron and quenching of singlet oxygen. aba effects on transcription multiple tfs are triggered by aba to initiate or inhibit signaling downstream. in our study, tfs that had the largest change in transcript abundance belonged to the ap2/erf superfamily. dreb is a subfamily of ap2/erf tfs that regulate the response of plants to abiotic stress conditions. ap2/erf tfs can crosstalk with other hormones such as ethylene, gibberellin, and salicylic acid. little is known about which particular drebs act as a sensor in each case. our results showed a unique and high expression of the vviddf gene in shoot tips, vvidreb2h specifically in roots, and vvierf in cell culture in response to aba. overexpression of the atddf in arabidopsis results in a dwarf phenotype, on account of a reduction of ga, which is consistent with our results in leaves where genes involved in ga biosynthesis were down regulated in response to aba. other tfs families such as bzip/abre, nac, and myc/myb also have a differential change in gene expression. transcript abundance of the vvimyb uniquely increased in roots in response to aba. this result is consistent with an arabidopsis study where expression of atmyb increased in response to aba treatment and only in roots; it also showed the greatest change in transcript abundance in response to osmotic stress. in leaves, transcript abundance of a vvinac had the highest increase in transcript abundance of any tf in response to aba. the ortholog of this gene in arabidopsis only shows a high expression in senescent leaves, and some nac tfs have been found to be part of the aba induction of leaf senescence. vvinac responds to aba and other defense-related hormones; it confers stress tolerance when overexpressed in arabidopsis. interestingly, the tf that had the highest transcript abundance in berries was vviabi the close ortholog of this gene in arabidopsis is found to be essential for seed maturation and is part of the aba signaling network. these tfs are good candidate genes to study the regulation of organ-specific responses to aba in grapevine. aba effects on protein modification genes involved in protein modification and metabolism were affected by aba in our study. posttranslational modifications such as phosphorylation, ubiquitination, and nitrosylation have been found to be involved in aba signaling pathways. phosphorylation is a key process for aba to trigger downstream targets. pp2c is a key protein that negatively regulates snrk2s via dephosphorylation. when snrk2s are phosphorylated they are active, resulting in the phosphorylation of downstream activator and repressor proteins. pp2cs had increased transcript abundance to slowdown the activation of the aba signaling pathway that occurs from a rapid increase in the amount of exogenously applied aba. these transcripts fall within the go category for protein amino acid phosphorylation process that was found to decrease in shoot tips, roots and cell cultures. ubiquitination is a rapid posttranslational modification that responds to various environmental stresses leading to protein degradation. in our study, we found a large increase in transcript abundance of a plant u-box domain-containing protein in cell culture and roots in response to aba. a previous study found that atpub gene expression was induced by aba and drought and it has e ligase activity. pub is a negative regulator in aba-mediated drought stress response; the atpub mutant showed more sensitivity to aba with enhanced tolerance to drought stress.  our transcriptomic analysis has revealed unique effects of aba in different grapevine organs. supporting our hypothesis that the response to a mild level of aba was complex and dependent on the organ involved. while there was not a common response to aba for any gene in all organs, there were common pathways or gene ontologies that were affected by applied aba, including transcription factor activities, aba metabolism and signaling, and flavonoid metabolism. this study provides aba-responsive candidate genes in each grapevine organ. identifying the differences in gene expression that regulate grapevine aba responses in individual organs is crucial for fully understanding hormone action and the physiological responses to water deficit in the whole plant. ultimately this knowledge can be utilized to manipulate the effects of aba in different organs to reach desirable outcomes such as enhanced drought tolerance and grape quality. 
abscisic acid regulates various developmental processes and stress responses over both short and longer time frames. the protein amino acid phosphorylation process was significantly overrepresented in shoot tips and roots treated with aba. the results indicated that these genes were involved in several biological processes such as flavonoid metabolism, response to reactive oxygen species, response to light, and response to temperature stimulus. transcriptomic analysis revealed genes whose transcript abundances varied significantly in a specific organ in response to aba treatment. no single gene exhibited the same changes in transcript abundance across all organs in response to aba. to elucidate the transcriptional regulation of early responses of grapevine responding to aba, different organs of grape were treated with 10 μm --aba for 2 h. nimblegen whole genome microarrays of vitis vinifera were used to determine the effects of aba on organ-specific mrna expression patterns. this study examined the short-term effects of aba on different organs of grapevine. understanding the aba responses in an organ-specific manner is crucial to fully understand hormone action and plant responses to water deficit. aba also significantly affected the expression of several transcription factors. the greatest number of significantly differentially expressed genes was observed in the roots followed by cell cultures, leaves, berries, and shoot tips, respectively. aba affected mrna abundance of genes associated with aba degradation, conjugation, and the aba signaling pathway.
 abscisic acid regulates various developmental processes and stress responses over both short and longer time frames. to elucidate the transcriptional regulation of early responses of grapevine responding to aba, different organs of grape were treated with 10 μm --aba for 2 h. nimblegen whole genome microarrays of vitis vinifera were used to determine the effects of aba on organ-specific mrna expression patterns.  transcriptomic analysis revealed genes whose transcript abundances varied significantly in a specific organ in response to aba treatment. no single gene exhibited the same changes in transcript abundance across all organs in response to aba. the biochemical pathways affected by aba were identified using the cytoscape program with the bingo plug-in software. the results indicated that these genes were involved in several biological processes such as flavonoid metabolism, response to reactive oxygen species, response to light, and response to temperature stimulus. aba affected ion and water transporters, particularly in the root. the protein amino acid phosphorylation process was significantly overrepresented in shoot tips and roots treated with aba. aba affected mrna abundance of genes associated with aba degradation, conjugation, and the aba signaling pathway. aba also significantly affected the expression of several transcription factors. the greatest number of significantly differentially expressed genes was observed in the roots followed by cell cultures, leaves, berries, and shoot tips, respectively. each organ had a unique set of gene responses to aba.  this study examined the short-term effects of aba on different organs of grapevine. the responses of each organ were unique indicating that aba signaling varies with the organ. understanding the aba responses in an organ-specific manner is crucial to fully understand hormone action and plant responses to water deficit. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords abscisic acid signalingorgan-specificitytranscriptomicsvitis vinifera lnevada agriculture experiment stationnev00345cramer grant r. issue-copyright-statement© the author 2016 