This family has been delimited into four ‘‘Classes’’, and 4 of the 17 members of the Class I AtHBs have been shown to be involved in ABA responses across diverse tissues . In addition, the expression of three AtHB6, 7, and 12, have been show to be up-regulated by ABA . An examination of the grape genome identified 10 orthologs that cluster with the Class 1 HBs . The PP2C protein phosphatases represent another large gene family being made up of 80 genes in Arabidopsis . Within this family group a cluster of genes containing many genes that have been characterized to function in the ABA-signaling pathway; most notably, the ABA-insensitive mutants, abi1 and abi2 . In addition, AtPP2C-A, AtHAB1, AtHAB2, and AtAHG1, also members of Group A, function in ABA signaling across diverse tissues . In Arabidopsis, all the members of this group are induced by ABA treatment. In grape, nine VvPP2Cs clustered in group A . The WRKY transcription factors are a large gene family, consisting of approximately 70 members making up three groups in Arabidopsis . The barley HvSUSIBA2, and AtWRKY2, and AtWRKY34, all fall within the same group consisting of 14 members in Arabidopsis . HvSUSIBA2 modulates the expression of a barley isoamylase gene during seed development via the binding of SURE elements . In addition, Sun et al. demonstrated that expression of HvSUSIBA2 is induced by exogenous sugar and its native expression profile during seed development correlates strongly with endogenous sucrose levels.
Hammargren et al. found that the sugar responsiveness of a nucleoside diphosphate kinase is altered in Atwrky2 and Atwrky34 mutant backgrounds. Grape contained 13 putative orthologs that fall within this group .Expression profiling was carried out in berry skins of fieldgrown Cabernet Sauvignon in order to identify those orthologs expressed during ripening. In addition, black plastic planting pots expression profiles under both control- and deficit-irrigated conditions were compared in order to identify orthologs whose expression pattern reflected the advancement of ripening under ED. Water deficits were applied continuously from fruit set until to the onset of ripening, resulting in an average difference in midday leaf water potential of 0.36 MPa before the onset of ripening and no difference during ripening . Of the 67 orthologous genes identified, 38 were expressed in grape berries. A summary of the expression profiles of all the genes examined in this study can be found in Figs. 4 and 5 while more detailed expression data is contained in Suppl. File 1. The majority of these genes were differentially regulated during berry development, with 26 exhibiting statistically significant changes with time in control and/or ED . There were few statistically significant differences in the magnitude of expression between control and ED . Six genes exhibited statistically significant differences between control and ED. Four of these instances, VvHB8, VvSnRK5, VvPP2C-3, and VvPP2C-7, all exhibit elevated levels of expression in ED at or just prior to the onset of ripening . Eight of the VvWRKYs selected for analysis were expressed in ripening grape. VvWRKY3, 5, and 6, were all differentially expressed during ripening and exhibited similar patterns of expression . They were up-regulated ranging from 4- to 16-fold at the onset of ripening. There were no significant differences in the expression of VvWRKY1, 2, 16, 18, and 19 across development or during water deficit . Of the 10 Class I, VvHB orthologs only four are expressed in fruit during ripening . Both VvHB4 and 8 were strongly up-regulated at the onset of ripening, exhibiting increases of [16-fold. VvHB8 is up-regulated much earlier under water deficit and high levels persist until late in ripening. VvHB4 is down-regulated early in development under ED. VvHB2 expression in controls generally decreased during development with a small up-regulation at the onset of ripening, although these changes were not statistically significant.
Under ED, however, this pattern of expression is more pronounced with a sharp eight fold decrease in expression at 81 DAA . VvHB3 was constitutively expressed during ripening with no significant changes over time or under ED. Six of the VvPP2Cs were expressed in grape berries. VvPP2C-3, 6, 7, and 9 were all differentially expressed during ripening, while VvPP2C-1 and 5 expression did not change significantly . VvPP2C-3, 6, 7, and 9 were all up-regulated strongly at the onset of ripening increasing as much as 16-fold. VvPP2C-3 and VvPP2C-7 expression were clearly induced earlier and to higher levels in ED. Both exhibited statistically significant two to fourfold greater levels of expression in green berries at the onset of ripening.In order to more directly test the effects of sugar and ABA on the onset of ripening, immature Cabernet Sauvignon berries were harvested from the field at 61 DAA and cultured in the presence of various combinations of sucrose and ABA until 84 DAA, a period of 23 days. The onset of ripening in the clusters from which these berries were collected in the field occurred at approximately 73 DAA, therefore the cultured berries were collected approximately 12 days prior to the onset of ripening. Ripening phenomena were induced in berry culture when treated with sucrose and ABA as evidenced by changes in color, softening, and gene expression. Berries treated with 10% sucrose and various ABA concentrations changed color while those treated with 2 or 10% sucrose alone remained green . 200 lM ABA and 2% sucrose ? 200 lM ABA treatments were included in our analyses but yielded no results because of a phenomenon where the berries exploded reproducibly . On average, cultured berries gained weight over the culturing period . Sucrose treatments of 2 and 10% showed the greatest weight gains corresponding to gains of 21 and 8%, respectively. Berries cultured in the presence of 10% sucrose with the addition of various ABA concentrations showed average weight gains of approximately 4%. Previous studies have found that a precipitous drop in grape berry elasticity occurs just prior to the onset of ripening in grape . In our current culture experiments, berry elasticity remained equal to that at T0 in the 2 and 10% sucrose treatments, while sharply decreasing with ABA treatment . Finally, the grape Myb transcription factor VvMybA1 was utilized as a molecular marker for the onset of ripening. VvMybA1 is responsible for activating anthocyanin biosynthesis and has a distinct pattern of expression; being absent prior to the onset of ripening at which time it is strongly up-regulated . In berry skins, VvMybA1 expression was completely absent from the 2 and 10% sucrose treatments and was strongly up-regulated in the 10% sucrose ? ABA treatments as expected . We hypothesized that orthologs of gene families regulated by sugar and ABA, whose expression was strongly up-regulated at the onset of ripening and advanced under ED, would be regulated similarly by sugar and/or ABA in cultured berries. To test this, changes in the expression of VvHB4, VvHB8, VvPP2C-3, and VvPP2C-6 were investigated in skins of cultured berries. In the field, all genes were strongly up-regulated at the onset of ripening and advanced under ED and in berry culture, expression was strongly induced in the presence of 10% sucrose ? ABA when compared with treatments of 2 and 10% sucrose alone . Among those genes analyzed, the magnitude of induction in the field versus in berry culture was variable. For example, when data from Fig. 7 was expressed as fold change both VvHB4 and VvPP2C-3 were induced 10-fold from 57 to 74 DAA in the field compared to 6- and 40-fold in culture, respectively.The transcriptional data in this study demonstrate that numerous sugar and ABA-signaling orthologs are expressed during ripening in grape, black plastic pots for plants and identify novel candidates in the control of non-climacteric fruit ripening. Several genes exhibited patterns of expression correlating with sugar and ABA accumulation at the onset of ripening in field-grown fruit. Changes in color, softening, and gene expression analogous to the onset of ripening in the field were induced in berry culture when treated with sucrose and ABA, demonstrating their role in controlling the onset of ripening. This study shows that many orthologous sugar and ABA-signaling components are regulated in fleshy fruit similar to their regulation originally characterized in model systems across diverse processes.These genes are easily delimited through nesting the currently available grape sequences comprising a gene family within their corresponding family in Arabidopsis. However, the current grape genome assembly, and its annotated proteome, certainly does not identify all the genes present in the grape genome so our analyses most likely failed to identify some orthologs. In the current study, we chose to use QPCR in our expression analyses instead of the current grape microarray for several reasons. First, the majority of the genes analyzed here are not present on the current Affymetrix Vitis vinifera gene chip since the chip was derived from ESTsand the present study utilizes the complete genome. Second, microarrays suffer from several limitations one of which is that they are particularly insensitive in quantifying low abundant transcripts .
Transcription factors are a largely low abundance transcripts, and a study in Arabidopsis comparing QPCR and Affymetrix microarray approaches found that the microarray could detect \55% of 1,400 transcription factors tested, compared to [85% via QPCR . Furthermore, cross-hybridization is common on microarrays, which is especially problematic when considering conserved gene families like those examined here. However, those genes represented on the chip were identified and expression profiles were compared with those determined via microarray analyses in two recent studies. Notably, Koyama et al. demonstrated via microarray that a VvHB transcription factor , identical to VvHB8 in this study, is also up-regulated at the onset of ripening and in response to exogenous sugar and ABA. For several other genes, expression profiles in the current study are nearly identical to those found by Deluc et al. . Many grape orthologs of genes shown previously to be modulated by sugar and/or ABA in model systems exhibited expression patterns during ripening, and in response to water deficit, consistent with their modulation by sugar and/or ABA in grape. More specifically, these genes are induced at the onset of ripening and induced earlier, and to higher levels, under water deficit. This characteristic pattern of expression is shared with many flavonoid pathway genes , and these correlations in expression throughout ripening suggest common regulatory mechanisms. Experiments in berry culture demonstrated that several sugar and ABA-signaling orthologs, and VvMybA1, a transcriptional activator of anthocyanin biosynthesis, are up-regulated by exogenous ABA in the presence of high sucrose. These data suggest that sugar and ABA play a predominant role in regulating the expression of a suite of genes at the onset of ripening, including those responsible for anthocyanin biosynthesis and components of their own signaling pathways. These results have interesting evolutionary implications demonstrating that some orthologs are consistently regulated by sugar and ABA across diverse developmental processes. Land plants, in general, have undergone abundant gene duplication through their evolutionary history , although there is debate on the exact nature and timing of these events across angiosperms and in grape specifically . Gene duplication is considered cornerstone to providing the raw material for evolution. A duplicate gene, now no longer essential, can undergo changes in its structure and/or regulation allowing for it to take up a novel role. The results of this study show that some of the Group A PP2Cs and Class I HBs have maintained their ABA responsiveness during fruit ripening in grape. At least with regard to ABA responsiveness, the nature of regulation has been conserved, but co-opted into a completely different developmental context. This may provide for the discovery of novel cis-regulatory elements through promoter sequence comparisons across species. In grape, advances in elucidating molecular mechanisms suffer from a lack of transgenic and related technologies on which most reverse genetic studies are based. This study demonstrates that model systems can provide fundamental knowledge and insight into function of other agronomically important plant species even in extremely divergent developmental processes. Equally, this suggests that Arabidopsis, with its wealth of tools available for facilitating reverse genetic studies, may provide a valuable system to characterize genes of interest from grape or other crop species. Already there are several examples of the successful characterization of grape genes in Arabidopsis and tobacco . This could prove especially useful considering the limitations of functional genetic analyses in perennial fruit crops where long propagation times areprohibitive. Future studies should include attempts to complement ABA and sugar signaling mutants in Arabidopsis and other model species with their orthologs implicated in ripening.