Distinct gibberellin functions during and after grapevine bud dormancy release.

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It has been hypothesized that (i) abscisic acid (ABA) represses bud-meristem activity; (ii) perturbation of respiration induces an interplay between ethylene and ABA metabolism, which leads to removal of repression; and (iii) gibberellin (GA)-mediated growth is resumed. The first two hypothesis have been formally supported. The current study examines the third hypothesis regarding the potential involvement of GA in dormancy release. We found that during natural dormancy induction, levels of VvGA3ox, VvGA20ox, and VvGASA2 transcripts and of GA1 were decreased. However, during dormancy release, expression of these genes was enhanced, accompanied by decreased expression of the bud-expressed GA-deactivating VvGA2ox. Despite indications for its positive role during natural dormancy release, GA application had inhibitory effects on bud break. Hydrogen cyanamide up-regulated VvGA2ox and down-regulated VvGA3ox and VvGA20ox expression, reduced GA1 levels, and partially rescued the negative effect of GA. GA had an inhibitory effect only when applied simultaneously with bud-forcing initiation. Given these results, we hypothesize that during initial activation of the dormant bud meristem, the level of GA must be restricted, but after meristem activation an increase in its level serves to enhance primordia regrowth.

Abscisic acid (ABA) regulates grape bud dormancy, and dormancy release stimuli may act through modification of ABA metabolism.

In warm-winter regions, induction of dormancy release by hydrogen cyanamide (HC) is mandatory for commercial table grape production. Induction of respiratory stress by HC leads to dormancy release via an uncharacterized biochemical cascade that could reveal the mechanism underlying this phenomenon. Previous studies proposed a central role for abscisic acid (ABA) in the repression of bud meristem activity, and suggested its removal as a critical step in the HC-induced cascade. In the current study, support for these assumptions was sought. The data show that ABA indeed inhibits dormancy release in grape (Vitis vinifera) buds and attenuates the advancing effect of HC. However, HC-dependent recovery was detected, and was affected by dormancy status. HC reduced VvXERICO and VvNCED transcript levels and induced levels of VvABA8'OH homologues. Regulation of these central players in ABA metabolism correlated with decreased ABA and increased ABA catabolite levels in HC-treated buds. Interestingly, an inhibitor of ethylene signalling attenuated these effects of HC on ABA metabolism. HC also modulated the expression of ABA signalling regulators, in a manner that supports a decreased ABA level and response. Taken together, the data support HC-induced removal of ABA-mediated repression via regulation of ABA metabolism and signalling. Expression profiling during the natural dormancy cycle revealed that at maximal dormancy, the HC-regulated VvNCED1 transcript level starts to drop. In parallel, levels of VvA8H-CYP707A4 transcript and ABA catabolites increase sharply. This may provide initial support for the involvement of ABA metabolism also in the execution of natural dormancy.

Functional characterization and developmental expression profiling of gibberellin signalling components in Vitis vinifera.

Gibberellins (GAs) regulate numerous developmental processes in grapevine (Vitis vinifera) such as rachis elongation, fruit set, and fruitlet abscission. The ability of GA to promote berry enlargement has led to its indispensable use in the sternospermocarpic ('seedless') table grape industry worldwide. However, apart from VvGAI1 (VvDELLA1), which regulates internode elongation and fruitfulness, but not berry size of seeded cultivars, little was known about GA signalling in grapevine. We have identified and characterized two additional DELLAs (VvDELLA2 and VvDELLA3), two GA receptors (VvGID1a and VvGID1b), and two GA-specific F-box proteins (VvSLY1a and VvSLY1b), in cv. Thompson seedless. With the exception of VvDELLA3-VvGID1b, all VvDELLAs interacted with the VvGID1s in a GA-dependent manner in yeast two-hybrid assays. Additionally, expression of these grape genes in corresponding Arabidopsis mutants confirmed their functions in planta. Spatiotemporal analysis of VvDELLAs showed that both VvDELLA1 and VvDELLA2 are abundant in most tissues, except in developing fruit where VvDELLA2 is uniquely expressed at high levels, suggesting a key role in fruit development. Our results further suggest that differential organ responses to exogenous GA depend on the levels of VvDELLA proteins and endogenous bioactive GAs. Understanding this interaction will allow better manipulation of GA signalling in grapevine.

Cytokinin-induced VvTFL1A expression may be involved in the control of grapevine fruitfulness.

Grapevine bud fruitfulness is determined by the differentiation of uncommitted meristem (UCM) into either tendril or inflorescence. Since tendril and inflorescence differentiation have long been considered sequential steps in inflorescence development, factors that control the progression of floral meristem development may regulate the final outcome of UCM differentiation, and thus affect fruitfulness. A comparison of the expression profiles of the master regulators of floral meristem identity (FMI) during development of fruitful and non-fruitful buds along the same cane allowed associating the expression of a homolog of terminal flower 1 (TFL1, a negative regulator of FMI) to fruitful buds, and the expression of positive FMI regulators to non-fruitful buds. Combined with (a) cytokinin-induced upregulation of VvTFL1A expression in cultured tendrils, which accompanied cytokinin-derived tendril transformation into branched, inflorescence-like structures, (b) positive regulation of VvTFL1A expression by cytokinin, which was demonstrated in transgenic embryonic culture expressing GUS reporter under the control of VvTFL1A promoter, and (c) a significantly higher level of active cytokinins in fruitful positions, the data may support the assumption of cytokinin-regulated VvTFL1A activity's involvement in the control of inflorescence development. Such activity may delay acquisition of FMI and allow an extended branching period for the UCM, resulting in the differentiation of inflorescence primordia.

Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement.

A grape-bud-oriented genomic platform was produced for a large-scale comparative analysis of bud responses to two stimuli of grape-bud dormancy release, hydrogen cyanamide (HC) and heat shock (HS). The results suggested considerable similarity in bud response to the stimuli, both in the repertoire of responding genes and in the temporary nature of the transcriptome reprogramming. Nevertheless, the bud response to HC was delayed, more condensed and stronger, as reflected by a higher number of regulated genes and a higher intensity of regulation compared to the response to HS. Integrating the changes occurring in response to both stimuli suggested perturbation of mitochondrial activity, development of oxidative stress and establishment of a situation that resembles hypoxia, which coincides with induction of glycolysis and fermentation, as well as changes in the interplay between ABA and ethylene metabolism. The latter is known to induce various growth responses in submerged plants and the possibility of a similar mechanism operating in the bud meristem during dormancy release is raised. The new link suggested between sub lethal stress, mitochondrial activity, hypoxic conditions, ethylene metabolism and cell enlargement during bud dormancy release may be instrumental in understanding the dormancy-release mechanism. Temporary increase of acetaldehyde, ethanol and ethylene in response to dormancy release stimuli demonstrated the predictive power of the working model, and its relevance to dormancy release was demonstrated by enhancement of bud break by exogenous ethylene and its inhibition by an ethylene signal inhibitor.

Abnormal Endogenous Repression of GA Signaling in a Seedless Table Grape Cultivar with High Berry Growth Response to GA Application.

Gibberellin (GA) application is routinely used in the table grape industry to increase berry size and cluster length. Although grapevine cultivars show a wide range of growth responsiveness to GA3 application, the reasons for these differences is unclear. To shed light on this issue, two commercial grapevine cultivars with contrasting berry response to GA were selected for comparative analysis, in which we tested if the differences in response: (1) is organ-specific or cultivar-related; (2) will be reflected in qualitative/quantitative differences in transcripts/proteins of central components of GA metabolism and signaling and levels of GA metabolites. Our results showed that in addition to the high response of its berries to GA, internodes and rachis of cv. Black finger (BF) presented a greater growth response compared to that of cv. Spring blush (SB). In agreement, the results exposed significant quantitative differences in GA signaling components in several organs of both cultivars. Exceptionally higher level of all three functional VvDELLA proteins was recorded in young BF organs, accompanied by elevated VvGID1 expression and lower VvSLY1b transcripts. Absence of seed traces, low endogenous GA quantities and lower expression of VvGA20ox4 and VvGA3ox3 were also recorded in berries of BF. Our results raise the hypothesis that, in young organs of BF, low expression of VvSLY1b may be responsible for the massive accumulation of VvDELLA proteins, which then leads to elevated VvGID1 levels. This integrated analysis suggests causal relationship between endogenous mechanisms leading to anomalous GA signaling repression in BF, manifested by high quantities of VvDELLA proteins, and greater growth response to GA application.

Similar mechanisms might be triggered by alternative external stimuli that induce dormancy release in grape buds.

The detection of genes having similar expression profiles following the application of different stimuli that trigger bud break may constitute potent tools for the identification of pathways with a central role in dormancy release. We compared the effects of heat shock (HS) and hydrogen cyanamide (HC) and demonstrated that HS leads to earlier and higher bud-break levels. Changes in transcript levels of catalase, alcohol dehydrogenase and pyruvate decarboxylase were induced following both treatments. However, timing and extent of changes in transcript level differed. Changes occurred earlier in HS-treated buds and were more intense in HC-treated buds. The changes in transcript levels after both treatments were temporary. The rapid and short-lasting changes in gene expression following HS treatment correlated with the faster and higher level of bud-break that this treatment exerted. This correlation may propose that the reported molecular events are mechanistically involved in dormancy release. To test the hypothesis that temporary oxidative stress is part of the mechanism inducing dormancy release, we analyzed the effect of HS and HC treatments on the expression of ascorbate peroxidase, glutathione reductase, thioredoxin h, glutathione S-transferase and sucrose synthase genes and found that they were induced by both treatments in a similar pattern. Taken together, these findings propose that similar cellular processes might be triggered by different stimuli that lead to dormancy release, and are consistent with the hypothesis that temporary oxidative stress and respiratory stress might be part of the mechanism that leads to bud break.

Involvement of calcium signalling in dormancy release of grape buds.

Artificial induction of grape bud dormancy release by hydrogen cyanamide (HC) serves as a reliable model system to explore the events occurring shortly after the induction of dormancy release. Recently, a group of genes with remarkable differences in expression level between HC-treated and control buds was identified. The identification of several calcium signalling-related genes within that group raised the hypothesis of the involvement of Ca(2+) signalling in grape bud dormancy release. Therefore, the effects of HC treatment on the expression profiles of several calcium sensors, the effect of the plasma membrane calcium channel blocker LaCl(3) and the calcium chelator EGTA on HC-induced and chilling-induced bud-break, and the effect of HC application on calcium-dependent protein phosphorylation activities in the bud tissue were studied. Here the HC-induced expression of Ca(2+)-ATPase is described, indicating that this treatment might evoke an increase in [Ca(2+)]cyt. Similar induction was confirmed for calmodulin, calmodulin-binding protein, and calcium-dependent protein kinase (CDPK). Both LaCl(3) and EGTA blocked the inducing effect of HC on bud-break, and their inhibitory effects were removed by supplying exogenous Ca(2+). Calcium-dependent histone phosphorylation was up to 70% higher in HC-treated buds. Endogenous protein phosphorylation assays detected four proteins exhibiting increased phosphorylation following HC treatment, of which two were phosphorylated in a calcium-dependent manner. One of these, a 47 kDa protein, presented strong and Ca(2+)-dependent phosphorylation only in HC-treated buds. The potential role of CDPK in the phosphorylation of this protein was supported by an immunoprecipitation assay. The data suggest, for the first time, that calcium signalling is involved in the mechanism of bud dormancy release.