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.

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Alterations in gene expression during early stages of dormancy release in grapevine buds were analyzed to facilitate the identification of gene products that may mediate the signal transduction of a dormancy-release signal, or derepression of meristematic activity. In the present report we describe the identification of GDBRPK, a transcript for an SNF-like protein kinase that is up-regulated upon chemical induction of dormancy release by hydrogen cyanamide (HC). Since SNF and SNF-like protein kinases are known as sensors of stress signals, we hypothesize that GDBRPK may be involved in the perception of a stress signal induced by HC. We also describe a simultaneous and remarkable induction of both PDC and ADH transcripts that was observed shortly after HC application, and was of a transient nature. These data may imply that HC application leads to a transient respiratory stress, which likely results in a temporary increase in the AMP/ATP ratio. Since AMP is known as a stress signal that is sensed by SNF-like kinases, we suggest that the SNF-like GDBRPK could serve as the sensor of this signal.

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.