Paradoxically, in eukaryotic cells, hydrogen peroxide (H₂O₂) accumulates in response to oxygen deprivation (hypoxia). The source of H₂O₂ under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H₂O₂ is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H₂O₂ in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H₂O₂ levels induced by KCN, suggesting that, under respiratory stress, H₂O₂ is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H₂O₂ induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H₂O₂, and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H₂O₂ production is stimulated by respiratory stress in grapevine buds, that H₂O₂ and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H₂O₂ levels by up-regulating the expression of VvCAT2.

It has been reported that low temperatures (LTs) and the plant hormone abscisic acid (ABA) induce the expression of CBF/DREB1 transcription factors in vegetative tissues and seedlings of Vitis vinifera and Vitis riparia and that foliar applications of ABA to V. vinifera increase the freezing tolerance or cold-hardiness of dormant buds. However, the combined effect of ABA and LTs on the expression of CBF/DREB1 transcription factors and on the acquisition of freezing tolerance in dormant grapevine buds has not been investigated. The objective of this study was to analyse the combined effect of ABA and LT treatments on the expression of CBF/DREB transcription factors and the acquisition of freezing tolerance. In vitro experiments with single-bud cuttings of grapevines were used to analyse the effect of ABA, ABA + LT and LT on the expression of CBF/DREB transcription factors, dehydrin and antioxidant genes, the acquisition of freezing tolerance and the endogenous content of ABA. Gene expression analysis was performed by quantitative real-time PCR and freezing tolerance was determined by measuring the low-temperature exotherm by differential thermal analysis. ABA levels were determined by gas chromatography coupled to an electron capture detector. The LT treatment and exogenous application of ABA to grapevine dormant buds increased the expression of the CBF/DREB1 transcription factors VvCBF2, VvCBF3, VvCBF4 and VvCBF6. The joint application of LT and ABA produced a huge increase in the expression of these transcription factors, which was greater than the sum of the increases produced by them individually, which indicates the existence of a synergistic effect between ABA and LT on the activation of these transcription factors. This synergic effect was also observed on the increase in bud cold-hardiness and on the expression of antioxidant and dehydrin genes. The synergy between ABA and LT on the expression of CBF/DREB1 transcription factors VvCBF2, VvCBF3, VvCBF4 and VvCBF6 plays a key role in cold acclimatization of grapevine buds. The results highlight the importance of the combination of stimuli in the improvement of genetic and physiological responses and help us to understand the adaption of plants to complex environments.

Recently, the plant hormone abscisic acid has been involved as key player in the regulation of endodormancy in grapevine buds (Vitis vinifera L). In this study, we show that in the vine, the expression of genes related to the biosynthesis of ABA (VvNCED1; VvNCED2) and the content of ABA are significantly higher at the latent bud than at the shoot-apex, while the expression of ABA catabolic gene (VvA8H3) showed no significant differences between both organs. A negative correlation between the content of ABA and transcript levels of the cell cycle genes (CCG) was found in both tissues. This result, suggested that ABA may regulate negatively the expression of the CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of the CCG was analyzed in two meristematic tissues of the vine, somatic embryos and shoot-apex. The results indicated that cell-cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding for cyclin-dependent kinases (VvCDKB1, VvCDKB2), for genes encoding cyclins type A (VvCYCA1, VvCYCA2, VvCYCA3), B (VvCYCB) and D (VvCYCD3.2a), and up-regulated the expression of VvICK5, a gene encoding for an inhibitor of CDKs. During the endodormancy the content of ABA increased and the expression of the CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC) reduced the content of ABA and up-regulated the expression of the CCG. Taken together, these results suggest that ABA by repressing the transcription of the CCG may modulate the development of endodormancy in grapevine buds.