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.

Hydrogen cyanamide (HC) is widely used to induce the breakage of endodormancy (ED) in grape and other deciduous fruit crop, though its mechanism of action is poorly understood. Applications of HC to grapevine buds produce oxidative stress and transient respiratory disturbances which are related to the breakage of ED. Moreover, since the expression and activity of catalase (Cat) is inhibited by HC, enhancements in the levels of H 2 O 2 have also been associated to the breakage of ED in grapevine buds. Here, we reported that increases in H 2 O 2 level in HC-treated grapevine buds are due to the inhibition of Cat activity and enhancement of the respiratory activity of buds. In addition, exogenous applications of H 2 O 2 partially reproduced the inducing effect of HC in the breakage of ED, thus providing further support for the hypothesis that H 2 O 2 mediates the effects of HC. On the other hand, Mit isolated from both control and HC-treated buds respired equally well when NADH was used as a respiratory substrate, but when succinate was used as an electron donor Mit respiration was non-detected, suggesting that the stimulatory effect of HC on bud respiration is related to metabolic alterations leading to increase of the concentration of NADH rather than to changes in Mit functionality.

• Background and Aims 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 coldhardiness 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. • Methods 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. • Key Results 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. • Conclusions 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.

Artículo de publicación ISIEndodormancy (ED) and cold hardiness (CH) are two strategies utilized by grapevine (Vitis vinifera L.) buds to survive unfavorable winter conditions. Each phenomenon is triggered by different environmental cues—ED by short-day (SD) photoperiod and cold hardiness (CH) by low temperatures. In grapevine buds, CH occurs mainly via the supercooling of intracellular water, a phenomenon associated with the low temperature exotherm (LTE). The seasonal dynamics of ED and CH were studied on grapevines buds by determining the BR50 (time required to reach 50 % of bud break under forced conditions) and the LTE, which measure the depth of ED and the level of CH, respectively. Overlapping BR50 and LTE curves revealed that CH began to develop in late April, when buds were fully endodormant and daily mean temperatures had started to drop below 14 C, suggesting that ED is a prerequisite for the acquisition of full CH. Increase in starch content and thickening of the cell wall (CW) of meristematic cells which occurs in dormant buds could be involved in structural and metabolic changes that favor CH subsequent acquisition. Interestingly, the thickening of the CW and the synthesis of starch which are associated with ED were induced by a SD-photoperiod, while the hydrolysis of starch, the accumulation of soluble sugars, and the upregulation of dehydrin genes, which are associated with CH, were induced by low temperatures. Overall, the results indicate that structural, metabolic, and transcriptional changes that occur during ED in grapevine buds are necessary for the further development of CH.FONDECYT Project 114031

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs.Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H 2 O 2 . The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H 2 O 2 in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plantextracts become unnecessary. To exemplified the high performance of the method, measurements of H 2 O 2 were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H 2 O 2 levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H 2 O 2 scavenging enzyme in leaves of grapevine.