Changes in the Activity of Catalase (EC 1.11.1.6) in Relation to the Dormancy of Grapevine (Vitis vinifera L.) Buds

Catalase activity in grapevine (Vitis vinifera L.) buds cv. `Perlette.' increased to a maximum in October and thereafter decreased within 3 months to less than half its maximal rate. The decrease in catalase activity coincided with the decline in temperature during winter. The rate of sprouting of buds forced at 23°C was negatively related to the activity of catalase. Artificial chilling of grapevine canes at 5°C resulted in a 25% decrease of catalase activity in the buds after 3 days and 31% after 17 days. The activity of catalase increased to the control level only 96 hours after removing canes from 5°C to room temperature. Efficient buddormancy breaking agents, such as thiourea and cyanamide decreased catalase activity to 64 and 50% of the controls respectively, while the activity of peroxidase remained the same under those conditions. A less efficient dormancy breaking agent dinitro-ortho-cresol, did not decrease catalase activity.     

Dormant Buds and Adventitious Root Formation by Vitis and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Dormant Buds and Adventitious Root Formation by <Emphasis Type="Italic">Vitis</Emphasis> and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

Expression analysis of phytochromes A, B and floral integrator genes during the entry and exit of grapevine-buds from endodormancy

A common molecular regulatory pathway that involves PHYA, PHYB and floral integrator genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) has been suggested to participate in the regulation of photoperiod dependent processes such as flowering and dormancy. In grapevines (Vitis vinifera L.), decreasing photoperiod and low temperatures trigger the transition of buds into endodormancy (ED), a process that is accompanied by drastic changes in gene expression of VvPHYA and B in leaves. To analyse the relationship of VvPHYA, VvPHYB, and grape homologues of Arabidopsis floral integrator genes VvCO, VvFT, VvMADS8, with ED, a comparative expression analysis of these genes was performed in grapevine-leaves and buds before, during and after the transition of buds into ED. The expression of all the above genes in the bud-tissue, and the fact that photoperiod regulates differently the expression of VvPHYA and B in buds than in leaves, suggests that the bud might be an autonomous or semi-autonomous organ in perceiving and transducing the photoperiod signal. On the other hand, the coordinated down-regulation of VvFT in leaves and buds during the transition of buds into ED, and its subsequent up-regulation following the application of dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, suggests that VvFT could play a key role in stimulating bud-growth by repressing their entry into ED.     

Is GABA-shunt functional in endodormant grapevine buds under respiratory stress?

It has been suggested that a respiratory stress is part of the mechanism through which the dormancy-breaking compounds, hydrogen cyanamide (HC) and sodium azide, induce the release of buds from the endodormancy (ED) in grapevines. The accumulation of metabolites like succinate, alanine (Ala) and γ-amino butyric acid (GABA), together with the activation of the GABA-shunt pathway, is a general feature of plants in response to oxygen deprivation and to respiratory stress. Unexpectedly, in a previous study, we found that GABA applied exogenously to grapevine buds, down-regulated the expression of most genes encoding for antioxidant enzymes, suggesting that its accumulation under respiratory stress conditions could be deleterious for the bud. In order to analyze whether GABA accumulates under respiratory stress conditions in grapevine buds, we analysed in this study, the effect of hypoxia, the respiration inhibitor KCN and the dormancy breaker compound HC, on the level of GABA, and on the expression levels of the GABA-shunt genes (VvGAD, VvGABA-T, VvSSADH). Additionally, genes from the Ala fermentative pathway (VvAlaAT, VvAspAT) were also analysed. The results revealed that although the three treatments mentioned above, up-regulated the expression of VvGAD1, the content of GABA remained constant, while Ala content increased. The lack of GABA accumulation under respiratory stress is an important physiological fact in grapevine buds, since it avoids the down-regulation of antioxidant genes, and promotes the incorporation of succinate into the TCA cycle, a fact that would be important in the release of buds from the ED.     

Genome-wide identification and analysis of the U-box family of E3 ligases in grapevine

E3 ubiquitin ligases play essential roles in determining the specificity of ubiquitination and subsequent protein degradation. The plant U-box (PUB) family of E3 ligases has been implicated in biotic and abiotic stress signaling and developmental events in various species. A comprehensive bioinformatics analysis identified 56 PUB genes in the grapevine (Vitis vinifera L.) genome. Based on conserved motifs, the VvPUB family was classified into seven subclasses. Expansion of this family was driven by chromosomal, segmental, and tandem duplications. Microarray expression profiling revealed that three PUB genes were preferentially expressed in pollen, four in leaf, and five in root. Moreover, a large number of PUB genes were differentially expressed under abiotic stresses and eight PUB genes likely participated in defense against powdery mildew. The microarray expression data were verified by RT-qPCR. Genome-wide identification of VvPUB genes and examination of their expression will give insights into the functions of U-box genes in grapevine.     

Secreted proteins produced by fungi associated with <Emphasis Type="Italic">Botryosphaeria</Emphasis> dieback trigger distinct defense responses in <Emphasis Type="Italic">Vitis vinifera</Emphasis> and <Emphasis Type="Italic">Vitis rupestris</Emphasis> cells

Grapevine trunk diseases (Eutypa dieback, esca and Botryosphaeria dieback) are caused by a complex of xylem-inhabiting fungi, which severely reduce yields in vineyards. Botryosphaeria dieback is associated with Botryosphaeriaceae. In order to develop effective strategies against Botryosphaeria dieback, we investigated the molecular basis of grapevine interactions with a virulent species, Neofusicoccum parvum, and a weak pathogen, Diplodia seriata. We investigated defenses induced by purified secreted fungal proteins within suspension cells of Vitis (Vitis rupestris and Vitis vinifera cv. Gewurztraminer) with putative different susceptibility to Botryosphaeria dieback. Our results show that Vitis cells are able to detect secreted proteins produced by Botryosphaeriaceae, resulting in a rapid alkalinization of the extracellular medium and the production of reactive oxygen species. Concerning early defense responses, N. parvum proteins induced a more intense response compared to D. seriata. Early and late defense responses, i.e., extracellular medium alkalinization, cell death, and expression of PR defense genes were stronger in V. rupestris compared to V. vinifera, except for stilbene production. Secreted Botryosphaeriaceae proteins triggered a high accumulation of δ-viniferin in V. vinifera suspension cells. Artificial inoculation assays on detached canes with N. parvum and D. seriata showed that the development of necrosis is reduced in V. rupestris compared to V. vinifera cv. Gewurztraminer. This may be related to a more efficient induction of defense responses in V. rupestris, although not sufficient to completely inhibit fungal colonization. Overall, our work shows a specific signature of defense responses depending on the grapevine genotype and the fungal species.     

Identification of pterostilbene as a phytoalexin from Vitis vinifera leaves

An inducible antifungal compound in grapevine leaves (Vitis vinifera L., cv Cabernet-Sauvignon) has been identified as trans-pterostilbene (3,5-dimethoxy-4′-hydroxy stilbene). It is only a minor component of the phytoalexin response of V. vinifera but its antifungal activity is relatively high by comparison with resveratrol and the viniferins, stress metabolites which have been identified previously in grapevine. Methods for the quantitative analysis of pterostilbene, resveratrol, ε- and α-viniferins by HPLC are described.