Characterization of Endogenous Levels of Brassinosteroids and Related Genes in Grapevines.

Agronomic breeding practices for grapevines (Vitis vinifera L.) include the application of growth regulators in the field. Brassinosteroids (BRs) are a family of sterol-derived plant hormones that regulate several physiological processes and responses to biotic and abiotic stress. In grapevine berries, the production of biologically active BRs, castasterone and 6-deoxocastasterone, has been reported. In this work, key BR genes were identified, and their expression profiles were determined in grapevine. Bioinformatic homology analyses of the Arabidopsis genome found 14 genes associated with biosynthetic, perception and signaling pathways, suggesting a partial conservation of these pathways between the two species. The tissue- and development-specific expression profiles of these genes were determined by qRT-PCR in nine different grapevine tissues. Using UHPLC-MS/MS, 10 different BR compounds were pinpointed and quantified in 20 different tissues, each presenting specific accumulation patterns. Although, in general, the expression profile of the biosynthesis pathway genes of BRs did not directly correlate with the accumulation of metabolites, this could reflect the complexity of the BR biosynthesis pathway and its regulation. The development of this work thus generates a contribution to our knowledge about the presence, and diversity of BRs in grapevines.

Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses.

Grapevine fruit development is a dynamic process that can be divided into three stages: formation (I), lag (II), and ripening (III), in which physiological and biochemical changes occur, leading to cell differentiation and accumulation of different solutes. These stages can be positively or negatively affected by multiple environmental factors. During the last decade, efforts have been made to understand berry development from a global perspective. Special attention has been paid to transcriptional and metabolic networks associated with the control of grape berry development, and how external factors affect the ripening process. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development. Several aspects will be considered, including seed development and the production of seedless fruits; veraison, at which anthocyanin accumulation begins in the berry skin of colored varieties; and hormonal regulation of berry development and signaling throughout ripening, focusing on the transcriptional regulation of hormone receptors, protein kinases, and genes related to secondary messenger sensing. Finally, berry responses to different environmental factors, including abiotic (temperature, water-related stress and UV-B radiation) and biotic (fungi and viruses) stresses, and how they can significantly modify both, development and composition of vine fruit, will be discussed. Until now, advances have been made due to the application of Omics tools at different molecular levels. However, the potential of these technologies should not be limited to the study of single-level questions; instead, data obtained by these platforms should be integrated to unravel the molecular aspects of grapevine development. Therefore, the current challenge is the generation of new tools that integrate large-scale data to assess new questions in this field, and to support agronomical practices.

Differences in respiration between dormant and non-dormant buds suggest the involvement of ABA in the development of endodormancy in grapevines.

Grapevine buds (Vitis vinifera L) enter endodormancy (ED) after perceiving the short-day (SD) photoperiod signal and undergo metabolic changes that allow them to survive the winter temperatures. In the present study, we observed an inverse relationship between the depth of ED and the respiration rate of grapevine buds. Moreover, the respiration of dormant and non-dormant buds differed in response to temperature and glucose, two stimuli that normally increase respiration in plant tissues. While respiration in non-dormant buds rose sharply in response to both stimuli, respiration in dormant buds was only slightly affected. This suggests that a metabolic inhibitor is present. Here, we propose that the plant hormone abscisic acid (ABA) could be this inhibitor. ABA inhibits respiration in non-dormant buds and represses the expression of respiratory genes, such as ALTERNATIVE NADH DEHYDROGENASE (VaND1, VvaND2), CYTOCHROME OXIDASE (VvCOX6) and CYTOCHROME C (VvCYTC), and induces the expression of VvSnRK1, a gene encoding a member of a highly conserved family of protein kinases that act as energy sensors and regulate gene expression in response to energy depletion. In addition to inducing ED the SD-photoperiod up-regulated the expression of VvNCED, a gene that encodes a key enzyme in ABA synthesis. Taken together, these results suggest that ABA through the mediation of VvSnRK1, could play a key role in the regulation of the metabolic changes accompanying the entry into ED of grapevine buds.

Relationship between endodormancy, FLOWERING LOCUS T and cell cycle genes in Vitis vinifera.

MAIN CONCLUSION: In grapevines, the increased expression of VvFT , genes involved in the photoperiodic control of seasonal growth ( VvAP1, VvAIL2 ) and cell cycle genes ( VvCDKA, VvCDKB2, VvCYCA1, VvCYCB, VvCYCD3.2 ) in the shoot apex relative to the latent bud, suggests a high mitotic activity of the apex which could prevent them to enter into endodormancy. Additionally, the up-regulation of these genes by the dormancy-breaking compound hydrogen cyanamide (H 2 CN 2 ) strongly suggests that VvFT plays a key role in regulating transcriptionally cell cycle genes. At the end of the growing season, short-day (SD) photoperiod induces the transition of latent grapevine buds (Vitis vinifera L) from paradormancy (PD) to endodormancy (ED), which allows them to survive the cold temperatures of winter. Meanwhile, the shoot apex gradually decreases its growth without entering into ED, and as a result of the fall of temperatures at the beginning of autumn, dies. To understand developmental differences and contrasting responses to environmental cues between both organs, the expression of cell cycle genes, and of genes involved in photoperiodic control of seasonal growth in trees, such as FLOWERING LOCUS T (FT), APETALA1 (AP1) and AINTEGUMENTA-like (AIL) was analyzed at the shoot apex and latent buds of vines during the transition from PD to ED. After shift to SD photoperiod, increased expression of cell cycle genes in the shoot apex suggests a high mitotic activity in this organ which could prevent them from entering into ED. Additionally, the increased expression of VvFT, VvAP1and VvAIL2 in the shoot apex, and the up-regulation of VvFT, VvAP1and cell cycle genes VvCDKA, VvCDKB2, VvCYCA.1, by the dormancy-breaking compound hydrogen cyanamide (H2CN2), strongly suggests that VvFT plays a key role in regulating transcriptionally cell cycle genes, giving thus, more support to the model for photoperiodic control of seasonal growth in trees. Furthermore, downregulation of VvFT by the SD photoperiod detected in leaves and buds of grapevines highlights the importance of VvFT in the induction of growth cessation and in ED development, probably by regulating the expression of cell cycle genes.

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene.

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) 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 H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) 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 H(2)O(2) 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, H(2)O(2), 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 H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.