Sucrose accumulation and endodormancy are synchronized events induced by the short-day photoperiod in grapevine buds.

At the end of the summer season, grapevine buds (Vitis vinifera L) grown in temperate climates enter a state of winter recess or endodormancy (ED), which is induced by the shortening of the photoperiod, and during this period, the buds accumulate sucrose. In this study, we investigated whether the shortening of the photoperiod regulates the accumulation of sucrose in the buds in the same way as it regulates its entry into the ED. Because sucrose accumulation is regulated by genes that control its transport and degradation, the effect of the SD photoperiod and the transition of buds from paradormancy (PD) to ED on the expression of sucrose transporter (VvSUTs) and invertase genes (VvINVs) was studied. To analyze the possible role of sucrose during ED development, its effect on bud swelling and sprouting was studied on dormant and nondormant buds under forced growth conditions. The results showed that the SD photoperiod upregulates the expression of the VvSUT genes and downregulates that of the VvINV genes in grapevine buds. Additionally, during the transition of buds from PD to ED, the sucrose content increased, the expression of the VvINV genes decreased, and the expression of the VvSUT genes did not change significantly. Sucrose delayed bud swelling and sprouting when applied to dormant buds but had no effect when applied to nondormant buds. Therefore, we concluded that ED development and sucrose accumulation were synchronized events induced by the SD photoperiod and that a sucrose peak marks the end of ED development in grapevine buds.

Hydrogen Peroxide Increases during Endodormancy and Decreases during Budbreak in Grapevine (Vitis vinifera L.) Buds.

Changes in the level of hydrogen peroxide (H2O2) is a good indicator to monitor fluctuations in cellular metabolism and in the stress responses. In this study, the changes in H2O2 content during bud endodormancy (ED) and budbreak were analysed in grapevine (Vitis vinifera L.). The results showed a gradual increase in the H2O2 content during the development of bud ED, which was mainly due to an increase in the activity of peroxidases (PODs). The maximum H2O2 content reached in the grapevine buds coincided with the maximum depth of bud ED. In contrast, during budbreak, the H2O2 content decreased. As the plant hormones cytokinin (CK) and auxin play an important role in budbreak and growth resumption in grapevine, the effect of exogenous applications of H2O2 on the expression of genes involved in CK and auxin metabolism was analysed. The results showed that H2O2 represses the expression of the CK biosynthesis genes VvIPT3a and VvLOG1 and induces the expression of the CK-inactivating gene VvCKX3, thus reducing potentially the CK content in the grapevine bud. On the other hand, H2O2 induced the expression of the auxin biosynthesis genes VvAMI1 and VvYUC3 and of the auxin transporter gene VvPIN3, thus increasing potentially the auxin content and auxin transport in grapevine buds. In general, the results suggest that H2O2 in grapevine buds is associated with the depth of ED and negatively regulates its budbreak.

VvDAM-SVPs genes are regulated by FLOWERING LOCUS T (VvFT) and not by ABA/low temperature-induced VvCBFs transcription factors in grapevine buds.

MAIN CONCLUSION: In deciduous fruit trees in which dormancy is induced by low temperatures, the expression of DORMACY-ASSOCIATED MADS-BOX genes (DAM) is regulated by CBF/DREB1 transcription factors. In Vitis vinifera, in which dormancy is induced by the photoperiod, VvDAM-SVPs gene expression is regulated by FLOWERING LOCUS T (VvFT). Using the sequences of the six peach (Prunus persica) DORMACY-ASSOCIATED MADS-box genes (DAM) as query, eight putative DAM genes belonging to the family of MADS-box transcription factors and related to the Arabidopsis floral regulators SHORT VEGETATIVE PHASE (SVP) and AGAMOUS LIKE 24 (AGL24) were identified in the V. vinifera genome. Among these, five belong to the subfamily SVP-like genes which have been associated with the regulation of flowering and dormancy in annual and perennial plants, respectively. It has been proposed that they play a direct role in the induction and maintenance of endodormancy (ED) through the regulation of the FLOWERING LOCUS T (FT) gene. In the present study, it is demonstrated that in V. vinifera: (1) VvDAM-SVPs genes are not regulated by ABA/low temperature-induced VvCBFs transcription factors as described for other species of deciduous fruit trees. (2) A contrasting expression pattern between VvDAM3-SVP and VvFT was found under different experimental conditions related to the entry and exit of grapevine buds from ED. (3) Overexpression of VvFT in somatic grapevine embryos (SGE) repressed the expression of VvDAM3-SVP and VvDAM4-SVP. Taken together, the results suggest that VvDAM3-SVP could be associated with ED in grapevine buds, and that its expression could be regulated by VvFT.

ABA promotes starch synthesis and storage metabolism in dormant grapevine buds.

In grapevine (Vitis vinifera L.) buds, the short day (SD)-photoperiod induces endodormancy and increases the level of ABA and the expression of ABA key biosynthesis genes, which suggests that ABA could be the mediator of the photoperiodic induction of endodormancy. In the present study, it was established that during the development of the endodormancy, the content of ABA and the accumulation of starch increased in parallel in the buds; however, these increases occurred after the buds were already in the state of endodormancy. Despite this finding the exogenous applications of ABA to single-bud cuttings increased the starch content and up-regulated the expression of starch synthesis genes (VvSS1 and VvSS3) and down-regulated the expression of sucrose metabolism genes, invertase (VvINV) and sucrose phosphate synthase (VvSUPS). In addition, the manipulation of the endogenous content of ABA in the grapevine buds by applications of hydrogen cyanamide and uniconazole-P, revealed that the depth of the endodormancy depends on the ABA levels. Taken together, the results indicate that the development of the endodormancy in grapevine buds is associated with the accumulation of starch and a shift in metabolism towards a storage metabolism; as ABA stimulates both processes, it must play an important role in the maintenance and release but not the induction of endodormancy in grapevine buds.

Abscisic acid (ABA) and low temperatures synergistically increase the expression of CBF/DREB1 transcription factors and cold-hardiness in grapevine dormant buds.

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 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. 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.

Sprouting of paradormant and endodormant grapevine buds under conditions of forced growth: similarities and differences.

MAIN CONCLUSION: Bud-break assays under forced growth conditions suggest that a drop in ABA content and an increase in sugars are common features in the sprouting of paradormant (PD) and endodormant (ED) grapevine buds. However, increases in cell division and in respiration are unique characteristics of the ED budding. In tropical and subtropical regions where the variations in day length and temperatures are minor throughout the year, the rupture of grapevine buds can be achieved during the current growing season given rise to a double-cropping system annually. However, it is unknown whether the breaking buds are in the paradormancy (PD) or endodormancy (ED) stage. In this study, we compared the breakage of PD and ED buds under conditions of forced growth. To do this, the expression of genes related to the metabolism of phytohormones and sugars, and of relevant physiological functions such as respiration and cell division was analyzed temporally throughout the incubation period in both types of buds. An early fall in the expression of the ABA biosynthesis gene (VvNCED1) and increases in genes related to sugar metabolism and transports were observed during the incubation period in both types of buds. However, while in the PD buds, the genes related to respiration and the cell cycle did not undergo significant changes in their expression during the incubation period, in the ED buds, the expression of these genes together with those related to auxin and cytokinin biosynthesis experienced a large increase. The results suggest that a drop in ABA content and an increase in sugars are early signals for the onset of bud break in both PD and ED vines, while the increase in respiration and cell division are unique characteristics of the ED buds, which reflect its transition from a resting state to a state of active growth.

ABA Represses the Expression of Cell Cycle Genes and May Modulate the Development of Endodormancy in Grapevine Buds.

Recently, the plant hormone abscisic acid (ABA) has been implicated as a key player in the regulation of endodormancy (ED) 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 in the latent bud than at the shoot apex, while the expression of an ABA catabolic gene (VvA8H3) showed no significant difference between either organ. A negative correlation between the content of ABA and transcript levels of cell cycle genes (CCG) was found in both tissues. This result suggested that ABA may negatively regulate the expression of CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of CCG was analyzed in two meristematic tissues of the vine: somatic embryos and shoot apexes. The results indicated that cell cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding cyclin-dependent kinases (VvCDKB1, VvCDKB2) and genes encoding cyclins of type A (VvCYCA1, VvCYCA2, VvCYCA3), B (VvCYCB), and D (VvCYCD3.2a) and up-regulated the expression of VvICK5, a gene encoding an inhibitor of CDKs. During ED, the content of ABA increased, and the expression of CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC) reduced the content of ABA and up-regulated the expression of CCG, this last effect was abolished when HC and ABA were co-applied. Taken together, these results suggest that ABA-mediated repression of CCG transcription may be part of the mechanism through which ABA modulates the development of ED in grapevine buds.

Cell cycle genes are activated earlier than respiratory genes during release of grapevine buds from endodormancy.

Single-bud cuttings of Vitis vinifera L exposed to forced growing conditions were used to investigate the involvement of phytohormones, abscisic acid (ABA), auxin (Aux) and cytokinin (CK) in the release of buds from the ED and in bud-sprouting. This artificial system imitates and hastens the natural sprouting that occurs in spring. Temporal expression analysis of genes related to phytohormones synthesis, showed an early drop in the expression of ABA biosynthesis gene that preceded an increase in Aux and CK biosynthesis genes. Bud-break is headed by the activation of all structures of the latent bud, especially the differentiation of the inflorescence and the development of the early stages of floral organs. Therefore, resumption of cell division and increases in respiration are essential for the activation of the bud. Temporal expression analysis of the cell cycle and respiration genes indicate that an increase in cell division go before the increase in respiration. These results, together with results indicating that the cell cycle genes are upregulated by Aux and CK, suggest that the events before the bud-break, start with a reduction in ABA content, followed by an increase in the content of Aux and CK, which activates the machinery of the cell cycle, which eventually would cause an increase in respiration.

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.

Short day-photoperiod triggers and low temperatures increase expression of peroxidase RNA transcripts and basic peroxidase isoenzyme activity in grapevine buds.

Expression of peroxidase (Prx) RNA transcripts was studied in grapevine cv. Thompson seedless through use of semi-quantitative RT-PCR. In roots, the three Vitis vinifera Prx ESTs, reported in the genomic facility database from the University of California (http://cgf.ucdavis.edu), were expressed as RNA transcripts, and three Prx isoenzymes, two basic (PrxB1) and (PrxB2) and a neutral Prx isoenzyme (PrxN1), were detected in root extracts. In buds, although one Prx EST (CB923206) was detected as a RNA transcript, three Prx activities were found in its extract, these same being the two basic isoenzymes found in the roots, and an acidic one (PrxA1) as well. In field grown grapevines, Prx RNA transcript was expressed transiently during bud endodormancy (ED) while under controlled conditions in a growth chamber, a short day (SD) photoperiod triggered expression of the Prx transcript in bud cuttings; low temperature, enhanced its expression level. Because environmental cues that trigger and enhance Prx RNA transcript expression in bud-tissue are the same as those that induce bud-ED in Vitis, the transient expression of Prx EST (CB923206) identified in buds could signal the full extent of ED in grapevines.