• 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.

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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.

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.