IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF MASTER REGULATORS OF THE ONSET OF BERRY RIPENING IN GRAPEVINE (Vitis vinifera L.)

Grapevine is one of the most important and cultivated fruit crops in the world. Its economic importance is especially related to winemaking and the production of high-quality grape is one of the major concerns of the viticulturists. In the last years, continuous temperatures increasing have caused an anticipation of the onset of berry ripening, called veraison, modifying the physiological characteristics of grape, its final quality and consequently wine quality. To prevent these negative effects, the interpretation of the molecular mechanisms controlling this process could provide allow the development of more specific and targeted intervention strategies. To this aim, many molecular studies have been performed. One of the most important is represented by the generation of the grapevine gene expression atlas; this study showed a transcriptomic reprogramming during the vegetative-to-mature transition, suggesting the existence of key regulator genes. Further studies showed that this phase transition seem to be regulated by specific genes, defined switch genes; they are mainly transcription factors and the identification of the functions of these genes could provide important details about the molecular mechanism controlling the maturation process in grapevine. Among these transcription factors, five of them, VviNAC33, VviNAC60, VviAGL15, VviWRKY19 and VvibHLH75, have been selected for functional characterization. Their functional analysis in grapevine has been performed using stable genetic transformation and transient gene expression approaches. The application, improvement and development of these approaches has supported the functional characterization of the five selected genes. Regarding the stable transformation, to identify a standard method, 3 different protocols in 3 different cultivars, using GFP as reporter gene, have been tested. The results showed that the regeneration of transgenic somatic embryos and plants occurred only in Shiraz and Garganega cultivars using embryogenic calli as transformation material, indicating that this complex process is cultivar-dependent. Stable genetic transformation was used for the functional analysis of both VviNAC33 and VviNAC60. In a previous work, both NAC genes have been overexpressed in grapevine plants; the overexpression of VviNAC33 has altered the chlorophyll metabolism, while the overexpression of VviNAC60 has caused stunted growth and anthocyanins leaf accumulation, indicating that both genes are involved in the regulation of vegetative-to-mature transition. In this PhD project, both NAC gene have been fused with EAR motif, the strongest transcriptional repression domain in plants, and stably expressed in Garganega and Shiraz plants. The results showed that some putative target genes of both NAC transcription factors are less expressed than WT plants, indicating that EAR motif represents a good approach to study the function of a transcription factor. Regarding transient gene expression, this method was used for the functional analysis of VviAGL15, VviWRKY19 and VvibHLH75. Leaf agroinfiltration was optimized using YFP as reporter gene and tested in different cultivars by a vacuum system. The analysis of YFP transient expression showed that the fluorescence signal is especially localized in the first and second leaf from apex. VviAGL15, VviWRKY19 and VvibHLH75 have been functionally characterized using the improved leaf agroinfiltration protocol. Each transcription factor was co-expressed with YFP gene: the visualization of its expression has allowed to select only agroinfiltrated leaves. Next microarray analysis of overexpressing leaves showed that many upregulated genes are involved in processes associate with ripening, and an exhaustive molecular interpretation of these preliminary results seem to indicate that VviAGL15, VviWRKY19 and VvibHLH75 are master regulators of the onset of berry ripening, controlling many aspects of the maturation programs