Exploring the participation of VviNAC factors in the transcriptional regulatory network which governs grapevine maturation processes

Grapevine (Vitis vinifera L.) is one of the most important fruit crops as it is widely cultivated, and the winemaking industry has a huge worldwide economic relevance. The global warming has affected viticulture altering the maturation processes; in particular, the anticipation of the onset of the berries ripening (veraison) has changed the physiological characteristics of grapes and, consequently, has negatively influenced the wine quality. In this contest, uncovering the molecular mechanisms of the ripening could provide the key in maintaining high quality grapes and wine. For all these reasons, the identification and characterization of master regulators controlling the transition from vegetative-to-mature growth are the challenging but fundamental tasks of this research project. At first, to provide insight into the transcriptional programs controlling the development of grapevine, a global gene expression atlas was generated (Fasoli et al., 2012). Combining this dataset with a berry-specific one (Massonnet et al., 2017), an integrated network analysis was performed (Palumbo et al., 2014) and a new category of genes (switch genes), which are significantly up regulated during the developmental shift and inversely correlated with many genes suppressed during the mature growth phase, was identified. Moreover, many transcription factors are present among them, strongly indicating that they could represent master regulators of the developmental phase transition; between them, the plant-specific NAC (NAM/ATAF/CUC) transcription factors represent an interesting family due to their key role in plant development processes and stress responses (Jensen et al., 2014). Fourteen VviNACs genes were selected for functional characterization as key candidates of the major transcriptome reprogramming during grapevine development: VviNAC01, VviNAC03 (D’Incà, 2017), VviNAC08, VviNAC11 (D’Incà, 2017), VviNAC13 (D’Incà, 2017), VviNAC15, VviNAC17, VviNAC18, VviNAC26, VviNAC33 (D’Incà, 2017), VviNAC38, VviNAC39, VviNAC60 (D’Incà, 2017) and VviNAC61. Different approaches have been carried out to investigate the function of VviNACs gene: the transient over expressions, DAP-seq (DNA Affinity Purification and sequencing, Bartlett et al., 2017) and, only for VviNAC60, the ChIP-seq (Chromatin Immunoprecipitation sequencing, Kaufmann et al., 2010). All of them are powerful techniques used to identify possible targets of the selected transcription factor regulations. VviNAC01 showed its important role in the ethylene pathways; VviNAC03 did not reveal a well-defined identity but seems to be plant growth related; VviNAC08 seems to have a possible role in the gibberellin-related and circadian mechanisms; VviNAC11 seems to be related to the control of the auxin pathways and the chlorophylls degradation; VviNAC13 revealed a probable action in the lignin and phenylpropanoid metabolic processes; VviNAC15, regulating many other TFs, highlighted its role in the regulation mechanisms orchestration; VviNAC17 appeared to be a regulator of the jasmonic acid-induced gene expression; VviNAC18 analyses reported its role in the chlorophyll degradation; VviNAC26 presented the regulation of many genes related to the sugars biosynthesis and the anthocyanin synthesis; VviNAC33 terminates the photosynthetic activity and organ vegetative growth; no data are available for VviNAC38; VviNAC39 resulted to up regulate the transport of sugar and lipids and the ubiquitin-conjugating; VviNAC60 revealed a lot of hormones related up regulated direct target genes and many transcription factors, highlighting again the important and major role of this transcription factor in the grapevine maturation processes; VviNAC61 revealed a predominant role in the regulation of the aromatic compounds biosynthesis. Concerning VviNAC60, ChIP-seq data were also obtained and one interesting gene, the SRG1- SENESCENCE-RELATED GENE 1 OXIDOREDUCTASE (VIT_10s0003g02400), was found. In order to define a regulation and co-regulation network between VviNACs, some candidate targets genes taken from the different DAP-seq datasets (VviNAC01, VviNAC05, VviNAC08, VviNAC34, VviNAC37 and VviNAC61) were tested by Dual Luciferase Reporter Assays to see by which of the selected TFs were actually regulated. The obtained results showed that VviNAC01 directly repressed VviNAC05 expression, whereas activated the VviNAC08 one; moreover, VviNAC01 was validated as repressor of its own transcription. VviNAC03 resulted a repressor of VviNAC05. VviNAC11 directly up regulated VviNAC34, VviNAC37 and VviNAC61. VviNAC13 resulted to regulate VviNAC34 and VviNAC37 expression. VviNAC15 activated VviNAC34. VviNAC17 acted as a repressor of VviNAC05, VviNAC08 and VviNAC61 expression. VviNAC18 resulted a direct activator of VviNAC05. VviNAC26 positively regulated the expression of VviNAC05 and directly down regulated the VviNAC08, VviNAC34 and VviNAC61 expression. VviNAC33 was found as a direct activator of VviNAC05, VviNAC08, VviNAC34, VviNAC37 and VviNAC61 expression. VviNAC60 showed to induced VviNAC05, VviNAC34 and VviNAC61 expression. This PhD thesis lights up the possible roles of some VviNACs in the grapevine development and presents a preliminary regulatory network between this family members; further analysis must be conducted to completely elucidate this complex regulation system.