Identificazione, caratterizzazione ed analisi funzionale di un fattore di trascrizione MYB di Vitis vinifera putativamente coinvolto nella regolazione della biosintesi dei benzenoidi volatili

The Vitis vinifera berry synthesizes the major determinants of the wine flavours, aromas, and colours. Flavours arise from volatile compounds, such as terpenes, norisoprenoids, and thiols stored as sugar or amino acid conjugates in the vacuoles of exocarp cells (Lund and Bohlmann, 2006). From the scent producing P. hybrida cv Mitchell was recently identified ODORANT1, an R2R3MYB-type transcription factor, which controls the synthesis of volatile benzenoids and regulates, at transcriptional level, shikimate pathway by the ability to activate EPSPs promoter (Verdonk et al, 2005; Ben Zvi et al, 2008). In this study we would like to identify genes involved in the synthesis of the principal volatile phenolic-benzenoids such as benzaldehyde (bitter almond taste in wine), phenylacetaldhyde, benzyll alcohol, 2-phenylethanol (rose) and vanilline (vanilla) that are found mainly in grape berry skin and that are involved in the primary aromas developing during berry ripening (Garcia et al, 2003). BlastP analyses were performed against the Genoscope Blast Server (www.genoscope.cns.fr) using the Petunia ODO1 sequence against the grapevine genome (French-Italian Public Consortium for Grapevine Genome Characterization, 2007). Three putative grapevine genes with the best amino acidic homology to PhODO1 were identified: VvODO3 (58% homology), VvODO2 (53% homology) and VvODO1 (51% homology). The expression level of each grapevine gene was analyzed in developing vegetative and reproductive organs of plants of V. vinifera cv. Corvina (clone 48) by Real-Time RT-PCR experiments. The results suggest that the three genes could be involved in the regulation of the synthesis of volatile benzenoids precursors in an organ specific way. The transcriptional profile of these regulatory genes was also studied during development, and withering of berries of V. vinifera cv. Corvina sampled in the season 2006. The results showed that the regulation of the volatile benzenoids synthesis seems to occur during the first phase of the berry development. VvODO1, VvODO2, VvODO3 were independently over-expressed in P. hybrida cv. Mitchell plants. Transgenic petunia plants and their flowers, expressing the heterologous genes, were analyzed for the expression levels of structural genes and their floral scent production. To analyze volatile compounds produced by petunia flowers in vivo, and to be able to follow volatile release during flower development, a Solid Phase Micro Extraction (SPME) device is placed in the floral headspace, which is subsequently analyzed by GC-MS. From the results of the analysis of the GC-MS spectra, it was clear that over-expressing VvODO3 increased the production levels of benzenoids molecules, despite the unchanged RNA levels of the major genes involved in the biosynthesis process.