Characterization of cis-acting elements regulating the expression of WOX transcription factors involved in Vitis vinifera somatic embryogenesis.

The family of Wuschel-related Homeobox (WOX) genes is a class of transcription factors involved in the early stages of embryogenesis and organ development in plants. In a previous study conducted in Vitis vinifera some of these genes (VvWOX1, VvWOX2, VvWOX4, VvWOX6, VvWOX9, VvWOX13B and VvWOX13C) showed different transcription levels in callus tissues induced from two different cultivars of V. vinifera ('Chardonnay' and 'Cabernet Sauvignon'). Therefore, we are investigating the genetic basis responsible of these differences. We have cloned and sequenced the promoter region (~2000 bp) proximal to the 5'-end of each VvWOX gene from 'Chardonnay' (CH) and 'Cabernet Sauvignon' (CS). The sequences were analysed in silico through ClustalW and PLACE software in order to identify firstly the nucleotide differences between the promoters isolated from the two cultivars and therefore the cis-acting elements present in these regions. In some of these promoters, we observed several differences between CH and CS characterized by the absence of some cis-acting elements potentially important for the gene regulation. Therefore, these promoters were introduced in Arabidopsis thaliana for the expression pattern characterization using the GUS reporter gene. The homozygous F3 generations of transgenic Arabidopsis were analyzed by GUS and qRT-PCR assays on several plant tissues collected in different developmental stages. The in silico analysis of the promoter pVvWOX4CS showed a long deletion of 60 nts in respect to the pVvWOX4CH promoter, although not decisive to induce variations in the number and type of cis-acting elements. Indeed, the GUS expression in transgenic Arabidopsis was localized in particular in young leaves and roots, but without differences between the two promoters isolated from CH and CS. The sequences of pVvWOX13CCH and pVvWOX13CCS showed instead small differences limited at few SNPs, concentrated particularly in a region proximal to 5'-end of the genes, but interestingly they are associated to 4 cis-acting elements present only in pVvWOX13CCS. Transgenic Arabidopsis containing the promoter pVvWOX13CCS showed a significant increase in the expression levels of the GUS gene in all organs in comparison to Arabidopsis transformed with pVvWOX13CCH. Likely, the promoter sequences responsible for such expression differences are related to only 4 SNPs in proximity to the 5'-end of the gene. Furthermore, studies are ongoing with truncated pVvWOX13C promoters to demonstrate these results. We are performing similar analyses for other pVvWOX promoters, and we are planning experiments of genetic transformation in grapevine to verify the most interesting data emerged from the characterization in Arabidopsis.