Regolazione del colore della bacca di vitis vinifera: identificazione ed analisi funzionale di fattori di trascrizione MYB e bHLH

Anthocyanins (from Greek: “ἀνθός ( anthos) = flower + κυανός (kyanos) = blue) are a class of water-soluble pigments belonging to the flavonoids family. The colour of anthocyanins can vary from red to blue and it depends on medium pH in which they are located and on the formation of salts with heavy metals present in various tissues. In Vitis vinifera the berry colour is determined by the accumulation of anthocyanins in vacuole cells during development. Delphinidin, cyanidin, petunidin, peonidin and malvidin are the most important pigments isolated from the Vitis vinifera berries (Beneytez-Garcia et al, 2002); they can be found mainly as 3-O-glucosides, and in smaller portion, such as acetic acid, caffeic acid and p-coumaric acid esters. The pigments composition is cultivar specific and it is an important biochemical marker to identify different Vitis vinifera varieties although anthocyanins biosynthesis depends on development stages and on environmental factors (Garcia-Beneytez et al., 2002). The absorption spectrum of anthocyanins depends on the pH, so the colour of the skin should also be influenced by pH levels of vacuolar cells of the tissue. Genetic and biochemical aspects of anthocyanins biosynthesis pathway and their role in the pigmentation of the tissues have been well characterized in Zea mais, Petunia hybrida, Antirrhinum major and Arabidopsis thaliana. The characterization of this phenomenon even in commercially important fruit species such as Vitis vinifera, would have a big impact application. Several studies have recently shown that anthocyanins biosynthesis pathway is regulated by MYB protein, bHLH and WD40 similar among different species, indicating that the function of this complex is highly conserved in plant proteins. In particular, in Petunia hybrida it was proposed a model that explains the presence of common elements between anthocyanins biosynthesis regulatory pathway and the vacuolar pH regulation pathway (Quattrocchio et al., 2006). In this system the PhAN1 gene, coding for a bHLH type transcription factor, regulates the anthocyanins synthesis through interaction with a MYB protein encoded by the PhAN2 gene, while vacuolar acidification is regulated by interaction with another MYB protein encoded by PhPH4 gene. In this model are also involved PhAN11, a protein belonging to the WD40 family, and PhJAF13, another bHLH family member . By Vitis vinifera genome analysis (French-Italian Public Consortium for Grapevine Genome Characterization, Nature 2007; Velasco et al.2007), it was possible to isolate and analyze genes sequences coding for transcription factors belonging to MYB, bHLH and WD40 protein families. The knowledge of regulative mechanism of Petunia hybrida could be the starting point to understand the control of berry colour and berry vacuolar pH during development. Phylogenetic analysis (Quattrocchio et al., 2006) have shown a high degree of sequence homology between the Petunia hybrida PhPH4 protein and two Vitis vinifera MYB proteins VvMYB5a (known in the literature also as VvMYBCS1) and VvMYB5b. Similar analysis have revealed the presence of three Vitis vinifera proteins, VvMYBA1, VvMYBA2 and VvMYBA3 (Kobayashi et al., 2002, 2004, Walker et al., 2007), showing a good sequence homology with PhAN2 (Quattrocchio et al., 2006). Initially, this work provides a biochemical characterization of vacuolar pH variations in skin, in flesh and whole berry of Vitis vinifera cv. Corvina in various developmental stages from 14 days after anthesis to ripening (approximately 120 days after anthesis), weekly sampled. Frequently, the function of a protein depends on its amino acid sequence, and so on nucleotide sequence of the encoding gene, although it may then be modified by some posttranslation regulations. Then, on the basis of this assumption, in this work have been isolated the Vitis vinifera cv. Corvina nucleotide sequences characterized by a greater homology with Petunia hybrida MYB, bHLH and WD40 genes involved in the transcriptional complex that regulates the anthocyanins biosynthesis and vacuolar pH acidification. Vitis vinifera functional genomics are very difficult because it is not simple to transform the grape so is very useful to use Petunia hybrida like model system. Petunia mutants for each MYB and bHLH transcription factors genes are available to express their homologue of grape gene. So, to prove whether homologous sequence are also functional homologous, we expressed ectopically in Petunia hybrida plants mutants for each gene their Vitis vinifera homologous sequence. The demonstration that the sequence homology between these genes corresponds also to an effective functional homology, can be achieved by the restore of missing function of the Petunia hybrida mutant by Vitis vinifera gene. Moreover it is very interesting to study the expression profiles of genes isolated in Vitis vinifera berry during fruit development by RT Real Time PCR analysis. Finally, to demonstrate whether the Petunia hybrida model may be valid also for Vitis vinifera it is necessary to study some protein-protein interactions between potential transcription factors we studied. For this demonstration we use BiFC analysis (Bimolecular Fluorescence Complementation, Walter et. At 2004).