Optimization of simultaneous flavanol, phenolic acid, and anthocyanin extraction from grapes using an experimental design: application to the characterization of champagne grape varieties.

Optimization of polyphenol extraction from grape skin, seed, and pulp was performed on Vitis vinifera L. cv. Pinot Noir, by response surface methodology using a Doehlert design. An acidified mixture of acetone/water/methanol was the best solvent for simultaneous extraction of major polyphenol groups from all berry parts, while optimum extraction times and solid-to-liquid ratios varied according to the part. The determined composition from the model agreed with independent experimental results. Analysis of the three Champagne grape varieties showed that proanthocyanidins were the major phenolic compounds in each part (60-93%). The total berry proanthocyanidin content was highest in Pinot Meunier (11 g kg(-1)) and lowest in Chardonnay (5 g kg(-1)), but Pinot Meunier pulp contained lower amounts of proanthocyanidins and phenolic acids (210 and 127 mg kg(-1) berry, respectively) than that of the other two varieties. The berry anthocyanin content was equivalent in both Pinot Noir and Pinot Meunier (632 and 602 mg kg(-1), respectively).

Molecular cloning and expression of cDNAs encoding alcohol dehydrogenases from Vitis vinifera L. during berry development.

Three full-length cDNAs (VvAdh1, VvAdh2, and VvAdh3) encoding alcohol dehydrogenases (EC 1.1.1.1) were obtained from grape berries (Vitis vinifera L.) by means of PCR and RACE. Pairwise comparisons at the nucleotide level showed that the three cDNAs displayed strong homology in the coding region, but were highly divergent in the 5' and 3' untranslated regions. VvAdh1 and VvAdh2 corresponded to the two previously characterised Adh genes from grapevine, but VvAdh3 was unrelated to known grapevine Adh sequences. The two first cDNAs presented a single ORF of 380 amino acids, whereas the last one has two additional residues. Moreover, the three encoded polypeptides possessed the 22 residues strictly conserved between Adh from different kingdoms. Expression pattern of the individual isogenes was investigated during fruit development. Specific primers were designed, and quantitative RT-PCR experiments were performed to increase the sensitivity of detecting isogenes with a low expression level. Results presented here revealed different developmental regulation of the three Adh isogenes during fruit ripening. VvAdh1 and VvAdh3 transcripts were temporarily accumulated in young, developing berry, whereas VvAdh2 was overexpressed later in fruit development, from the onset of ripening (véraison). Expression analysis also indicated that VvAdh2 accounted for most of the Adh mRNAs present in berries during development. The increased ADH activity detected in berries correlated with the expression pattern of VvAdh2 transcripts. The VvAdh2 and VvAdh3 encoded enzymes were purified from overexpressing E. coli cells. Comparison of kinetic properties of the two ADH enzymes showed a difference in affinity with either ethanol or acetaldehyde as substrates. Significance of multiple Adh expressed in berries is discussed.

Cloning and characterization of Vine-1, a LTR-retrotransposon-like element in Vitis vinifera L., and other Vitis species.

We report the organization of a grapevine chimeric gene Adhr-Vine-1, composed by an Adhr gene, into which a retroelement, Vine-1, was inserted. Sequence analysis revealed that Adhr is a member of the Adh multigene family, but does not correspond to any other grapevine Adh described to date. Vine-1, albeit defective, is the most complete LTR (long terminal repeat)-retrotransposon-like element described in Vitis vinifera L. It is 2392 bp long, with two almost identical LTRs (287 bp) in the same orientation, and flanked by direct repeats of a 5 bp host DNA. This element presents other features, characteristic of retroviruses and retrotransposons including inverted repeats, a primer binding site, and a polypurine tract. It has a single open reading frame (ORF) of 581 amino acids, potentially encoding for a gag protein and parts of the protease and integrase proteins. Vine-1 is most likely related to the copia-like type family, but with no significant similarity to any previously described plant retrotransposon or inserted element, nor to any eukaryotic element described to date. Vine-1 element has been found in Adhr at the same location in different V. vinifera cultivars, but not in some other analyzed Vitis species. These data suggest that Vine-1 insertion in Adhr is specific to V. vinifera, and has occurred after the Adh isogene separation, but prior to cultivar development. Sequences related to Vine-1 were revealed in multiple copies in the V. vinifera genome and, to a lesser extent, in other analyzed Vitis species. The polymorphism observed prompts us to question the role played by transposition in the evolution of the Vitis genus.