Analysis of genes expressed in grape berries

Daniel Lex Ean Waters

Microarray analysis of developing grape berries (Vitis vinifera cv. Shiraz) has revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week two post flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and veraison, a period of rapid berry growth and is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at veraison and remained up-regulated until 13 weeks post flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function. Although it is apparent that changes in gene express10n levels play a role in initiating ripening, it is unknown to what extent gene expression levels differ between cultivars during ripening. In order to gauge the extent to which transcript levels differ between wine grape cultivars in the berry skin during ripening, a comparison of a selection of wine grape cultivars using cDNA microarray was undertaken. The microarray slides were hybridised with Cy3 and Cy5 labelled mRNA derived from the skin of ripening berries of the cultivars Cabernet Sauvignon, Pinot Noir, Shiraz, Chardonnay, Riesling, Sauvignon Blanc and Semillon. Only small differences in the levels of expression of a small proportion of the genes within the berry skin were detected after ripening commenced. Most of the differences appear to arise from environmental signals rather than genome differences. The colour of grape berries are determined by the amount and type of anthocyanin that are synthesised in the berry skin. Three enzymes, flavonoid 3 'hydroxylase (F3 'H), flavonoid 3',5'hydroxylase (F3',5'H) and dihydroflavanol reductase (DFR) are key enzymes of anthocyanin biosynthesis. F3 'H and F3 ',5'H determine the level of hydroxylation of the Bring which is a major determinant of anthocyanin colouration while DFR is known to affect the range of anthocyanins found within any one plant via its substrate specificity. cDNA clones from grape berry skin (Vitis vinifera cv. Shiraz) have been isolated that are likely to code for proteins with F3'H, F3',5'H and DFR activity. Data obtained from 5'-RACE suggests both the F3'H and F3',5'H cDNA clones are full-length and two alleles ofF3',5'H and a single allele of F3 'H are transcribed in grape berry skin. Northern blot and EST data suggests both genes are expressed at low levels in grape berry skin. Sequence analysis of grape DFR confirms the DFR sequence reported by Sparvoli and co-workers in the key region of the gene which determines substrate specificity and suggests the absence of anthocyanins with a mono-hydroxylated B-ring in grape is not due to the inability of DFR to reduce dihydrokaempferol but rather results from F3 'H and F3 ',5'H competing more efficiently for available substrate.

Analysis of genes expressed in grape berries

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Analysis of genes expressed in grape berries

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