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We investigated the effect of exogenous abscisic acid (ABA) application on the transcriptome as well as the phenolic profiles in the skins of Vitis vinifera cv. Cabernet Sauvignon grape berries grown on the vine and cultured in vitro. ABA application rapidly induced the accumulation of anthocyanin and flavonol. Correlatively, the structural genes in the phenylpropanoid and flavonoid pathways, their transcriptional regulators, as well as genes considered to be involved in the acylation and transport of anthocyanin into the vacuole, were upregulated by ABA treatment. The Genechip analysis showed that the ABA treatment significantly up- or downregulated a total of 345 and 1,482 transcripts in the skins of berries grown on the vine and cultured in vitro, respectively. Exogenous ABA modulated the transcripts associated with osmotic responses, stress responses, cell wall modification, auxin and ethylene metabolism and responses, in addition to the induction of anthocyanin biosynthetic genes, and reduced those associated with photosynthesis; approximately half of these transcripts were identical to the previously reported ripening-specific genes.

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A new regulator of proanthocyanidin (PA) biosynthesis in grapes was found by screening genes coordinately expressed with PA accumulation under different light conditions using a substantially improved method of serial analysis of gene expression (SuperSAGE). This R2R3-MYB transcription factor, VvMYBPAR, shows high protein sequence similarity with PA biosynthesis-regulating plant MYBs, such as VvMYBPA2 and TRANSPARENT TESTA2. Its transcript levels were relatively high in the skins of young berries, whereas the levels were higher in the seeds and at a maximum around veraison. In addition to its response to modified light conditions, the gene responded to abscisic acid application in the skins of cultured berries. Among the PA-specific branch genes, this transcript profile was not correlated with that of VvANR and VvLAR1 but was closely related to that of VvLAR2, suggesting different regulation of PA-specific branch genes from that of a known PA regulator, VvMYBPA2. The PA-specific regulation of VvMYBPAR was confirmed by VvMYBPAR constitutive expression in Arabidopsis in which the transgene specifically induced PA biosynthetic genes and resulted in PA accumulation in plants grown on sucrose-supplemented media to induce anthocyanin synthesis. A transient reporter assay using grapevine cells showed that VvMYBPAR activated the promoters on PA-specific branch genes and candidate genes associated with modification and transport of monomeric PA precursors, as well as the promoters of VvCHS3 and VvF3'5'Hd in the common flavonoid pathway, but not that of VvUFGT on the anthocyanin-specific branch. This new factor suggests the polygenic regulation of PA biosynthesis in grapes by closely related MYB transcription factors.

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The effect of bunch shading during early development (before the onset of ripening) and/or during ripening on the phenolic composition of grape skins (Vitis vinifera L. cv. Cabernet Sauvignon) as well as on the mRNA levels of the biosynthetic genes on the flavonoid pathway was examined. Shading during early development resulted in decreased proanthocyanidin (PA) concentrations. The PA concentrations decreased during ripening, and the decrease of the concentrations was lower in berries shaded during early development than that in the exposed berries. Thus, no significant effect of shading during early development was observed at harvest. Shading during ripening did not influence this decline in the PAs. On the other hand, shading during early development induced changes in the composition such as a decrease of the trihydroxylated subunits within PAs, which agreed with the relative decrease of VvF3′5′H expression. The anthocyanin concentrations were remarkably reduced when the bunches were shaded during ripening, which was in accordance with the decreased transcription of several anthocyanin biosynthetic genes and transcriptional factors. Shading during early development did not influence the anthocyanin concentrations at harvest; however, it decreased the proportion of trihydroxylated anthocyanins. Thus, shading during early development also had an influence on the compounds biosynthesized during ripening.

Necdin is a 325-amino-acid residue protein encoded by a cDNA clone isolated from neurally differentiated embryonal carcinoma cells. Ectopic expression of necdin induces growth arrest of proliferative cells. Necdin binds to major transcription factors E2F1 and p53, suggesting that necdin exerts its functions through the interactions with these cell-cycle-regulating factors. However, information about precise localization of endogenous necdin protein is currently lacking. A rabbit polyclonal antibody was raised against a bacterially expressed recombinant protein of necdin (amino acids 83–325). Immunoblot analysis revealed that necdin protein was expressed almost exclusively in the brain of adult mice. A relative molecular mass of endogenous necdin was estimated at approximately 43,000. In developing mouse brain, necdin was most abundant during fetal and neonatal periods. Necdin was highly enriched in the cytoplasm of hypothalamic neurons in fetal and adult mice. The subcellular fractionation analysis revealed that necdin was concentrated in the cytosol fraction of brain cells. These results suggest that endogenous necdin protein is localized predominantly in the cytoplasm of differentiated neurons and moves into the nucleus under specific conditions.