Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

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As a result of natural hybridization and human selection over millennia, the skin colors of grapes have become greatly diversified. The color is determined by the quantity and composition of anthocyanins. Color-skinned cultivars accumulate anthocyanins in their skins, whereas white-skinned cultivars do not. Myb-related transcription-factor genes such as VvmybA1 regulate anthocyanin biosynthesis. VvMYBA2r, VlmybA1-1, VlmybA1-2, and VlmybA2, which are homologs of VvmybA1, also regulate anthocyanin biosynthesis. In this study, we isolated a novel Myb-related sequence, VlmybA1-3, from cultivars of Vitis labruscana (Vitis vinifera x Vitis labrusca) by means of inverse PCR, and confirmed by means of transient gene expression assay that the gene regulates anthocyanin biosynthesis in grape berry skin. Seedlings of V. labruscana with two functional haplotypes at a region of berry color loci accumulated more anthocyanins than seedlings with a single functional haplotype. In addition, we investigated the haplotypes at the region in 35 cultivars (both V. vinifera and V. labruscana), and found certain typical characteristics. These findings will contribute to the selection of seedlings with high anthocyanin quantities in breeding programs for wine and table grapes, and will help elucidate the origin and evolution of Vitis species.

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