Ayako Ikegami

DkMyb4 Is a Myb Transcription Factor Involved in Proanthocyanidin Biosynthesis in Persimmon Fruit

Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of the plant and also to the taste of the fruit, mainly through astringency. Persimmon (Diospyros kaki) is unique in being able to accumulate abundant PAs in the fruit flesh. Fruits of the nonastringent (NA)-type mutants lose their ability to produce PA at an early stage of fruit development, while those of the normal astringent (A) type remain rich in PA until fully ripened. The expression of many PA pathway genes was coincidentally terminated in the NA type at an early stage of fruit development. The five genes encoding the Myb transcription factor were isolated from an A-type cultivar (Kuramitsu). One of them, DkMyb4, showed an expression pattern synchronous to that of the PA pathway genes in A- and NA-type fruit flesh. The ectopic expression of DkMyb4 in kiwifruit (Actinidia deliciosa) induced PA biosynthesis but not anthocyanin biosynthesis. The suppression of DkMyb4 in persimmon calluses caused a substantial down-regulation of the PA pathway genes and PA biosynthesis. Furthermore, analysis of the DNA-binding ability of DkMyb4 showed that it directly binds to the MYBCORE cis-motif in the promoters of the some PA pathway genes. All our results indicate that DkMyb4 acts as a regulator of PA biosynthesis in persimmon and, therefore, suggest that the reduction in the DkMyb4 expression causes the NA-type-specific down-regulation of PA biosynthesis and resultant NA trait.

Inhibition of flavonoid biosynthetic gene expression coincides with loss of astringency in pollination-constant, non-astringent (PCNA)-type persimmon fruit

Summary Pollination-constant and non-astringent (PCNA)-type persimmon fruit lose their astringency as the fruit develops on the tree, while pollination-constant and astringent (PCA)-type persimmon fruit remain astringent even during fruit maturation. The main reason for the loss of astringency in PCNA-type fruit is sudden termination of tannin accumulation in the fruit at an early stage of fruit development. Astringency of persimmon fruit is due to condensed tannins (CTs) synthesised via the flavonoid biosynthetic pathway. We investigated seasonal patterns of gene expression involved in flavonoid biosynthesis in PCNA- and PCA-type persimmon fruit as a first step to elucidate the mechanism for the sudden termination of tannin accumulation in PCNA-type fruit. Partial DNA sequences of nine structural genes in the flavonoid biosynthetic pathway (PAL, C4H, 4CL, CHS, CHI, F3H, F3’H, F3’5’H, and DFR) were determined using PCR products amplified with degenerate primers for these genes. These sequences were then used as probes for northern blot analysis of the seasonal expression patterns of these genes in the PCNA cvs. ‘Suruga’ and ‘Hanagosho’, and the PCA cvs. ‘Kuramitsu’ and ‘Yokono’. In the early stages of fruit development, when both types of fruit show high astringency, all these genes were expressed at high levels in both types of fruit. However, as fruit developed, expression of all nine genes declined and became undetectable in PCNA-type cvs. ‘Suruga’ and ‘Hanagosho’, coincident with the termination of tannin accumulation. By contrast, in PCA-type cvs. ‘Kuramitsu’ and ‘Yokono’, all nine genes were expressed at high levels until a late stage of fruit development, coincident with high tannin accumulation in the fruit. PCNA-type persimmon appears to be defective in a primary or regulatory step in flavonoid biosynthesis, so that expression of all nine genes involved in biosynthesis became undetectable at an early stage of fruit development.

Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.)

Fruits of persimmon (Diospyros kaki Thunb.) accumulate large amounts of proanthocyanidins (PAs) in the early stages of development. Astringent (A)-type fruits remain rich in soluble PAs even after they reach full-mature stage, whereas non-astringent (NA)-type fruits lose these compounds before full maturation. As a first step to elucidate the mechanism of PA accumulation in this non-model species, we used suppression subtractive hybridization to identify transcripts accumulating differently in young fruits of A- and NA-type. Interestingly, only a few clones involved in PA biosynthesis were identified in A–NA libraries. Represented by multiple clones were those encoding a novel 1-Cys peroxiredoxin and a new member of family 1 glycosyltransferases. Quantitative RT-PCR analyses confirmed correlation of the amount of PAs and accumulation of transcripts encoding these proteins in young persimmon fruits. Furthermore, the new family 1 glycosyltransferase was produced in Escherichia coli and shown to efficiently catalyze galactosylation at 3-hydroxyl groups of several anthocyanidins and flavonols. These findings suggest a complex mechanism of PA accumulation in persimmon fruits.

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.

The Expression Profile of Lectin Differs from That of Seed Storage Proteins in Castanea crenata Trees

Using Northern blot analysis, the expression of the Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) gene was compared with that of its seed storage protein (SSP) gene. After cDNA cloning of SSP, the expression profile of SSP mRNA and CCA mRNA were compared. SSP mRNA was seed-specific, while CCA mRNA was expressed in the stems and flowers (both male and female) as well as in the seeds. Whereas extracts from all organs observed using Western blot analysis exhibited positive signals, in seeds, large expressions of SSP mRNA were restricted to the late maturation and harvest stages. Levels were maintained during the dormant period. No expression was observed during the germination stage. In contrast, CCA mRNA expression was maintained at a high level during development, was at a relatively low level during dormancy, and showed subsequent high expression during germination. These results suggest that one of the physiological roles of CCA is to act as a vegetative storage protein. But since protein expression did not coincide with that of mRNA, the expression of CCA may be regulated both at the transcription and the translation levels.

Screening of genes encoding isoforms of lectin in Japanese chestnut (Castanea crenata) trees

Screening of a cDNA library revealed that Castanea crenata agglutinin (CCA) consists of two homologous protomers: CCA, which has been reported previously, and its isoform, designated CCA-Q, which contains an additional glutamine residue inserted at position 153 of CCA. PCR-Southern and subsequent analyses of the genomic sequence indicated that both CCA and CCA-Q should be translated from the same gene, CCA, which consists of four exons and three introns. Therefore, the difference may be caused by a type of alternative splicing. PCR-Southern analysis also indicated the existence of another gene, sCCA, which showed high identity to exon 1, intron 1 and exon 4 of CCA. In addition, the entire sCCA gene was transcribed into an mRNA with no obvious open reading frame, although the amount of transcribed product was less than 1/100 of the level of the CCA transcript. Thus, sCCA is estimated to be a pseudogene. These results suggest that no CCA isoforms encoded by different genes are present in Japanese chestnut trees, except for the presence of a homolog pseudogene.