Keizo Yonemori

Isolation of LEAFY and TERMINAL FLOWER 1 homologues from six fruit tree species in the subfamily Maloideae of the Rosaceae

Flowering is an essential stage of fruit production. To understand the molecular mechanisms controlling flowering in maloid fruit tree species, we isolated and analyzed genes homologous to Arabidopsis LEAFY (LFY; flower meristem identity gene) and TERMINAL FLOWER 1 (TFL1; inflorescence meristem identity gene) from six fruit tree species in the subfamily Maloideae of the Rosaceae; apple (Malus × domestica), Japanese pear (Pyrus pyrifolia), European pear (Pyrus communis), quince (Cydonia oblonga), Chinese quince (Chaenomeles sinensis), and loquat (Eriobotrya japonica). Two LFY homologues and two TFL1 homologues were cloned from all six maloid species by rapid amplification of 3′ and 5′ cDNA ends, reverse transcription-PCR, and PCR with genomic DNA. Phylogenetic analysis by the neighbor-joining method showed that the two LFY homologues and two TFL1 homologues were classified into two distinct clades. The presence of multiple copies of LFY and TFL1 homologues is discussed with reference to the polyploid origin of the subfamily Maloideae.

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit

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.

On the nature of coagulated tannins in astringent-type persimmon fruit after an artificial treatment of astringency removal

Abstract There are many reports concerning the astringency removal of persimmon fruit by ethanol or carbon dioxide treatment. However, none are concerned with the chemical characteristics of tannins after astringency removal, so the chemical properties of coagulated tannins after the treatment were investigated. For this purpose, the astringency of ‘Hiratanenashi’ persimmon ( Diospyros kaki Thunb.) fruit was removed by enclosing them for several days on the tree in polyethylene bags containing 5 ml of 5% ethanol in water. On analyzing these fruit, we found that the coagulated tannins caused by this treatment can be resolubilized in 1% HC1-MeOH (methanol containing 1% hydrochloric acid) with about 80% efficiency. However, as the browning reaction of coagulated tannins in the fruit flesh proceeded gradually after astringency removal, the degree of resolubilization decreased to 20%. Enzymatically isolated tannin cells placed in 1% HCl-MeOH solution revealed that coagulated brown tannin cells did not show any changes, while coagulated colorless tannin cells swelled spherically by resolubilization of tannins. Furthermore, gel chromatography of tannins resolubilized with 1% HCl-MeOH from ethanol-treated fruit showed much smaller molecular size when compared with tannins extracted with 80% MeOH in water from untreated astringent fruit. Tannins extracted with 1 % HCl-MeOH from untreated astringent fruit also showed smaller molecular size than tannins extracted with 80% MeOH in water from the same fruit. The involvement of noncovalent bonds such as hydrogen bonding in tannin coagulation is discussed.

DkMyb2 wound-induced transcription factor of persimmon (Diospyros kaki Thunb.), contributes to proanthocyanidin regulation

Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of a plant against biotic and abiotic stresses. Persimmon (Diospyros kaki) accumulates abundant PAs in each plant organ, and some potential Myb-like transcription factors (Myb-TFs) involved in the production of PAs have been isolated. In this study, we aimed to molecularly characterize one of them, DkMyb2, which was placed in a subclade including a PA regulator of Arabidopsis (Arabidopsis thaliana), TRANSPARENT TESTA2 (TT2), and was co-induced with PA pathway genes after wound stress. Ectopic DkMyb2 overexpression caused significant up-regulation of PA pathway genes in transgenic persimmon calluses and significant accumulation of PA, and increased mean degree of polymerization of PAs in transgenic kiwifruit calluses. Analysis of the DNA-binding ability of DkMyb2 by electrophoretic mobility shift assays showed that DkMyb2 directly binds to the AC-rich cis-motifs known as AC elements in the promoters of the two PA pathway genes in persimmon, DkANR, and DkLAR. Furthermore, a transient reporter assay using a dual-luciferase system demonstrated direct transcriptional activation of DkANR and DkLAR by DkMyb2. We also discuss subfunctionalization of two PA regulators in persimmon, DkMyb2 and DkMyb4, as well as PA regulators in other plant species from the viewpoint of their ability to bind to cis-motifs and their functions in transcriptional activation. Our results provide insight into the multiple regulatory mechanisms that control PA metabolism by Myb-TFs in persimmon.

Phylogenetic relationships of Mangifera species revealed by ITS sequences of nuclear ribosomal DNA and a possibility of their hybrid origin

Abstract. The phylogenetic relationships among 14 Mangifera L. species of Thailand were analyzed by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Parsimony and neighbor joining (NJ) analyses revealed that the common mango (M. indica L.) was closely related to M. laurina Bl., M. sylvatica Roxb., and M. oblongifolia Hook. f. Mangifera foetida Lour. and M. odorata Griff. were also related to M. indica in both parsimonious and NJ trees, although these two species are classified into a different subgenus (subgenus Limus) from the subgenus Mangifera to which M. indica belongs. ITS sequence analysis revealed that several species have nucleotide additivity (two different nucleotides at the same locus) at several sites in the ITS region. Also, M. indica had several polymorphisms among cultivars. This finding may suggest a possibility of hybrid origin of Mangifera species, although Mangifera species are all assumed to be diploid having chromosome number of 2n=2x=40.

Seasonal Abscisic Acid Signal and a Basic Leucine Zipper Transcription Factor, DkbZIP5, Regulate Proanthocyanidin Biosynthesis in Persimmon Fruit

Proanthocyanidins (PAs) are secondary metabolites that contribute to plant protection and crop quality. Persimmon (Diospyros kaki) has a unique characteristic of accumulating large amounts of PAs, particularly in its fruit. Normal astringent-type and mutant nonastringent-type fruits show different PA accumulation patterns depending on the seasonal expression patterns of DkMyb4, which is a Myb transcription factor (TF) regulating many PA pathway genes in persimmon. In this study, attempts were made to identify the factors involved in DkMyb4 expression and the resultant PA accumulation in persimmon fruit. Treatment with abscisic acid (ABA) and an ABA biosynthesis inhibitor resulted in differential changes in the expression patterns of DkMyb4 and PA biosynthesis in astringent-type and nonastringent-type fruits depending on the development stage. To obtain an ABA-signaling TF, we isolated a full-length basic leucine zipper (bZIP) TF, DkbZIP5, which is highly expressed in persimmon fruit. We also showed that ectopic DkbZIP5 overexpression in persimmon calluses induced the up-regulation of DkMyb4 and the resultant PA biosynthesis. In addition, a detailed molecular characterization using the electrophoretic mobility shift assay and transient reporter assay indicated that DkbZIP5 recognized ABA-responsive elements in the promoter region of DkMyb4 and acted as a direct regulator of DkMyb4 in an ABA-dependent manner. These results suggest that ABA signals may be involved in PA biosynthesis in persimmon fruit via DkMyb4 activation by DkbZIP5.

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.

Synthetic cytokinins control persimmon fruit shape, size and quality

SummaryThe effect of three synthetic cytokinins on fruit development of persimmon (Diospyros kaki L.) ‘Hiratanenashi’ was studied by spraying flowers or young fruitlets with 10 or 100 ppm 4PU-30 and TDZ, and 100 or 1000 ppm BA. The most effective for promoting fruit growth was 4PU-30 at 100 ppm, followed by TDZ at 100 ppm and 4PU-30 at 10 ppm. BA at 100 or 1000 ppm and TDZ at 10 ppm had no effect on the fruit enlargement. At 100 ppm, 4PU-30 produced flatter fruit, by promoting transverse growth. Both 4PU-30 and TDZ retarded ripening, as evidenced by delayed chlorophyll degradation and low sugar accumulation. Comparison between prebloom and postbloom applications on cvs Tonewase and Saijo revealed that the former treatment produced flatter, heavier fruit than did the latter. The treated fruit also contained less sugars, especially the reducing sugars, than did the control fruits, indicating that the ripening process was also retarded in these cultivars.

Chemical structures of the condensed tannins in the fruits of Diospyros species

The structural variety of the condensed tannins (proanthocyanidins) in the fruits of 16Diospyros species are reported. Eleven species contained condensed tannins mostly consisting of a mixture of catechin (CA) and gallocatechin (GCA) repeating units; the other five species did not. The GCA content in the CA-GCA total varied from 0.3% to 84.6%. The number of esterified gallic acid per one flavan repeating unit (degree of galloylation, DG) ranged from 0.01 to 0.89. The GCA content was found to be proportional to the DG values. Thus, 16Diospyros species tested may be classified into five groups by the analytical data of their condensed tannins. It may be interesting to compare their structural characteristics with those of the condensed tannins in other fruits, leaves, woods, and barks from the viewpoint of their biosynthesis and function in the plants.