Takashi Akagi

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.

Functional and Expressional Analyses of PmDAM Genes Associated with Endodormancy in Japanese Apricot

Bud endodormancy in woody plants plays an important role in their perennial growth cycles. We previously identified a MADS box gene, DORMANCY-ASSOCIATED MADS box6 (PmDAM6), expressed in the endodormant lateral buds of Japanese apricot (Prunus mume), as a candidate for the dormancy-controlling gene. In this study, we demonstrate the growth inhibitory functions of PmDAM6 by overexpressing it in transgenic poplar (Populus tremula × Populus tremuloides). Transgenic poplar plants constitutively expressing PmDAM6 showed growth cessation and terminal bud set under environmental conditions in which control transformants continued shoot tip growth, suggesting the growth inhibitory functions of PmDAM6. In the Japanese apricot genome, we identified six tandemly arrayed PmDAM genes (PmDAM1–PmDAM6) that conserve an amphiphilic repression motif, known to act as a repression domain, at the carboxyl-terminal end, suggesting that they all may act as transcriptional repressors. Seasonal expression analysis and cold treatment in autumn indicated that all PmDAMs were repressed during prolonged cold exposure and maintained at low levels until endodormancy release. Furthermore, PmDAM4 to PmDAM6 responses to a short period of cold exposure appeared to vary between low- and high-chill genotypes. In the high-chill genotype, a short period of cold exposure slightly increased PmDAM4 to PmDAM6 expression, while in the low-chill genotype, the same treatment repressed PmDAM4 to PmDAM6 expression. Furthermore, PmDAM4 to PmDAM6 expression was negatively correlated with endodormancy release. We here discuss the genotype-dependent seasonal expression patterns of PmDAMs in relation to their involvement in endodormancy and variation in chilling requirements.

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.

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.

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.

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.

Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb.

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.

Fine genotyping of a highly polymorphic ASTRINGENCY-linked locus reveals variable hexasomic inheritance in persimmon (Diospyros kaki Thunb.) cultivars

Persimmon (Diospyros kaki Thunb.) is one of the major tree crops in East Asia and is generally hexaploid. A single ASTRINGENCY (AST) locus controls the astringency/non-astringency (A/NA) trait of persimmon fruit, one of the most important traits for consumption, on each of the six corresponding chromosomes. Although several molecular approaches are in progress to elucidate the molecular mechanisms of astringency trait in persimmon, the distinct polysomic behavior of the AST locus remains to be solved. The aim of this study was to perform fine genotyping of a highly polymorphic marker locus linked to the AST locus, detect the allele pairing in ten segregated F1 lines derived from hybridization of A-type × NA-type cultivars, and identify the basis of hexaploid inheritance at the AST locus in persimmon. The results showed that persimmon cultivars frequently produce aneuploid offspring bearing an extra chromosome with the AST locus, with the incidence of aneuploidy varying among the cultivars. On the examination of hexasomic behavior in persimmon cultivars, the ratios of individuals bearing each allele pair segregated from A-type parents showed a good fit to the expected ratios in an autohexaploid inheritance model, except for cvs. Luo-tian-tian-shi and Sa-gok-shi which fitted to an autoallohexaploid inheritance model. These results suggest variable hexasomic behavior among persimmon cultivars.

Effects of seasonal temperature changes on DkMyb4 expression involved in proanthocyanidin regulation in two genotypes of persimmon (Diospyros kaki Thunb.) fruit

Persimmon fruits accumulate a large amount of proanthocyanidin (PA). Fruits of the mutant non-astringent (NA) type lose their ability to accumulate PA at an early stage of fruit development, whereas fruits of the normal astringent (A) type sustain PA accumulation until ripening. This allelotype is determined by the genotype of a single ASTRINGENCY (AST) locus. It is possible that the reduction in PA accumulation in NA-type fruits is due to phenological down-regulation of DkMyb4 (a PA regulator) and the resultant down-regulation of structural genes in the PA pathway. In this study, attempts were made to identify the regulatory mechanisms of phenological PA accumulation in A- and NA-type fruits, focusing particularly on the effects of ambient temperature. Continuous cool temperature conditions caused sustained expression of DkMyb4 in NA-type fruits, as well as in A-type fruits, resulting in increased expression of PA pathway genes and PA accumulation. However, the expression of some A/NA phenotypic marker genes was not significantly affected by the cool temperature conditions. In addition, PA composition in NA-type fruits exposed to cool temperatures differed from that in A-type fruits. These results indicate that a cool ambient temperature may have induced DkMyb4 expression and resultant PA accumulation, but did not directly affect the expression of the AST gene.

Genome-wide view of genetic diversity reveals paths of selection and cultivar differentiation in peach domestication

Domestication and cultivar differentiation are requisite processes for establishing cultivated crops. These processes inherently involve substantial changes in population structure, including those from artificial selection of key genes. In this study, accessions of peach (Prunus persica) and its wild relatives were analysed genome-wide to identify changes in genetic structures and gene selections associated with their differentiation. Analysis of genome-wide informative single-nucleotide polymorphism loci revealed distinct changes in genetic structures and delineations among domesticated peach and its wild relatives and among peach landraces and modern fruit (F) and modern ornamental (O-A) cultivars. Indications of distinct changes in linkage disequilibrium extension/decay and of strong population bottlenecks or inbreeding were identified. Site frequency spectrum- and extended haplotype homozygosity-based evaluation of genome-wide genetic diversities supported selective sweeps distinguishing the domesticated peach from its wild relatives and each F/O-A cluster from the landrace clusters. The regions with strong selective sweeps harboured promising candidates for genes subjected to selection. Further sequence-based evaluation further defined the candidates and revealed their characteristics. All results suggest opportunities for identifying critical genes associated with each differentiation by analysing genome-wide genetic diversity in currently established populations. This approach obviates the special development of genetic populations, which is particularly difficult for long-lived tree crops.