Isolation and characterization of a Laccase gene potentially involved in proanthocyanidin polymerization in oriental persimmon (Diospyros kaki Thunb.) fruit

Proanthocyanidins (PAs, condensed tannins) are important health-promoting phytochemicals that are abundant in many plants. Oriental persimmon (Diospyros kaki Thunb.) is an excellent source of PAs because of its unique ability to accumulate large quantities of these compounds in its young fruit. There are two different spontaneous mutant phenotypes of oriental persimmons which lose their astringent taste naturally on the tree; while plants without these mutations remain rich in soluble PAs until the fruit fully ripened. The mutations are referred to as pollination-constant non-astringent genotypes named J-PCNA and C-PCNA, and are from Japan and China respectively. In this work we speculated that the loss of astringency in C-PCNA fruit is due to the soluble PAs transferred into insoluble upon polymerization, which was quite different from that of the J-PCNA. A DkLAC1 gene was isolated by the homology-based clone method. The predicted protein product of this gene showed that the DkLAC1 is a plant laccase which is phylogenetically related to the known enzyme AtLAC15 involved in the polymerization of PAs. Expression patterns of PAs biosynthetic genes associated with soluble PAs contents in three types of Oriental persimmons. Expression levels of DkLAC1 in C-PCNA type plants were linked with the reduction of soluble PAs in the flesh of the fruit. In addition the cis-elements in the DkLAC1 promoter regions indicated that the gene might also be regulated by the DkMYB4 as is seen with other well-known structural genes in Oriental persimmon. We conclude that DkLAC1 is potentially involved in PA polymerization in C-PCNA during normal ripening in C-PCNA persimmon.     

<Emphasis Type="Italic">ALDH2</Emphasis> genes are negatively correlated with natural deastringency in Chinese PCNA persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

Chinese pollination-constant and non-astringent persimmon (C-PCNA) has important application values in the genetic improvement of PCNA for its trait of natural deastringency controlled by a single dominant gene. However, the key genes and the regulatory networks are still not fully understood. The process of C-PCNA natural deastringency may be associated with the acetaldehyde-mediated coagulation of soluble tannins, but the functions of ALDH2 genes related to the metabolism of acetaldehyde are not clear. In this work, three types of persimmon cultivars, ‘Eshi 1’ and ‘Luotian Tianshi’ (C-PCNA type), ‘Youhou’ (J-PCNA type), and ‘Mopanshi’ (non-PCNA type), were sampled. Two members of ALDH2 family genes, DkALDH2a and DkALDH2b, were isolated from ‘Eshi 1’ persimmon fruit. Gene expression patterns indicated that they may be involved in “coagulation effect”, which leads to natural deastringency in C-PCNA persimmon fruit. Transient expression in ‘Eshi 1’ leaves further demonstrated that their expression can reduce the consumption of soluble tannins and inhibit the astringency removal process. Therefore, DkALDH2a and DkALDH2b are negatively correlated with natural deastringency in C-PCNA persimmon.     

<Emphasis Type="Italic">ADH</Emphasis> and <Emphasis Type="Italic">PDC</Emphasis> genes involved in tannins coagulation leading to natural de-astringency in Chinese pollination constant and non-astringency persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

Pollination constant non-astringency (PCNA)-type persimmons are the most desirable cultivar because the fruit loses astringency naturally and does not require any treatments for edibility. The mechanism of natural astringency loss in Chinese PCNA (C-PCNA)-type persimmon is probably related to the coagulation of soluble tannins into insoluble tannins, which is quite different from that in the Japanese PCNA (J-PCNA) type. In this work, three types of persimmon cultivars were sampled: ‘Luotian-tianshi’ (C-PCNA), ‘Maekawa-jirou’ (J-PCNA), and ‘Mopanshi’ (pollination constant astringent (PCA)) were sampled. Three DkADH and four DkPDC genes were isolated from C-PCNA plants. Three candidate genes for soluble tannins coagulation identified in C-PCNA fruit (DkADH1, DkPDC1, and DkPDC2) were characterized through combined analysis of spatiotemporal expression patterns and tannin and acetaldehyde contents during fruit development. Transient over-expression in persimmon leaves showed that DkADH1 and DkPDC2 led to a significant decrease in the levels of soluble tannins in infiltrated leaves. These results indicated that DkADH and DkPDC genes should be considered key genes for natural astringency loss in C-PCNA types.     

SSR-based molecular profiling of 237 persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.) germplasms using an <Emphasis Type="Italic">ASTRINGENCY</Emphasis>-linked marker

Pollination-constant non-astringent (PCNA) trait is desirable in persimmon production because it confers natural astringency loss in mature persimmon fruit. Expression of the PCNA trait requires six homozygous recessive PCNA (ast) alleles at the single ASTRINGENCY (AST) locus in hexaploid persimmon. When crossing non-PCNA accessions to breed PCNA offspring, knowledge of ast and non-PCNA (AST) allele dosage in the parental accessions is important, because more PCNA offspring can segregate from a non-PCNA parent with more ast and fewer AST alleles. Previously, we have demonstrated that a region linked to the AST locus has numerous fragment size polymorphisms with varying numbers of simple sequence repeats. Here, we reveal the polymorphisms in this region in a broad collection of persimmon germplasms. Among 237 accessions, we distinguished 21 AST- and 5 ast-linked fragments with different sizes. Based on the number of fragments detected per individual, we identified 21 non-PCNA accessions with three different ast alleles; by crossing these with a PCNA parent, we obtain PCNA offspring under autohexaploid inheritance. Furthermore, AST and ast allelic combination patterns in hexaploid persimmon were shown to be applicable to cultivar identification of non-PCNA accessions. We directly sequenced ast-linked fragments from 48 accessions with one-size peak of ast-linked fragment and found two distinctive groups of fragments based on single nucleotide polymorphisms. This result suggests that a bottleneck event occurred during ast allele development. We conclude that our fragment size profile can be used to accelerate PCNA breeding that uses non-PCNA parents and to study ast allele accumulation in persimmon.     

Characterization of a highly polymorphic region closely linked to the <Emphasis Type="Italic">AST</Emphasis> locus and its potential use in breeding of hexaploid persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

The pollination-constant, non-astringent (PCNA) type of persimmon is ideal for production because its fruits lose astringency at harvest regardless of seed formation. The PCNA trait in Japanese persimmons is controlled by a single locus, AST, and is recessive to the non-PCNA trait. Because cultivated persimmon is hexaploid, only the homozygous genotype with six recessive alleles is PCNA. A region tightly linked to AST has been used as a DNA marker for breeding. Three non-PCNA (A) alleles have been reported. Here, we show that the region linked to AST is highly polymorphic and includes microsatellites. By analyzing the size of PCR-amplified fragments, we distinguished 12 different A alleles from 14 non-PCNA cultivars and a Chinese PCNA ‘Luotian-tianshi.’ Then, using A fragment size, we assessed A allele inheritance in six non-PCNA × PCNA populations by analyzing segregation of each A allele in a population and segregation of progeny genotypes. By using A allele segregation analysis, we were able to estimate the copy number of each A allele in five non-PCNA parents but not in ‘Amahyakume.’ By analyzing progeny genotype segregation, we were able to estimate the ‘Amahyakume’ genotype. Our approach can be used not only for the selection of PCNA individuals in populations, but also for estimation of the copy number of A alleles in a possible non-PCNA parent. This would enable us to select non-PCNA parents with fewer A alleles, which would segregate more PCNA individuals in crosses with PCNA cultivars.     

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> 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.     

Effects of seasonal temperature changes on <Emphasis Type="Italic">DkMyb4</Emphasis> expression involved in proanthocyanidin regulation in two genotypes of persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> 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.     

Volatile compounds associated to the loss of astringency in persimmon fruit revealed by untargeted GC–MS analysis

High resolution volatile profiling (67 compounds identified) of fruits from 12 persimmon cultivars was established and used to characterize the different astringency types of persimmon fruit before and after deastringency treatment. Analysis of the volatile profile of fruit enables us to differentiate between cultivars that at the moment of harvest produced non-astringent fruit (Pollination Constant Non Astringent—PCNA-type) from astringent ones (non-PCNA-type). Fruit failing to accumulate astringent compounds naturally (PCNA fruit) showed high levels of 3(2H)-benzofuranone, while this compound was not detected in any astringent type fruit (non-PCNA). In addition to this, PCNA cultivars also showed at harvest higher accumulation of benzeneacetaldehyde and lipid-derived aldehydes (hexanal, heptanal, octanal and decanal) than non-PCNA fruit. The application of postharvest deastringency treatment to all non-PCNA cultivars resulted on an important insolubilization of tannins. In general the CO₂-treatment enhanced the levels of acetaldehyde, however those cultivars showing high levels of dihydrobenzofuran at harvest did not present an increment of acetaldehyde. In contrast, all non-PCNA cultivars exhibited an important accumulation of lipid-derived aldehydes due to CO₂-treatment. Therefore, we propose that lipid-derived aldehydes (mainly decanal, octanal and heptanal) may be playing a role in the astringency loss. Our results suggest that 3(2H)-benzofuranone, benzeneacetaldehyde and lipid-derived aldehydes could be used as markers for both natural and artificial loss of astringency.     

Isolation and expression of gene encoding leucoanthocyanidin reductase from <Emphasis Type="Italic">Diospyros kaki</Emphasis> during fruit development

Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidin to (+)-catechin, a precursor of proanthocyanidins abundant in Japanese persimmon (Diospyros kaki Thunb.) fruits. A putative LAR gene (DkLAR) was isolated by rapid amplification of cDNA ends from young fruits. The full-length cDNA of DkLAR gene was 1 356 bp long and encoded an open reading frame of 349 residues. The deduced DkLAR protein was closely related to the homolog in other plant species. The expression of the DkLAR gene in Chinese pollination-constant non-astringent (PCNA) genotype was coincident with the tannin cell development, but was not in Japanese PCNA and Chinese pollination-variant astringent (PCA) genotypes.