Cytological, Phytohormone, and Transcriptome Analyses Provide Insights into Persimmon Fruit Shape Formation (Diospyros kaki Thunb.).

Fruit shape is an important external feature when consumers choose their preferred fruit varieties. Studying persimmon (Diospyros kaki Thunb.) fruit shape is beneficial to increasing its commodity value. However, research on persimmon fruit shape is still in the initial stage. In this study, the mechanism of fruit shape formation was studied by cytological observations, phytohormone assays, and transcriptome analysis using the long fruit and flat fruit produced by 'Yaoxianwuhua' hermaphroditic flowers. The results showed that stage 2-3 (June 11-June 25) was the critical period for persimmon fruit shape formation. Persimmon fruit shape is determined by cell number in the transverse direction and cell length in the longitudinal direction. High IAA, GA4, ZT, and BR levels may promote long fruit formation by promoting cell elongation in the longitudinal direction, and high GA3 and ABA levels may be more conducive to flat fruit formation by increasing the cell number in the transverse direction and inhibiting cell elongation in the longitudinal direction, respectively. Thirty-two DEGs related to phytohormone biosynthesis and signaling pathways and nine DEGs related to cell division and cell expansion may be involved in the persimmon fruit shape formation process. These results provide valuable information for regulatory mechanism research on persimmon fruit formation.

Comparative transcriptome analysis reveals regulatory network and regulators associated with proanthocyanidin accumulation in persimmon.

BACKGROUND: Proanthocyanidins (PAs) are important plant secondary metabolites that confer flavor, nutritional value, and resistance to pathogens. Persimmon is one of the PA richest crops. Mature fruits can be inedible because of the astringency caused by high PA levels and need to go through a de-astringency treatment before consumption. The molecular basis for PA accumulation is poorly known, particularly transcriptional regulators. We characterised three genotypes ('Luotiantianshi' (LT), 'Mopanshi' (MP), and 'Youhou' (YH)) with different PA accumulation patterns using an approach that combined PacBio full-length sequencing and Illumina-based RNA sequencing to build high-quality full-length transcriptomes. Additionally, we analysed transcriptome dynamics of the three genotypes (LT, MP, and YH) at four key fruit developmental stages. RESULTS: A total of 96,463 transcripts were obtained. We identified 80,075 protein-coding sequences (CDSs), 71,137 simple sequence repeats (SSRs), and 27,845 long noncoding RNAs (lncRNAs). Pearson correlation coefficient (PCC), principal component analysis (PCA), and differentially expressed transcripts (DETs) analyses indicated that the four different developmental stages within a genotype exhibited similar transcriptome activities. A total of 2,164 transcripts specific to each fruit developmental stage were detected. The transcripts specific to early stages were attributed to phenylpropanoid and flavonoid biosynthesis. Co-expression network analyses revealed MEbrown and MEblue modules were strongly associated to PA accumulation. From these two modules, 20 hub TFs are potential regulators for PA accumulation. Among them, Cluster_78388 (SBP protein), Cluster_63454 (bZIP protein), and Cluster_66595 (MYB protein) appear to involve in the PA biosynthesis in Chinese genotypes. CONCLUSIONS: This is the first high-quality reference transcriptome for commercial persimmon. Our work provides insights into the molecular pathways underlying PA accumulation and enhances our global understanding of transcriptome dynamics throughout fruit development.

Comparative transcriptome analysis reveals the mechanism involving ethylene and cell wall modification related genes in Diospyros kaki fruit firmness during ripening.

Rapid loss of firmness is a major handicap for persimmon (Diospyros kaki Thunb.) transportation and retail. The present study employed a comparative transcriptomic approach to elucidate the mechanism involving ethylene and cell wall modification related genes in fruit firmness control of two cultivars during post harvest ripening. In contrast to the short shelf life cultivar (Mopan), the long shelf life cultivar (Yoho) kept high firmness during ripening. Extensive loss of firmness in Mopan drove an intense transcriptional activity. Globally, Mopan and Yoho shared very few common differentially expressed structural genes and regulators. Yoho strongly repressed the expression of ACC synthase and several classes of cell wall degradation genes at the onset of ripening and only induced them during late ripening period. Various ERF, WRKY, MYB, bHLH transcription factors were found highly active during fruit ripening. Overall, this study generates novel gene resources as important tools for extending persimmon shelf life.

Influence of ripening stage and de-astringency treatment on the production of dehydrated persimmon snacks.

BACKGROUND: Seasonal persimmon (Diospyros kaki L.) crops have steadily increased in Spain; this has been linked to a significant increase in the postharvest production waste. Therefore, development of valorized products is of great interest. In this study, a hot air-drying technique was used to obtain persimmon snacks. Slices from astringent and non-astringent persimmons (submitted to de-astringency treatment) at three different ripening stages were dried at 40 and 60 °C to reach 15 ± 3% water content. RESULTS: After the drying treatment, dehydrated samples were harder, turned into a more orange hue angle, and had a reduced soluble tannin content. Dehydrated samples obtained from the astringent fruit at the most advanced ripening stage had similar soluble tannin content as the samples obtained from non-astringent fruit, especially at 60 °C. Besides, a high correlation was observed between the level of astringency perceived by consumers and the decrease of soluble tannin content. Although, in the first ripening stage, consumers preferred the snacks obtained from non-astringent fruits; in the last ripening stage, snacks produced from astringent fruits were equally accepted than the non-astringent ones. CONCLUSION: Therefore, well-accepted persimmon snacks are obtained from both astringent and non-astringent fruits when advanced ripening stages of persimmon are used. © 2020 Society of Chemical Industry.

Epigenetic Flexibility Underlies Somaclonal Sex Conversions in Hexaploid Persimmon.

Epigenetic regulation adds a flexible layer to genetic variations, potentially enabling long-term, but reversible, changes to a trait, while maintaining genetic information. In the hexaploid Oriental persimmon (Diospyros kaki), genetically monoecious cultivars bearing male flowers require the Y-encoded small RNA (smRNA) gene, OGI. This gene represses the expression of its autosomal counterpart gene, MeGI, as part of the canonical male production system. However, a D. kaki cultivar, Saijo, which lacks the OGI gene and originally bears only female flowers, occasionally produces somaclonal mutant male and revertant female (RF) branches. In this study, we investigated the mechanisms underlying these somaclonal sex conversions in persimmon. Specifically, we aimed to unravel how a genetically female tree without the OGI gene can produce male flowers and RF flowers. Applying multi-omics approaches, we revealed that this noncanonical male production system is basically consistent with the canonical system, in which the accumulation of smRNA targeting MeGI and the considerable DNA methylation of MeGI are involved. The epigenetic status of MeGI on CGN and CHG was synchronized to the genome-wide methylation patterns, both in transition to and from the male production system. These results suggest that the somaclonal sex conversions in persimmon are driven by the genome-wide epigenetic regulatory activities. Moreover, flexibility in the epigenetic layers of long-lived plant species (e.g. trees) is important for overcoming genetic robustness.

Antioxidant and the Dwarfing Candidate Gene of "Nantongxiaofangshi" (Diospyros kaki Thunb.).

The aims of this work were to identify genes related to dwarfing for subsequent dwarfing-related research in persimmon and evaluate the relationship between antioxidant activity, dwarf, and hormones of persimmon trees for analyzing the possible dwarf mechanism oxidation factors. In the present study, a transcriptome analysis of "Nantongxiaofangshi" was used to identify and clone 22 candidate genes related to gibberellin signal transduction pathways and synthetic pathway. The expression of these genes was assessed in two persimmon cultivars, "Dafangshi" and "Nantongxiaofangshi," by RT-qPCR at different phenological stages and in response to the exogenous application of GA3 (GA treatment) and PAZ (paclobutrazol, a plant growth inhibitor, also called PP333). The results revealed differential expression of 14 of these 22 genes in the two varieties. Subsequently, endogenous hormone levels were assessed of the two varieties, along with the number of internodes and internode length. The results suggested that the persimmon could be used as a valuable and powerful natural candidate for providing information on the functional role of dwarfing.

Anti-inflammatory activities of astringent persimmons (Diospyros kaki Thunb.) stalks of various cultivar types based on the stages of maturity in the Gyeongnam province.

BACKGROUND: Natural products play a significant role in human health in relation to the prevention and treatment of inflammatory conditions. One of the plants with great medicinal potentials is Diospyros kaki which is mainly cultivated in Asian countries including Korea, Japan, and China. Astringent D. kaki is a wild species with an astringent taste until they are Ripened. kaki calyx is a traditional Korean medicine (TKM) made from the stalks of astringent D. kaki and is used in treating bed-wetting, vomiting, and hiccupping. The present study was designed to investigate the potential anti-inflammatory activities of astringent D. kaki stalks based on cultivar types and stages of maturity. METHODS: The anti-inflammatory effects of the stalk extracts of local astringent D. kaki cultivar species were evaluated on RAW 264.7 cells. Cell viability was measured using a Cell Counting Kit-8 (CCK8) method. The anti-inflammatory effects were determined by measuring the nitric oxide (NO) concentration of the supernatant. Cellular signaling pathways were determined by quantitative polymerase chain reactions of inducible nitric oxide synthase (iNOS). Protein expression of iNOS and phospho-p65 was determined using western blot, and the nuclear localization of p65 was determined using confocal imaging in RAW 264.7 cells. RESULTS: We found that the stage 1 (8-9 month) samples all showed a high percentage of tannic acid content and Gojongsi (Hamyang) stalks had the highest content. The stage 1 samples also showed the highest inhibition of NO production. Decreases in the expression of iNOS and phosphorylated p65, and in the nuclear localization of p65, were dose-dependent. All the extracts were nontoxic under 100 µg/ml concentration. CONCLUSION: This study provides insight into the changes in tannic acid content in astringent D. kaki and their anti-inflammatory effects, in relation to their stage of maturity. These results are expected to be useful in the verification of the efficacy of oriental medicine and the timing of proper harvest for medical use.

Copper and zinc in vineyard and orchard soils at millimeter vertical resolution.

Intensive uses of agrochemicals and soil amendments often cause the elevation of Cu and Zn concentrations in vineyard (VY) and orchard soils. The concentration and speciation of Cu and Zn in the soils at millimeter resolution is critical to understanding the risk of transport of these metals via surface runoff and infiltration. The objective of this study was to investigate the concentration and chemical species of Zn and Cu in VY and persimmon (PS) soils at millimeter vertical resolution. The soils were collected with 5 mm increments down to 5 cm depth and with 5 cm increments down to 25 cm depth. The total concentration and chemical species of Zn and Cu were determined by total digestion and X-ray absorption fine structure (XAFS) spectroscopy, respectively. The Zn concentration of VY soil reached a maximum of 290 mg kg-1 at the uppermost layer of the profile (0.5-1.0 cm). The Cu concentration of VY soil reached a maximum of 201 mg kg-1 (10-15 cm). These Zn and Cu concentrations were greater than background levels. Zinc K-edge XAFS spectroscopy determined that the uppermost layer of VY soil (0-0.5 cm) contained 42% Zn associated with humus and lesser extent of Zn associated with gibbsite (37%) and kaolinite (21%). Zinc associated with humus was not observed in the VY soil profiles below 0.5 cm, whereas Zn associated with gibbsite and kaolinite contributed >83% of total Zn species. Copper K-edge XAFS spectroscopy determined the presence of Cu bonded with humus (40-67%) and Cu adsorbed on kaolinite (26-45%) in the entire soil profile. Our study found the remarkable variation of Cu and Zn concentration and speciation within several centimeters from the soil surface in vineyard and orchard landscapes.

Persimmon orchards harbor an abundant and well-established predatory mite fauna.

Traditionally persimmon cultivation has been considered a minor crop in Spain, but in recent years this crop has experienced an important increase in both cultivated area and production. This increase has been mainly attributed to the widespread adoption of a new postharvest treatment which considerably extends the fruit commercialization period. The sudden expansion of this crop has not allowed time to correctly develop an integrated pest management (IPM) program. Consequently, chemical treatments have become the main strategy to lessen the impact of pests. Given the importance of phytoseiids in other Mediterranean fruit crops, where they are the basis of IPM, we sought to determine whether they could be similarly employed in persimmon crops. For this, we studied the predatory mite complex, the phytoseiid population dynamics and the potential prey for them during three consecutive seasons in four persimmon orchards, two of which managed conventionally, and two organically. Phytoseiids were abundant throughout the season, found on average at a density of more than 1 predatory mite per leaf. The most abundant species was Euseius stipulatus (57.3%) followed by Typhlodromus phialatus (24.8%), Amblyseius andersoni (17.1%) and Paraseiulus talbii (0.8%). Persimmon leaves provided a diversity of prey for predatory mites throughout the year, the most abundant being mealybugs, coccids, whiteflies and thrips. The abundance of predatory mites was significantly correlated to the abundance of potential prey available. From our results we anticipate that phytoseiids will be key actors in the development of persimmon IPM. Their role in this crop is discussed, as well as how to conserve their populations.

Gene networks orchestrated by MeGI: a single-factor mechanism underlying sex determination in persimmon.

Separating male and female sex organs is one of the main strategies used to maintain genetic diversity within a species. However, the genetic determinants and their regulatory mechanisms have been identified in only a few species. In dioecious persimmons, the homeodomain transcription factor, MeGI, which is the target of a Y chromosome-encoded small-RNA, OGI, can determine floral sexuality. The basic features of this system are conserved in the monoecious hexaploid Oriental persimmon, in which an additional epigenetic regulation of MeGI determines floral sexuality. The downstream regulatory pathways of MeGI remain uncharacterized. In this study, we examined transcriptomic data for male and female flowers from monoecious persimmon cultivars to unveil the gene networks orchestrated by MeGI. A network visualization and cistrome assessment suggested that class-1 KNOTTED-like homeobox (KNOX)/ovate family protein (OFP)/growth regulating factors (GRFs) and short vegetative phase (SVP) genes mediate the differences in gynoecium and androecium development between male and female flowers, respectively. The expression of these genes is directly controlled by MeGI. The gene networks also suggested that some cytokinin, auxin, and gibberellin signaling genes function cooperatively in the KNOX/OFP/GRF pathway during gynoecium differentiation. Meanwhile, SVP may repress PI expression in developing androecia. Overall, our results suggest that MeGI evolved the ability to promote gynoecium development and suppress androecium development by regulating KNOX/OFP/GRF and SVP expression levels, respectively. These insights may help to clarify the molecular mechanism underlying the production of unisexual flowers, while also elucidating the physiological background enabling a single-factor system to establish dioecy in plants.

Hormonal control of parthenocarpic fruit set in 'Rojo Brillante' persimmon (Diospyros kaki Thunb.).

In persimmon (Diospyros kaki Thunb.), one to three waves of fruit abscission can occur. The parthenocarpic cv. Rojo Brillante may abscise close to 50% of flowers, which implies a major economic losses. In order to study this process, 700 flowers were labelled, 600 had the lobes of the calyx removed at three stages to promote abscission. Half of them were also treated with gibberellic acid (GA3; 10 mg l-1) to counteract the effect, and 100 were used as control. In the second year, GA3 (25 mg l-1) was applied to whole trees. Calyx lobe removal (CLR) reduced fruit growth rates and advanced and increased fruitlet abscission, whereas GA3 counteracted this effect. Furthermore, when GA3 was applied to the whole tree, fruit set was increased. The time-course of fruit abscission paralleled a decreased in hormonal and carbohydrate contents. Control fruit showed a peak of gibberellin (GA1 and GA4) and IAA concentration at anthesis. Hexose concentrations remained almost constant from flower bud to fruit set, whereas that of sucrose diminished with time. A peak in ethylene production occurred at anthesis, which increased when CLR was performed prior to or at anthesis, but not when performed at fruit set, when ethylene was markedly smaller. GA3 also counteracted it. Accordingly, we suggest that fruit set depends on the induction of gibberellin (GA) and IAA responses in the persimmon, and since there is no shortage of hormones or carbohydrates at anthesis, ethylene production at anthesis seems the most plausible cause of the physiological fruitlet abscission.

Functional characterization of persimmon ß-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation.

Cell wall metabolism during fruit ripening is a highly organized process that involves complex interplay among various cell wall hydrolases. Among these cell wall hydrolases, ß-galactosidase has been identified to participate in cell wall metabolism via its ability to catalyze galactosyl metabolism from the large and complex side chains of cell walls. In this study, the galactose content in the pericarp increased during persimmon fruit ripening, but cell wall galactosyl residues decreased, indicating a relationship between galactose metabolism and persimmon fruit ripening. Expression of a previously isolated ß-galactosidase gene, DkGAL1, increased 25.01-fold during fruit ripening. Heterologous expression of DkGAL1 under the CaMV 35S promoter in tomato accelerated on-plant and postharvest fruits ripening. The fruit firmness of one of transgenic line, OE-18, was 23.83% lower than that of WT at the breaker stage. The transgenic fruits produced more ethylene by promoting the expression of ethylene synthesis-related genes and cell wall degradation-related genes. Overexpression of DkGAL1 in tomato also reduced cell-to-cell adhesion and promoted both wider intercellular spaces and less cell compaction in transgenic fruit structures. Moreover, DkGAL1 was involved in seed germination and radicle elongation in transgenic tomato seeds. These results confirm the role of DkGAL1 in fruit ripening and suggest that this gene alters galactose metabolism in the fruit, which can promote ripening and reduce cellular adhesion. In addition, the role of DkGAL1 is not limited to fruit softening; DkGAL1 was also involved in seed germination and radicle elongation in transgenic tomato seeds.

Role of Brassinosteroids in Persimmon ( Diospyros kaki L.) Fruit Ripening.

Brassinosteroids (BRs) are phytohormones that regulate numerous processes including fruit ripening. In this study, persimmon ( Diospyros kaki L.) fruits were treated with 24-epibrassinolide (EBR) or brassinazole (Brz, a BR biosynthesis inhibitor) and then stored at ambient temperature. The results show that endogenous BR contents gradually increased during persimmon fruit ripening. EBR treatment significantly increased both the content of water-soluble pectin and the activities of polygalacturonase, pectate lyase, and endo-1,4-beta-glucanase but significantly reduced the content of acid-soluble pectin and cellulose, resulting in rapid fruit softening. The EBR treatment also promoted ethylene production and respiration rate. In contrast, Brz treatment delayed persimmon fruit ripening. qRT-PCR analysis showed that DkPG1, DkPL1, DkPE2, DkEGase1, DkACO2, DkACS1, and DkACS2 were up-regulated (especially a 38-fold increase in DkEGase1) in the fruit of the EBR-treated group. These results suggest that BRs are involved in persimmon fruit ripening by influencing cell-wall-degrading enzymes and ethylene biosynthesis.

Review on the significance of chlorine for crop yield and quality.

The chloride concentration in the plant determines yield and quality formation for two reasons. First, chlorine is a mineral nutrient and deficiencies thereof induce metabolic problems that interfere with growth. However, due to low requirement of most crops, deficiency of chloride hardly appears in the field. Second, excess of chloride, an event that occurs under chloride-salinity, results in severe physiological dysfunctions impairing both quality and yield formation. The chloride ion can effect quality of plant-based products by conferring a salty taste that decreases market appeal of e.g. fruit juices and beverages. However, most of the quality impairments are based on physiological dysfunctions that arise under conditions of chloride-toxicity: Shelf life of persimmon is shortened due to an autocatalytic ethylene production in fruit tissues. High concentrations of chloride in the soil can increase phyto-availability of the heavy metal cadmium, accumulating in wheat grains above dietary intake thresholds. When crops are cultivated on soils that are moderately salinized by chloride, nitrate fertilization might be a strategy to suppress uptake of chloride by means of an antagonistic anion-anion uptake competition. Overall, knowledge about proteins that catalyse chloride-efflux out of the roots or that restrict xylem loading is needed to engineer more resistant crops.

Characterization and influence of subtropical persimmon cultivars on juice and jelly characteristics

ABSTRACT To increase the availability to consumers and add more value to persimmon (Diospyros kaki L.), which is a very perishable and seasonal fruit and in order to identify which cultivars grown in subtropical regions are more suitable for processing in the form of juice and jelly, as well as understand what the consumer profile is for these products, the objective of this study was to evaluate the influence of different persimmon cultivars (Rama Forte, Mel, Guiombo and Taubaté) grown in subtropical regions of Brazil on the physicochemical characteristics, rheological properties and sensory acceptance of the resulting juice and jelly in order to identify cultivars with the greatest potential for industrial use. The different studied persimmon cultivars had different physical and physicochemical characteristics which resulted in juices and jellies with different physicochemical, rheological and sensory characteristics. Based on sensory acceptance and productivity/adaptability of persimmon crop in Brazil, the most suitable persimmon cultivars for processing are Rama Forte and Guiombo. In this study it was found that the consumer prefers a more acidic persimmon juice and a less firm or softer, less sweet, clearer and more intense yellow color persimmon jelly.

CHARACTERIZATION AND INFLUENCE OF SUBTROPICAL PERSIMMON CULTIVARS ON JUICE AND JELLY CHARACTERISTICS.

To increase the availability to consumers and add more value to persimmon (Diospyros kaki L.), which is a very perishable and seasonal fruit and in order to identify which cultivars grown in subtropical regions are more suitable for processing in the form of juice and jelly, as well as understand what the consumer profile is for these products, the objective of this study was to evaluate the influence of different persimmon cultivars (Rama Forte, Mel, Guiombo and Taubaté) grown in subtropical regions of Brazil on the physicochemical characteristics, rheological properties and sensory acceptance of the resulting juice and jelly in order to identify cultivars with the greatest potential for industrial use. The different studied persimmon cultivars had different physical and physicochemical characteristics which resulted in juices and jellies with different physicochemical, rheological and sensory characteristics. Based on sensory acceptance and productivity/adaptability of persimmon crop in Brazil, the most suitable persimmon cultivars for processing are Rama Forte and Guiombo. In this study it was found that the consumer prefers a more acidic persimmon juice and a less firm or softer, less sweet, clearer and more intense yellow color persimmon jelly.

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de-astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia-responsive ethylene response factors (ERFs) participate in anaerobic de-astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO2 (95%) was ameliorated by adding the ethylene inhibitor 1-MCP (1 µL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO2 -induced softening involves ethylene signalling. Among the hypoxia-responsive genes, expression of eight involved in fruit cell wall metabolism (Dkß-gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual-luciferase assay indicated that DkERF8/16/19 could trans-activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C-repeat/dehydration-responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia-responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1-MCP. Fruit of the Japanese cultivar 'Tonewase' provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO2 treatment without 1-MCP and changes in cell wall enzymes and ERFs did not occur.

An integrated analysis based on transcriptome and proteome reveals deastringency-related genes in CPCNA persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs) during development. PAs cause a dry or puckering sensation due to its astringency. Pollination constant and non-astringent (PCNA) persimmon fruits can lose astringency during fruit ripening. However, little is known about the mechanism of natural de-astringency of Chinese PCNA (CPCNA). To gain insight into the molecular events of CPCNA natural de-astringency, we used mRNA-seq and iTRAQ-based quantitative proteomic analysis to measure changes in genes and proteins expression at two key stages of natural astringency removal (i.e. 10 and 20 weeks after bloom) and water-treated (i.e. 40 °C·12 h) de-astringency fruits. Our analyses show that the three predominantly process in CPCNA de-astringency: (1) water treatment strongly up-regulates glycolysis/acetaldehyde metabolism, (2) expression of genes/proteins involved in PA biosynthetic pathway was remarkably reduced in natural and water-treated de-astringency, (3) sugar metabolism and ethylene related pathway were quite abundant in natural de-astringency. We also found ethylene-related TFs were quite abundant in natural de-astringency, followed by WRKY and NAC transcription factors. These results provide an initial understanding of the predominantly biological processes underlying the natural de-astringency and "coagulation effect" in CPCNA.

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes and diverse roles of isoenzymes during persimmon fruit development and postharvest softening.

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit.

Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing.

BACKGROUND: microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified. RESULTS: In this study, two small RNA libraries established using 'Eshi No. 1' persimmon (Diospyros kaki Thunb.; CPCNA) fruits collected at 15 and 20 weeks after flowering (WAF) were sequenced through Solexa platform in order to identify miRNAs involved in deastringency of persimmon. A total of 6,258,487 and 7,634,169 reads were generated for the libraries at 15 and 20 WAF, respectively. Based on sequence similarity and hairpin structure prediction, 236 known miRNAs belonging to 65 miRNA families and 33 novel miRNAs were identified using persimmon transcriptome data. Sixty one of the characterized miRNAs exhibited pronounced difference in the expression levels between 15 and 20 WAF, 17 up-regulated and 44 down-regulated. Expression profiles of 12 conserved and 10 novel miRNAs were validated by stem loop qRT-PCR. A total of 198 target genes were predicted for the differentially expressed miRNAs, including several genes that have been reported to be implicated in proanthocyanidins (PAs, or called tannin) accumulation. In addition, two transcription factors, a GRF and a bHLH, were experimentally confirmed as the targets of dka-miR396 and dka-miR395, respectively. CONCLUSIONS: Taken together, the present data unraveled several important miRNAs in persimmon. Among them, miR395p-3p and miR858b may regulate bHLH and MYB, respectively, which are influenced by SPL under the control of miR156j-5p and in turn regulate the structural genes involved in PA biosynthesis. In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors. All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.