Changes of microbial communities and metabolites in the fermentation of persimmon vinegar by bioaugmentation fermentation.

To evaluate the effects of bioaugmentation fermentation inoculated with one ester-producing strain (Wickerhamomyces anomalus ZX-1) and two strains of lactic acid bacteria (Lactobacillus plantarum CGMCC 24035 and Lactobacillus acidophilus R2) for improving the flavor of persimmon vinegar, microbial community, flavor compounds and metabolites were analyzed. The results of microbial diversity analysis showed that bioaugmentation fermentation significantly increased the abundance of Lactobacillus, Saccharomyces, Pichia and Wickerhamomyces, while the abundance of Acetobacter, Apiotrichum, Delftia, Komagataeibacter, Kregervanrija and Aspergillus significantly decreased. After bioaugmentation fermentation, the taste was softer, and the sensory irritancy of acetic acid was significantly reduced. The analysis of HS-SPME-GC-MS and untargeted metabolomics based on LC-MS/MS showed that the contents of citric acid, lactic acid, malic acid, ethyl lactate, methyl acetate, isocitrate, acetoin and 2,3-butanediol were significantly increased. By multivariate analysis, 33 differential metabolites were screened out to construct the correlation between the differential metabolites and microorganisms. Pearson correlation analysis showed that methyl acetate, ethyl lactate, betaine, aconitic acid, acetoin, 2,3-butanediol and isocitrate positively associated with Wickerhamomyces and Lactobacillus. The results confirmed that the quality of persimmon vinegar was improved by bioaugmentation fermentation.

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.

Larvicidal Activity against Spodoptera frugiperda of some Constituents from two Diospyros Species. In silico Pesticide-likeness Properties, Acetylcholinesterase Activity and Molecular Docking.

This report informs for the first time the chemical constituents of Diospyros xolocotzii and Diospyros digyna, the pesticidal and the acetylcholinesterase (AChE) inhibition potential of some compounds calculated by in silico approaches, the larvicidal activity against Spodoptera frugiperda of available compounds, the AChE inhibition of selected compounds, and the results of the molecular docking of the most active ones with this receptor. From the aerial parts of D. xolocotzii were isolated pentacyclic triterpenes (1-4, 6, 10, 11-13), phytosterols (15-17), and isodiospyrin (18), whereas the analysis of aerial parts of D. digyna conducted to the isolation of pentacyclic triterpenes (4, 5, 7-9, 11-14), (4S)-shinanolone (19), and scopoletin (20). For comparison purposes, origanal (21) was chemically prepared from 11. The in silico analysis showed that the tested compounds have pesticide potential. The larvicidal activities of 11>13>12 indicated that the increase of the oxidation degree at C-28 increases their bioactivity. Compounds 11 and 21 presented the higher inhibition in the acetylcholinesterase assay, and the higher binding energies, and for the interactionswith AChE by molecular docking. Both Diospyros species are sources of triterpenes with pesticidal potential and the molecular changes in lupane triterpenes correlate with the observed bioactivity and molecular docking.

Reduced graphene oxide-persimmon tannin/Pt@Pd nanozyme-based cascade colorimetric sensor for detection of 1,5-anhydroglucitol.

1,5-anhydroglucitol (1,5-AG) is of considerable clinical relevance as a biochemical marker of glucose metabolism in the assessment and monitoring of diabetes. Herein, a simple colorimetric biosensor was constructed for the identification and detection of 1,5-AG by using pyranose oxidase (PROD) enzyme cascaded with reduced graphene oxide/persimmon tannin/Pt@Pd (RGO-PT/Pt@Pd NPs) nanozyme. The as-prepared RGO-PT/Pt@Pd NPs had excellent peroxidase-like activity and can be applied as a nanozyme. First, PROD enzyme reacts with the target 1,5-AG, decomposing 1,5-AG into 1,5-anhydrofuctose (1,5-AF) and H2O2. At this point, the highly catalytic RGO-PT/Pt@Pd NPs nanozyme produces a cascade with PROD enzyme which catalyzes the decomposition of H2O2 to produce O2. This in turn oxidizes the substrate 3,3',5,5'-tetramethylbenzidine (TMB) and produces a color change in the solution. Finally, the detection of 1,5-AG was achieved by measuring the absorption peak at 652 nm with an ultraviolet visible (UV-vis) spectrophotometer. Under optimal conditions, the linear operating range of the 1,5-AG enzyme cascade colorimetric sensor was 1.0-100.0 µg/mL, and the limit of detection (LOD) was 0.81 µg/mL. The proposed colorimetric biosensor was successfully applied to detect 1,5-AG in spiked human serum samples with the recoveries of 97.2-103.9% and RSDs of 1.94-4.48%. It provides a promising developmental assay for clinical detection of 1,5-AG.

Comparative Metabolomic and Transcriptomic Analyses Reveal Distinct Ascorbic Acid (AsA) Accumulation Patterns between PCA and PCNA Persimmon Developing Fruit.

Persimmon fruit has a high nutritional value and significantly varies between pollination-constant astringent (PCA) and pollination-constant non-astringent (PCNA) persimmons. The astringency type affects sugar, flavonoids, and tannin accumulation and is well known in persimmon fruit. However, the impact of the fruit astringency type on ascorbic acid (AsA) accumulation is limited. In this study, typical PCA varieties ('Huojing' and 'Zhongshi5') and PCNA varieties ('Yohou' and 'Jiro') of persimmon fruit were sampled at four developing stages (S1-S4) to provide valuable information on AsA content variation in PCA and PCNA persimmon. Persimmon fruit is rich in ascorbic acid; the AsA content of the four varieties 'Zhongshi5', 'Huojing', 'Jiro', and 'Youhou' mature fruit reached 104.49, 48.69, 69.69, and 47.48 mg/100 g. Fruit of the same astringency type persimmon showed a similar AsA accumulation pattern. AsA content was significantly higher in PCA than PCNA fruit at S1-S3. The initial KEGG analysis of metabolites showed that galactose metabolism is the major biosynthetic pathway of AsA in persimmon fruit. There were significant differences in galactose pathway-related metabolite content in developing PCA and PCNA fruit, such as Lactose, D-Tagatose, and D-Sorbitol content in PCA being higher than that of PCNA. Combined gene expression and WGCNA analyses showed that the expression of the GME (evm.TU.contig4144.37) gene was higher in PCA-type than in PCNA-type fruit in S1-S3 and exhibited the highest correlation with AsA content (r = 690 **, p < 0.01). Four hub genes, including the DNA methylation gene, methyltransferase gene, F-box, and Actin-like Protein, were identified as potential regulators of the GME gene. These results provide basic information on how astringency types affect AsA accumulation and will provide valuable information for further investigation on AsA content variation in persimmon fruit.

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon.

Proanthocyanidins (PAs) are specialized metabolites that influence persimmon fruit quality. Normal astringent (A)-type and non-astringent (NA)-type mutants show significant variation in PA accumulation, but the influencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by DkDTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pattern of DkDTX5/MATE5 was correlated with PA accumulation in NA-type fruit. Upregulation and downregulation of DkDTX5/MATE5 promoted and inhibited PA accumulation, respectively, in the NA-type fruit. Interestingly, transporter assays of Xenopus laevis oocytes indicated that DkDTX5/MATE5 preferentially transported the PA precursors catechin, epicatechin, and epicatechin gallate, resulting in their increased ratios relative to the total PAs, which was the main source of variation in PA accumulation between the A-type and NA-type. The allele lacking Ser-84 in DkDTX5/MATE5 was identified as a dominantly expressed gene in the A-type and lost its transport function. Site-directed mutagenesis revealed that DkDTX5/MATE5 binds to PA precursors via Ser-84. These findings clarify the association between the transporter function of DkDTX5/MATE5 and PA variation, and can contribute to the breeding of new cultivars with improved fruit quality.

Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Transcriptome and Metabolome Reveal Distinct Sugar Accumulation Pattern between PCNA and PCA Mature Persimmon Fruit.

Persimmon (Diospyros kaki) fruit have significant variation between pollination-constant non-astringent (PCNA) and pollination-constant astringent (PCA) persimmons. The astringency type affects not only the soluble tannin concentration but also the accumulation of individual sugars. Thus, we comprehensively investigate the gene expression and metabolite profiles of individual sugars to resolve the formation of flavor differences in PCNA and PCA persimmon fruit. The results showed that soluble sugar, starch content, sucrose synthase, and sucrose invertase were significantly different between PCNA and PCA persimmon fruit. The sucrose and starch metabolism pathway was considerably enriched, and six sugar metabolites involving this pathway were significantly differentially accumulated. In addition, the expression patterns of diferentially expressed genes (such as bglX, eglC, Cel, TPS, SUS, and TREH genes) were significantly correlated with the content of deferentially accumulated metabolites (such as starch, sucrose, and trehalose) in the sucrose and starch metabolism pathway. These results indicated that the sucrose and starch metabolism pathway maintained a central position of sugar metabolism between PCNA and PCA persimmon fruit. Our results provide a theoretical basis for exploring functional genes related to sugar metabolism and provide useful resources for future studies on the flavor differences between PCNA and PCA persimmon fruit.

Diospyros malabarica fruit preparation mediates immunotherapeutic modulation and epigenetic regulation to evoke protection against non-small cell lung cancer (NSCLC).

ETHNOPHARMACOLOGICAL RELEVANCE: Diospyros malabarica is an ethnomedicinal plant with hypoglycaemic, anti-bacterial, and anti-cancer properties and it belongs to the Ebenaceae family which is well known for its medicinal uses since ancient times and application of its bark and unripened fruit has been significantly mentioned in Ayurvedic texts. The Diospyros malabarica species which is known as the Gaub in Hindi and Indian Persimmon in English is native to India, however, it is distributed throughout the tropics. AIM OF THE STUDY: As Diospyros malabarica fruit preparation (DFP) possesses medicinal values, the study aims to evaluate its role as natural, non-toxic, and cost-effective dendritic cells (DCs) maturing immunomodulatory agent and also as an epigenetic regulator to combat Non-small cell lung cancer (NSCLC) which is a type of lung cancer whose treatment options such as chemotherapy, radiation therapy, etc. are accompanied with some adverse side effects. Thus, immunotherapeutic strategies are in high demand to evoke tumor protective immunity against NSCLC without causing such side effects. MATERIALS AND METHODS: Peripheral Mononuclear Cells (PBMCs) derived monocytes of normal subjects and NSCLC patients were utilized to generate DCs matured with either LPS (LPSDC) or DFP (DFPDC). Mixed Lymphocyte Reaction (MLR) was carried out with the differentially matured DCs co-culturing T cells and cytotoxicity of lung cancer cells (A549) was measured through LDH release assay and cytokine profiling was carried out via ELISA respectively. PBMCs of normal subjects and NSCLC patients have transfected separately in vitrowith CRISPR-activation plasmid of p53 and CRISPR-Cas9 knockout plasmid of c-Myc to analyze epigenetic mechanism(s) in the presence and absence of DFP. RESULTS: Diospyros malabarica fruit preparation (DFP) treated DC upregulates the secretion of T helper (TH)1 cell specific cytokines (IFN-γ and IL-12) and signal transducer and activator of transcription molecules (STAT1 and STAT4). Furthermore, it also downregulates the secretion of TH2-specific cytokines (IL-4 and IL-10). Diospyros malabarica fruit preparation (DFP) enhances p53 expression by reducing methylation levels at the CpG island of the promoter region. Upon c-Myc knockout, epigenetic markers such as H3K4Me3, p53, H3K14Ac, BRCA1, and WASp were enhanced whereas H3K27Me3, JMJD3, and NOTCH1 were downregulated. CONCLUSION: Diospyros malabarica fruit preparation (DFP) not only increases the expression of type 1 specific cytokines but also augments tumor suppression modulating various epigenetic markers to evoke tumor protective immunity without any toxic activities.

Diospyros malabarica (Desr.) Kostel fruits extract attenuated acute and chronic inflammation through modulation of the expression of pro- and anti-inflammatory biomarkers in rat models.

Rheumatoid arthritis is a chronic inflammatory disorder of polyarticular tissues, characterised by progressive synovitis. Its prolonged treatment imparts a huge burden on the healthcare system and results in toxicity, which necessitates the search for safe, efficacious and cost-effective therapies. Diospyros malabarica (Desr.) Kostel is traditionally used for anti-inflammatory purposes; however, to the best of our knowledge, there is no detailed study reporting the in vivo anti-inflammatory potential of this plant. Therefore, in the current study, the methanol extract of D. malabarica (Desr.) Kostel fruit (mDMF) was evaluated for its antioxidant, anti-inflammatory and anti-arthritic potentials, along with its underlying mechanisms. The antioxidant activity was evaluated by DPPH assay. Total phenolic and flavonoid contents were estimated via colorimetric and high-performance liquid chromatography (HPLC) methods. Different doses (250, 500 and 750 mg/kg) of mDMF were used to evaluate the anti-inflammatory and anti-arthritis actions in acute inflammatory (carrageenan and histamine-induced paw oedema) and Freund's complete adjuvant (FCA)-induced arthritis rat models. Levels of various pro- and anti-inflammatory biomarkers were estimated using ELISA and RT-PCR techniques. Paw samples were used for different histopathological and radiographic studies. Qualitative phytochemical and HPLC analyses indicated the presence of various polyphenolic compounds in mDMF, which exhibited marked antioxidant activity in the DPPH assay. mDMF showed time-dependent anti-inflammatory and anti-arthritic effects in in vivo models. ELISA assay data showed significant (p < 0.05) reduction in the serum levels of C-reactive protein and rheumatoid factor in the mDMF treatment groups. RT-PCR data showed significant (p < 0.05) down-regulation of various pro-inflammatory markers (TNF-α, NF-κB, COX-2, IL-1ß and IL-6) and up-regulation of anti-inflammatory markers (IκB, IL-4 and IL-10) in serum samples of rats treated with mDMF. The histopathology of the ankle joints showed reduced pannus formation, joint swelling and synovial hyperplasia in mDMF-treated animals when compared with the untreated disease control group. Overall, it may be concluded that the antioxidant, anti-inflammatory and anti-arthritis properties of mDMF are due to its flavonoid and phenolic constituents. Further studies using a stable oral dosage form of D. malabarica (Desr.) Kostel fruits extract are warranted to explore its effects in other inflammatory disorders, including irritable bowel syndrome, appendicitis and hepatitis.

The antioxidant system response to drought-stressed Diospyros lotus treated with exogenous melatonin.

Drought is one of the major abiotic stresses adversely impacting the growth of persimmon, which is a widely cultivated traditional fruit tree in North China. Melatonin is a bio-stimulator involved in mediating plant responses to drought. The role of exogenous melatonin application in the drought tolerance of Diospyros lotus was examined under drought stress with different doses of melatonin (0, 10, 50, and 100 µM). Exogenous melatonin application significantly mitigated the adverse effects of drought stress on chlorophyll fluorescence, lipid peroxidation, reactive oxygen species (ROS) accumulation and nitric oxide (NO) content. The 100-µM melatonin application produced the most beneficial impacts against drought stress. The melatonin-enhanced tolerance could be attributed to improved antioxidant enzymes, reduced drought-induced ROS accumulation, and lipid peroxidation. Melatonin application activated major antioxidant enzymes such as superoxide dismutase, catalase, peroxidase, glutathione reductase, and ascorbate peroxidase. Interestingly, NO concentration was significantly higher in 10 and 50 µM melatonin treatments and lower in 100 µM melatonin treatment compared to the control. Moreover, exogenous melatonin application affected the mRNA transcript levels of several genes involved in ROS metabolism, including DlRBOHA, DlSOD, DlCAT, and DlPOD. Hence, the responses of Diospyros lotus to drought varied with different doses of melatonin. Our results provide a concrete insight into the effects of melatonin with varying doses in alleviating drought as well as a platform for its potential application in the related fields.

DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

Nonlinear molecular dynamics of quercetin in Gynocardia odorata and Diospyros malabarica fruits: Its mechanistic role in hepatoprotection.

Liver performs number of critical physiological functions in human system. Intoxication of liver leads to accumulation of free radicals that eventually cause damage, fibrosis, cirrhosis and cancer. Carbon tetrachloride (CCl4) belongs to hepatotoxin is converted to a highly reactive free radical by cytochrome P450 enzymes that causes liver damage. Plant extracts derived quercetin has substantial role in hepatoprotection. This study highlights the possible mechanism by which quercetin plays significant role in hepatoprotection. HPLC analysis revealed the abundance of quercetin in the fruit extracts of Gynocardia odorata and Diospyros malabarica, were isolated, purified and subjected to liver function analysis on Wistar rats. Post quercetin treatment improved liver function parameters in the hepatotoxic Wistar rats by augmenting bilirubin content, SGOT and SGPT activity. Gene expression profile of quercetin treated rats revealed down regulation of HGF, TIMP1 and MMP2 expressed during CCl4 induction. In silico molecular mechanism prediction suggested that quercetin has a high affinity for cell signaling pathway proteins BCL-2, JAK2 and Cytochrome P450 Cyp2E1, which all play a significant role in CCl4 induced hepatotoxicity. In silico molecular docking and molecular dynamics simulation have shown that quercetin has a plausible affinity for major signaling proteins in liver. MMGBSA studies have revealed high binding of quercetin (ΔG) -41.48±11.02, -43.53±6.55 and -39.89±5.78 kcal/mol, with BCL-2, JAK2 and Cyp2E1, respectively which led to better stability of the quercetin bound protein complexes. Therefore, quercetin can act as potent inhibitor against CCl4 induced hepatic injury by regulating BCL-2, JAK2 and Cyp2E1.

Persimmon-derived tannin has antiviral effects and reduces the severity of infection and transmission of SARS-CoV-2 in a Syrian hamster model.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread across the world. Inactivating the virus in saliva and the oral cavity represents a reasonable approach to prevent human-to-human transmission because the virus is easily transmitted through oral routes by dispersed saliva. Persimmon-derived tannin is a condensed type of tannin that has strong antioxidant and antimicrobial activity. In this study, we investigated the antiviral effects of persimmon-derived tannin against SARS-CoV-2 in both in vitro and in vivo models. We found that persimmon-derived tannin suppressed SARS-CoV-2 titers measured by plaque assay in vitro in a dose- and time-dependent manner. We then created a Syrian hamster model by inoculating SARS-CoV-2 into hamsters' mouths. Oral administration of persimmon-derived tannin dissolved in carboxymethyl cellulose before virus inoculation dramatically reduced the severity of pneumonia with lower virus titers compared with a control group inoculated with carboxymethyl cellulose alone. In addition, pre-administration of tannin to uninfected hamsters reduced hamster-to-hamster transmission of SARS-CoV-2 from a cohoused, infected donor cage mate. These data suggest that oral administration of persimmon-derived tannin may help reduce the severity of SARS-CoV-2 infection and transmission of the virus.

The regulatory role of gibberellin related genes DKGA2ox1 and MIR171f_3 in persimmon dwarfism.

'Nantongxiaofangshi' (Diospyros kaki Thunb., D. kaki Thunb.) is a local cultivar of persimmon with dwarf-like traits in Jiangsu, China. Closely spaced planting afforded by dwarfism is usually one of the most important ways to promote fruit cultivation and production. However, the understanding of dwarfism in D. kaki Thunb. is very limited at the molecular level, which hinders the further increase of the fruit production. In this work, a persimmon transgenic system was successfully established, and the field experiments of grafting phenotype were carried out. The results showed that D. kaki Thunb. could be used as an interstock to induce dwarfing in grafted scions, and the dwarf character was better when interstock lengths were between 20 and 25 cm. Furthermore, the key genes related to dwarfism in D. kaki Thunb. were screened and verified, and subsequently, the regulatory role of related genes in persimmon dwarfism was figured out. It was found that the gene encoding gibberellin 2-oxidase-1 (DkGA2ox1) involved in GA biosynthesis was associated with the dwarfing in D. kaki Thunb. Overexpression of DkGA2ox1 in Diospyros lotus resulted in a typical dwarf phenotype. Meanwhile, the microRNA data showed that the miR171f_3 demonstrated the active involvement in GA pathway response in persimmon dwarfism. DkGA2ox1 and MIR171f_3, as two highly expressed genes in D. kaki Thunb. interstock, could be used as stimulus signals to affect the content of GA in scion, however, the specific transmission mechanism still needs to be further explored. Ultimately, the bioactive GA level was decreased, resulting in the scion dwarfism.

Transcription factors DkBZR1/2 regulate cell wall degradation genes and ethylene biosynthesis genes during persimmon fruit ripening.

BRASSINAZOLE RESISTANT (BZR) transcription factors are critical components of the brassinosteroid signalling pathway, but their possible roles in fruit ripening have rarely been reported. In this study, four BZR sequences were isolated from persimmon fruit. Among the four BZR genes, DkBZR1/2 were expressed in persimmon fruit; DkBZR1 protein amount decreased and dephosphorylated DkBZR2 gradually accumulated during the storage period. DkBZR1/2 proteins were localized in both the nucleus and cytoplasm and accumulated in the nucleus after 24-epibrassinolide treatment. DkBZR1 suppressed the transcription of Diospyros kaki endo-1,4-betaglucanase 1 (DkEGase1) and 1-aminocyclopropane-1-carboxylate synthase 1 (DkACS1) by binding to the BR response element (BRRE) in their promoters, and DkBZR2 activated the transcription of pectate lyase 1 (DkPL1) and 1-aminocyclopropane-1-carboxylate oxidase 2 (DkACO2) by binding to the E-box motif in their promoters. Transient overexpression of DkBZR2 promoted the conversion of acid-soluble pectin to water-soluble pectin and increased ethylene production in persimmon fruit. Our findings indicate that DkBZR1 and DkBZR2 serve as repressors and activators of persimmon fruit ripening, respectively.

DkMYB14 is a bifunctional transcription factor that regulates the accumulation of proanthocyanidin in persimmon fruit.

Proanthocyanidins (PAs) are phenolic secondary metabolites that contribute to the protection of plant and human health. Persimmon (Diospyros kaki Thunb.) can accumulate abundant PAs in fruit, which cause a strong sensation of astringency. Proanthocyanidins can be classified into soluble and insoluble PAs; the former cause astringency but the latter do not. Soluble PAs can be converted into insoluble PAs upon interacting with acetaldehydes. We demonstrate here that DkMYB14, which regulates the accumulation of PA in persimmon fruit flesh, is a bifunctional transcription factor that acts as a repressor in PA biosynthesis but becomes an activator when involved in acetaldehyde biosynthesis. Interestingly, both functions contribute to the elimination of astringency by decreasing PA biosynthesis and promoting its insolubilization. We show that the amino acid Gly39 in the R2 domain and the ethylene response factor-associated amphiphilic repression-like motif in the C-terminal of DkMYB14 are essential for the regulation of both PA and acetaldehyde synthesis. The repressive function of DkMYB14 was lost after the mutation of either motif, and all activities of DkMYB14 were eliminated following the mutation of both motifs. Our results demonstrate that DkMYB14 functions as both a transcriptional activator and a repressor, directly repressing biosynthesis of PA and promoting its insolubilization, resulting in non-astringency in persimmon.

Effects of inside-out heat-shock via microwave on the fruit softening and quality of persimmon during postharvest storage.

Rapid postharvest softening largely limits the shelf-life of persimmon (Diospyros kaki L.) fruit. Microwave is a new environmental-friendly inside-out heat-shock approach, whose effect on the fruit softening and quality has not yet been investigated. The current study applied two kinds of microwave treatments (low-power long-time, LPLT, or high-power short-time, HPST) to persimmon fruit with comparison to the hot water (HW) treatments. The results showed both microwave treatments maintained firmness, facilitated the deastringency, and increased soluble solid contents (SSC) and sugar-acid ratio of persimmon fruit. The microwave treatments reduced the cellulose and pectin degradation, and inhibited the cellulase activity, resulting in a significantly higher firmness than HW treatment and control after 2 and 4 days of storage. Moreover, application of HPST treatment down-regulated gene expression of DkPG1, DkPE2 and DkEGase1 compared with untreated fruits. These results indicated that microwave treatment is a promising soft-delaying method for the preservation of persimmon fruit.

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.

Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO2 Treatment.

Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO2 treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO2 (95% CO2) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO2. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 µg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.