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.

A novel dual ATM/DNA-PK inhibitor, XRD-0394, potently radiosensitizes and potentiates PARP and topoisomerase I inhibitors.

A majority of cancer patients receive radiation therapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiation therapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage-response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. Additionally, in cells lacking BRCA1/2 XRD-0394 shows single agent activity and synergy in combination with PARP inhibitors. A Phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with RT has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with RT, PARP inhibitors and targeted delivery of topoisomerase I inhibitors.

Genetic insights into the dissolution of dioecy in diploid persimmon Diospyros oleifera Cheng.

BACKGROUND: Dioecy, a sexual system of single-sexual (gynoecious/androecious) individuals, is rare in flowering plants. This rarity may be a result of the frequent transition from dioecy into systems with co-sexual individuals. RESULTS: In this study, co-sexual expression (monoecy and hermaphroditic development), previously thought to be polyploid-specific in Diospyros species, was identified in the diploid D. oleifeara historically. We characterized potential genetic mechanisms that underlie the dissolution of dioecy to monoecy and andro(gyno)monoecy, based on multiscale genome-wide investigations of 150 accessions of Diospyros oleifera. We found all co-sexual plants, including monoecious and andro(gyno)monoecious individuals, possessed the male determinant gene OGI, implying the presence of genetic factors controlling gynoecia development in genetically male D. oleifera. Importantly, discrepancies in the OGI/MeGI module were found in diploid monoecious D. oleifera compared with polyploid monoecious D. kaki, including no Kali insertion on the promoter of OGI, no different abundance of smRNAs targeting MeGI (a counterpart of OGI), and no different expression of MeGI between female and male floral buds. On the contrary, in both single- and co-sexual plants, female function was expressed in the presence of a genome-wide decrease in methylation levels, along with sexually distinct regulatory networks of smRNAs and their targets. Furthermore, a genome-wide association study (GWAS) identified a genomic region and a DUF247 gene cluster strongly associated with the monoecious phenotype and several regions that may contribute to andromonoecy. CONCLUSIONS: Collectively, our findings demonstrate stable breakdown of the dioecious system in D. oleifera, presumably also a result of genomic features of the Y-linked region.

Shoot Organogenesis and Regeneration from Leaf Seedlings of Diospyros oleifera Cheng.

Persimmons (Diospyros) are economically important trees that are widely cultivated for wood production in China, and Diospyros oleifera Cheng is the main persimmon grafting stock. However, an efficient tissue culture system has not been perfected for D. oleifera due to the limits of proliferation and rooting cultures. Therefore, this study examined the effects of different plant growth regulators and concentrations on the primary culture of young embryos, induction of leaf callus, differentiation of adventitious shoots, and rooting culture of D. oleifera. The optimal formula for young embryo germination was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg/L gibberellic acid (GA3); after 25 days, the sprouting rate of the young embryos was 67.3%. The best medium for leaf callus induction was 1/2MS medium containing 2.0 mg/L of 6-benzylaminopurine (6-BA) and 0.5 mg/L of naphthaleneacetic acid (NAA), and the callus induction rate was 88.9%. Then, the callus was transferred to 1/2MS medium containing 2.0 mg/L of zeatin (ZT), 0.5 mg/L of NAA, and 2.0 mg/L of thidiazuron (TDZ) to induce adventitious shoots; after 25 days, 5.4 buds were produced per explant, and the induction rate of the adventitious shoots was 88.3%. The adventitious shoots were transferred to 1/2MS medium containing 2.0 mg/L of ZT, 2.0 mg/L of 6-(γ,γ-dimethylallylamino)purine (2iP), and 0.1 mg/L of indole acetic acid (IAA) for the proliferation culture, for which the multiplication coefficient approached 7.5. After multiplication, the adventitious shoots were inoculated into 1/2MS medium containing 1.0 mg/L of indole butyric acid (IBA), 0.5 mg/L of NAA, and 1.0 mg/L of kinetin (KT); the rooting rate was 60.2%, and the average number of roots was 6.9.

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.

Regioselective and Diastereoselective Halofunctionalization of Alkenes Promoted by Organophotocatalytic Solar Catalysis.

A visible-light metal-free photocatalytic regioselective and enantioselective alkene halofunctionalization reaction under mild conditions is reported. Various terminal and internal alkenes were transformed to their α-halogenated and α,ß-dibrominated derivatives in good to excellent yields within reaction time as short as 5 min. Water can be used as the "green" nucleophile and solvent in the halohydroxylation and halo-oxidation reactions. Different types of products can be obtained by adjusting the reaction conditions. In addition, sunlight is proved to produce products with similar yields, representing a practical example of solar synthesis and providing an opportunity for solar energy utilization.

Allele-aware chromosome-level genome assembly of the autohexaploid Diospyros kaki Thunb.

Artificially improving persimmon (Diospyros kaki Thunb.), one of the most important fruit trees, remains challenging owing to the lack of reference genomes. In this study, we generated an allele-aware chromosome-level genome assembly for the autohexaploid persimmon 'Xiaoguotianshi' (Chinese-PCNA type) using PacBio CCS and Hi-C technology. The final assembly contained 4.52 Gb, with a contig N50 value of 5.28 Mb and scaffold N50 value of 44.01 Mb, of which 4.06 Gb (89.87%) of the assembly were anchored onto 90 chromosome-level pseudomolecules comprising 15 homologous groups with 6 allelic chromosomes in each. A total of 153,288 protein-coding genes were predicted, of which 98.60% were functionally annotated. Repetitive sequences accounted for 64.02% of the genome; and 110,480 rRNAs, 12,297 tRNAs, 1,483 miRNAs, and 3,510 snRNA genes were also identified. This genome assembly fills the knowledge gap in the autohexaploid persimmon genome, which is conducive in the study on the regulatory mechanisms underlying the major economically advantageous traits of persimmons and promoting breeding programs.

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.

Comparative transcriptomic and metabolomic analyses reveal differences in flavonoid biosynthesis between PCNA and PCA persimmon fruit.

The fruit of the persimmon (Diospyros kaki.) has high economic and nutritional value and is rich in flavonoids. Flavonoids are essential secondary metabolisms in plants. The association between persimmon astringency and changes in the proanthocyanidins (a flavonoid subclass) content is well-known. However, information on the relationships between different astringency types and other flavonoid subclasses and biosynthetic genes is more limited. In this study, an initial correlation analysis between total flavonoids and fruit astringency type, and KEGG analysis of metabolites showed that flavonoid-related pathways were linked to differences between mature pollination-constant non-astringent (PCNA) varieties ('Jiro' and 'Yohou') and pollination-constant astringent (PCA) fruit varieties ('Zhongshi5' and 'Huojing'). Based on these findings, variations in the expression of genes and metabolites associated with flavonoid biosynthesis were investigated between typical PCNA ('Jiro') and PCA ('Huojing') persimmons during fruit development. The flavonoid concentration in 'Huojing' fruit was significantly higher than that of 'Jiro' fruit, especially, in levels of proanthocyanin precursor epicatechin and anthocyanin cyanidin derivatives. Combined WGCNA and KEGG analyses showed that genes such as PAL, C4H, CHI, CHS, F3H, F3'5'H, FLS, DFR, ANR, ANS, and UF3GT in the phenylpropanoid and flavonoid biosynthesis pathways may be significant factors impacting the proanthocyanin precursor and anthocyanin contents. Moreover, interactions between the R2R3MYB (evm.TU.contig7272.598) and WD40 (evm.TU.contig3208.5) transcription factors were found to be associated with the above structural genes. These findings provide essential information on flavonoid biosynthesis and its regulation in the persimmon and lay a foundation for further investigation into how astringency types affect flavor components in PCNA and PCA persimmons.

Regulatory mechanism of MeGI on sexuality in Diospyros oleifera.

Dioecy system is an important strategy for maintaining genetic diversity. The transcription factor MeGI, contributes to dioecy by promoting gynoecium development in Diospyros lotus and D. kaki. However, the function of MeGI in D. oleifera has not been identified. In this study, we confirmed that MeGI, cloned from D. oleifera, repressed the androecium development in Arabidopsis thaliana. Subsequently, chromatin immunoprecipitation-sequencing (ChIP-seq), DNA affinity purification-sequencing (DAP-seq), and RNA-seq were used to uncover the gene expression response to MeGI. The results showed that the genes upregulated and downregulated in response to MeGI were mainly enriched in the circadian rhythm-related and flavonoid biosynthetic pathways, respectively. Additionally, the WRKY DNA-binding protein 28 (WRKY28) gene, which was detected by ChIP-seq, DAP-seq, and RNA-seq, was emphasized. WRKY28 has been reported to inhibit salicylic acid (SA) biosynthesis and was upregulated in MeGI-overexpressing A. thaliana flowers, suggesting that MeGI represses the SA level by increasing the expression level of WRKY28. This was confirmed that SA level was lower in D. oleifera female floral buds than male. Overall, our findings indicate that the MeGI mediates its sex control function in D. oleifera mainly by regulating genes in the circadian rhythm, SA biosynthetic, and flavonoid biosynthetic pathways.

Prevalence of genotoxic bacteria in men undergoing biopsy for prostate cancer.

BACKGROUND: New evidence suggests that bacteria-produced DNA toxins may have a role in the development or progression of prostate cancer. To determine the prevalence of these genes in a noninfection (i.e., colonized) state, we screened urine specimens in men before undergoing a biopsy for prostate cancer detection. METHODS: We developed a multiplex polymerase chain reaction using three of the most described bacterial genotoxin gene primers: Colibactin (polyketone synthase [pks] gene island: clbN and clbB), cytotoxic necrotizing factor (cnf1) toxin, and cytolethal distending toxin B (cdtB) represented gene islands. After calibration on Escherichia coli samples of known genotypes, we used a training and validation cohort. We performed multiplex testing on a training cohort of previously collected urine from 45 men undergoing prostate biopsy. For the validation cohort, we utilized baseline urine samples from a previous randomized clinical trial (n = 263) with known prostate cancer outcomes. RESULTS: The prevalence of four common bacterial genotoxin genes detected in the urine before prostate biopsy for prostate cancer is 8% (25/311). The prevalence of pks island (clbN and clbB), cnf1, and cdt toxin genes are 6.1%, 2.4%, and 1.7%, respectively. We found no association between urinary genotoxins and prostate cancer (p = 0.83). We did identify a higher proportion of low-grade cancer (92% vs. 44%) in those men positive for urinary genotoxin and higher-grade cancer in those genotoxin negative (8% vs. 56%, p = 0.001). CONCLUSIONS: The prevalence of urinary genotoxins is low and does not correspond to a prostate cancer diagnosis. The urine was taken at one point in time and does not rule out the possibility of previous exposure.

Transcriptomic profiling analysis to identify genes associated with PA biosynthesis and insolubilization in the late stage of fruit development in C-PCNA persimmon.

PA-enhanced content causes astringency in persimmon fruit. PCNA persimmons can lose their astringency naturally and they become edible when still on the tree, which allows for conserves of physical and financial resources. C-PCNA persimmon originates in China. Its deastringency trait primarily depends on decreased PA biosynthesis and PA insolubilization at the late stage of fruit development. Although some genes and transcription factors that may be involved in the deastringency of C-PCNA persimmon have been reported, the expression patterns of these genes during the key deastringency stage are reported less. To investigate the variation in PA contents and the expression patterns of deastringency-related genes during typical C-PCNA persimmon 'Xiaoguo-tianshi' fruit development and ripening, PA content and transcriptional profiling were carried out at five late stages from 70 to 160 DAF. The combinational analysis phenotype, PA content, and DEG enrichment revealed that 120-140 DAF and 140-160 DAF were the critical phases for PA biosynthesis reduction and PA insolubilization, respectively. The expression of PA biosynthesis-associated genes indicated that the downregulation of the ANR gene at 140-160 DAF may be associated with PA biosynthesis and is decreased by inhibiting its precursor cis-flavan-3-ols. We also found that a decrease in acetaldehyde metabolism-associated ALDH genes and an increase in ADH and PDC genes might result in C-PCNA persimmon PA insolubilization. In addition, a few MYB-bHLH-WD40 (MBW) homologous transcription factors in persimmon might play important roles in persimmon PA accumulation. Furthermore, combined coexpression network analysis and phylogenetic analysis of MBW suggested that three putative transcription factors WD40 (evm.TU.contig1.155), MYB (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203), might connect and co-regulate both PA biosynthesis and its insolubilization in C-PCNA persimmon. The present study elucidated transcriptional insights into PA biosynthesis and insolubilization during the late development stages based on the C-PCNA D. kaki genome (unpublished). Thus, we focused on PA content variation and the expression patterns of genes involved in PA biosynthesis and insolubilization. Our work has provided additional evidence on previous knowledge and a basis for further exploration of the natural deastringency of C-PCNA persimmon.

Agrobacterium tumefaciens-Mediated Transformation of Candida glabrata.

The use of broad-spectrum antimycotic therapy, immunosuppressive therapy, and indwelling medical devices has contributed to the increased frequency of mucosal and systemic infections caused by Candida glabrata. A major concern for C. glabrata and other Candida spp. infections is the increase in drug resistance. To address these issues, additional molecular tools for the study of C. glabrata are needed. In this investigation, we developed an Agrobacterium tumefaciens transformation system for C. glabrata. A number of parameters were investigated to determine their effect on transformation frequency, and then an optimized protocol was developed. The optimal conditions for the transformation of C. glabrata were found to be an infection incubation temperature of 26 °C, 0.2 mM acetosyringone in both induction media and co-culture media, 0.7% agar concentration, and a multiplicity of infection of 50:1 A. tumefaciens to C. glabrata. Importantly, the frequency of multiple integrations was low (5%), demonstrating that A. tumefaciens generally integrates at single sites in C. glabrata, which is consistent with other fungal A. tumefaciens transformation systems. The development of this system in C. glabrata adds another tool for the molecular manipulation of this increasingly important fungal pathogen.

Effects of Different Chemicals on Sexual Regulation in Persimmon (Diospyros kaki Thunb.) Flowers.

Research on crop sexuality is important for establishing systems for germplasm innovation and cultivating improved varieties. In this study, androecious persimmon trees were treated with various concentrations of ethrel (100, 500, and 1,000 mg/L) and zeatin (1, 5, and 10 mg/L) to investigate the morphological, physiological, and molecular characteristics of persimmon. Ethrel at 1,000 mg/L and zeatin at 10 mg/L both significantly reduced the stamen length and pollen grain diameter in androecious trees. Ethrel treatment also led to reduced stamen development with degenerated cellular contents; zeatin treatment promoted the development of arrested pistils via maintaining relatively normal mitochondrial morphology. Both treatments altered carbohydrate, amino acid, and endogenous phytohormone contents, as well as genes associated with hormone production and floral organ development. Thereafter, we explored the combined effects of four chemicals, including ethrel and zeatin, as well as zebularine and 5-azacytidine, both of which are DNA methylation inhibitors, on androecious persimmon flower development. Morphological comparisons showed that stamen length, pollen viability, and pollen grain diameter were significantly inhibited after combined treatment. Large numbers of genes involving in carbohydrate metabolic, mitogen-activated protein kinase (MAPK) signaling, and ribosome pathways, and metabolites including uridine monophosphate (UMP) and cyclamic acid were identified in response to the treatment, indicating complex regulatory mechanisms. An association analysis of transcriptomic and metabolomic data indicated that ribosomal genes have distinct effects on UMP and cyclamic acid metabolites, explaining how male floral buds of androecious persimmon trees respond to these exogenous chemicals. These findings extend the knowledge concerning sexual differentiation in persimmon; they also provide a theoretical basis for molecular breeding, high-yield cultivation, and quality improvement in persimmon.

Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Genomics-based discrimination of 2n gamete formation mechanisms in polyploids: a case study in nonaploid Diospyros kaki 'Akiou'.

Unreduced gametes (2n gametes), possessing double the haploid genome, whatever ploidy that happens to be, are a common source of ploidy variation in plant populations. First and second division restitution (FDR and SDR) are the dominant mechanisms of 2n gamete production; all else being equal, FDR gametes have a higher degree of heterozygosity, thus they are advantageous in breeding. The discrimination of these mechanisms from the consequence of hybridization is challenging, especially in higher polyploids, and usually requires information on centromere location. In this study, we propose a genotyping-based strategy to uncover the mechanisms of 2n gamete formation in progeny that has a higher ploidy than its parents. Simulation of 2n gamete production revealed that FDR and SDR pathways can be discriminated based on allele transmission patterns alone without information on centromere location. We applied this strategy to study the formation mechanism of a nonaploid Diospyros kaki 'Akiou', which was bred via hybridization between D. kaki hexaploid cultivars. The result demonstrated that 'Akiou' was derived from the fertilization of a normal female gamete by a 2n male gamete and that this 2n gamete was produced through FDR. Consequently, the distinct duplex transmission pattern in the FDR gamete enabled us to infer the genomic characteristics of polyploid persimmon. The method could be tested only for the plant being polypoid, which allows for the ability to discriminate causes of 2n gamete formation using allele dosage in progeny, and will be useful in future studies of polyploid genomics.

Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens.

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

The beneficial effects of polysaccharide obtained from persimmon (Diospyros kaki L.) on the proliferation of Lactobacillus and gut microbiota.

The objective of this study was to investigate the effect of polysaccharide extracts from persimmon (PPE) on the proliferation of Lactobacillus and the gut microbiota of mice. Lactobacillus strains were cultured in medium containing PPE, and differential gene expression was evaluated using transcriptomics. In addition, 16S rDNA was employed to analyze the abundance and diversity of fecal colonies in mice, and the influence of PPE on the intestinal flora in mice was further examined. The results showed that Lactobacillus acidophilus NCFM and Lactobacillus acidophilus CICC 6075 could proliferate in PPE medium. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis indicated that glucose metabolism-related genes, such as phosphoyruvate hydratase (eno) and PTS mannose transporter subunit IIAB (manX), were up-regulated. The metabolic pathways of fructose and mannose were also significantly up-regulated. After gavage of mice with PPE, 16S rDNA sequencing of mouse feces indicated that the beneficial bacteria in the intestines proliferated and the abundance of harmful bacteria was reduced. PPE can maintain the balance of intestinal microorganisms in mice. Therefore, PPE has a significant positive effect on both Lactobacillus proliferation and gut microbiota of mice.

Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic floral buds of andromonoecious Diospyros kaki Thunb.

BACKGROUND: Persimmon (Diospyros kaki Thunb.) has various labile sex types, and studying its sex differentiation can improve breeding efficiency. However, studies on sexual regulation patterns in persimmon have focused mainly on monoecy and dioecy, whereas little research has been published on andromonoecy. In order to reveal the sex differentiation regulation mechanism of andromonoecious persimmon, we performed histological and cytological observations, evaluated OGI and MeGI expression and conducted phytohormones assays and mRNA and small RNA transcriptome analyses of the male and hermaphroditic floral buds of the andromonoecious persimmon 'Longyanyeshi 1'. RESULTS: Stages 2 and 4 were identified as the critical morphological periods for sex differentiation of 'Longyanyeshi 1' by histological and cytological observation. At both stages, OGI was differentially expressed in male and hermaphroditic buds, but MeGI was not. This was different from their expressions in dioecious and monoecious persimmons. Meantime, the results of phytohormones assays showed that high IAA, ABA, GA3, and JA levels at stage 2 may have promoted male floral bud differentiation. However, high JA levels at stage 4 and high ZT levels at stages 2 and 4 may have promoted hermaphroditic floral bud differentiation. In these phytohormone biosynthesis and signaling pathways, 52 and 54 differential expression genes (including Aux/IAA, ARFs, DELLA, AHP, A-ARR, B-ARR, CYP735A, CRE1, PP2C, JAZ, MYC2, COI1, CTR1, SIMKK, ACO, and MPK6) were identified, respectively. During the development of male floral buds, five metacaspases genes may have been involved in pistil abortion. In addition, MYB, FAR1, bHLH, WRKY, and MADS transcription factors might play important roles in persimmon floral bud sex differentiation. Noteworthy, miR169v_1, miR169e_3, miR319_1, and miR319 were predicted to contribute to phytohormone biosynthesis and signaling pathways and floral organogenesis and may also regulate floral bud sex differentiation. CONCLUSION: The present study revealed the differences in morphology and phytohormones content between male and hermaphroditic floral buds of 'Longyanyeshi 1' during the process of sex differentiation, and identified a subset of candidate genes and miRNAs putatively associated with its sex differentiation. These findings can provide a foundation for molecular regulatory mechanism researching on andromonoecious persimmon.

Study of Sexual-Linked Genes (OGI and MeGI) on the Performance of Androecious Persimmons (Diospyros kaki Thunb.).

It is reported that the production of floral sexual phenotype in hexaploid monoecious persimmon (Diospyros kaki) is closely related to a pseudogene called OGI, and a short interspersed nuclear element (SINE)-like insertion (named Kali) in the OGI promoter leads to the gene silence. As a result, DNA methylation level of MeGI promoter determines the development of male or female flowers. However, the molecular mechanism in androecious D. kaki, which only bear male flowers, remains elusive. Here, real-time quantitative polymerase chain reaction (RT-qPCR), molecular cloning, and bisulfite PCR sequencing technique were carried out using 87 materials, including 56 androecious resources, 15 monoecious, and 16 gynoecious cultivars, to investigate the performance of OGI and MeGI on the specific androecious type of D. kaki in China. In conclusion, the Kali insertion was exactly located in the OGI promoter region, and the OGI gene and the Kali sequence were existing and conserved in androecious D. kaki. Meanwhile, we also demonstrated that the MeGI gene was widespread in our investigated samples. Ultimately, our result convincingly provided evidence that the low expression of OGI is probably ascribed to the presence of Kali displaying strong methylation in the OGI promoter, and low expression of MeGI, as well as high DNA methylation level, in the promoter was closely connected with the production of male flowers; this result was consistent with the monoecious persimmon model. Our findings provide predominant genetic aspects for investigation into androecious D. kaki, and future perfecting the sex-determining mechanisms in persimmon.