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Abstract Flowering plant (angiosperm) mitochondrial genomes are remarkably dynamic in their structures. We present the complete mitochondrial genome of hawthorn (Crataegus pinnatifida Bunge), a shrub that bears fruit and is celebrated for its extensive medicinal history. We successfully assembled the hawthorn mitogenome utilizing the PacBio long-read sequencing technique, which yielded 799,862 reads, and the Illumina novaseq6000 sequencing platform, which producing 6.6 million raw paired reads. The C. pinnatifida mitochondria sequences encompassed a total length of 440,295 bp with a GC content of 45.42%. The genome annotates 54 genes, including 34 that encode proteins, 17 that encode tRNA, and three genes for rRNA. A fascinating interplay was observed between the chloroplast and mitochondrial genomes, which share 17 homologous sequences sequences that rotal 1,933 bp. A total of 134 SSRs, 22 tandem repeats and 42 dispersed repeats were identified in the mitogenome. Four conformations of C. pinnatifida mitochondria sequences recombination were verified through PCR experiments and Sanger sequencing, and C. pinnatifida mitogenome is more likely to be assembled into three circular-mapping chromosomes. All the RNA editing sites that were identified C-U edits, which predominantly occurred at the first and second positions of the codons. Phylogenetic and collinearity analyses identified the evolutionary trajectory of C. pinnatifida, which reinforced the genetic identity of the hawthorn section. This unveiling of the unique multi-partite structure of the hawthorn mitogenome offers a foundational reference for future study into the evolution and genetics of C. pinnatifida.

IntroductionThe basic helix-loop-helix (bHLH) transcription factor family is one of the largest gene families in plants, extensively involved in plant growth, organ development, and stress responses. However, limited studies of this family are available in sea buckthorn (Hippophae rhamnoides).MethodsIn this study, we identified 144 bHLH genes in H. rhamnoides (HrbHLH) through a genome-wide search method, then explored their DNA and protein sequences and physicochemical properties.Results and discussionAccording to the sequence similarities, we classified them into 15 groups with specific motif structures. To explore their expressions, we performed gene expression profiling using RNA-Seq and identified 122 HrbHLH mRNAs were highly expressed, while the remaining 22 HrbHLH genes were expressed at very low levels in all 21 samples. Among these HrbHLH genes, HrbHLH47, HrbHLH74, HrbHLH90, HrbHLH131 showed the highest expression level in the root nodule, root, leaf, stem and fruit tissues. Furthermore, eleven HrbHLH genes displayed increased expressions during the fruit development process of sea buckthorn. Finally, we validated the expression patterns of HrbHLH genes using reverse transcription quantitative real-time PCR (QPCR). This comprehensive analysis provides a useful esource that enables further investigation of the physiological roles and molecular functions of the HrbHLH TFs.

Abstract 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.

Long-distance transfer of genetic material and metabolites between rootstock and scions is well documented in homo-grafted hybrids but has rarely been reported in genetically-distant grafts where the rootstock and scion belong to different families. In this study, we grafted Vitis vinifera scions onto Schisandra chinensis stocks and obtained 20 vegetative hybrids, Vitis vinifera/Schisandra chinensis (Vs). After 25 years of growth, we found that the phenotypes of the leaves, internodes, and fruits of the Vs hybrids above the graft union resembled an intermediate phenotype between V. vinifera and S. chinensis, and the new traits were stable when propagated vegetatively. We further analyzed genetic differences between Vv plants and Vs hybrids using high-throughput sequencing, while metabolomes were analyzed by liquid chromatography-mass spectrometry (LC-MS). We found a total of 2113 differentially expressed genes (DEGs). GO annotation and KEGG pathway enrichment analysis showed that these DEGs enriched mainly in oxidation-reduction and metabolic processes. Seventy-nine differentially expressed miRNAs (DEMs) containing 27 known miRNAs and 52 novel miRNAs were identified. A degradation analysis detected 840 target genes corresponding to 252 miRNAs, of which 12 DEMs and their corresponding target gene expression levels were mostly negatively correlated. Furthermore, 1188 differential metabolic compounds were identified. In particular, in Vs hybrids, the abundance of the metabolites schizandrin and gomisin as the main medicinal ingredients in S. chinensis were down-regulated and up-regulated, respectively. Our data demonstrated the effects of interfamily grafts on the phenotype, transcript profile and metabolites of the scion, and also provided new insight into the genetic, phenotypic, and metabolic plasticity associated with genetically distant grafted hybrids.

The ecological habitats of Chinese quince (Chaenomeles speciosa Nakai) fruits affect their phenotype. Currently, limited or no rapid method exists for classifying Chinese quince fruit from different ecosystems. This study developed a partial least squares discriminant analysis (PLS-DA) classification model to effectively and nondestructively classify 663 Chinese quince fruit samples from six environments in 2020. PLS-DA models and other variable selection approaches were used in this study. The near-infrared spectroscopy (NIRs) absorption spectra of raw Chinese quince fruit samples from six habitats showed a similar shape. The spectra of each environment showed little variance. The raw fruit spectra varied significantly among habitat categories after the first derivative preprocessing phase. The uninformative variable elimination (UVE) variable selection approach had greater calibration and validation set specificity of 0.93 and 0.98. This study found the best classification specificity using the UVE variable selection approach compared to other methods including the PLS-DA model without variable selection. The UVE approach improved Yunnan habitat categorization specificity from 86% to 88% when integrated with PLS-DA. Additionally, the validation set for quinces originating from Anhui, Chongqing, Hubei, Shandong, and Zhejiang achieved an ideal classification score of 100%. The findings of the study indicated that PLS-DA can serve as an alternative approach for classifying the habitats of Chinese quince fruits. When used in conjunction with other methods, this technique can assist researchers, scientists, and industry professionals in identifying the main factors responsible for significant variations in the habitats, composition, and quality of Chinese quince fruits.

Abstract 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.

Abstract 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.

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.

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.

Silvicultural practices greatly improve the economic value of wood products from forests. Stem dimensions, wood density, and stem form are closely linked to end-product performance. This research aimed to examine the effects of stand density and stem height on variables that reflect ring growth and wood properties of Sassafras tzumu stands during the self-thinning phase. Between the ages of 10 and 40 years, the number of stems per hectare has declined from 1,068 to 964 due to density-dependent mortality. As the relative stand density decreased, there were significant reductions in the average tree ring width (5.07–3.51 mm) and increases in latewood proportions (49.88–53.49%) and the density of the annual growth ring (165.60–708.58 kg/m3). Therefore, ring density, earlywood density, and latewood density increased with decreasing relative stand density after self-thinning occurred. Ring width, earlywood width, and latewood width significantly increased from the base to the apex of the stem. Stand density and stem height had additive effects on S. tzumu wood properties during the self-thinning phase. A shift in the growth allocation along the longitudinal stem in response to self-thinning resulted in decreasing radial growth, increasing wood density, and improved stem form. In summary, we found a significant influence of stand density on tree ring growth, wood quality, and stem form of S. tzumu trees during the self-thinning phase.

Abstract 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.

Basic helix–loop–helix (bHLH) proteins belong to one of the largest families involved in plant growth, development, signal transduction, and secondary metabolism. Although bHLH genes have been previously identified in persimmon (Diospyros kaki), systematic studies have not been reported. A total of 59 bHLH family members have been identified from the “Xiaoguotianshi” persimmon transcriptome. These proteins were clustered into 12 groups from I to XII based on their phylogenetic relationships with Arabidopsis thaliana. Combined with the phylogenetic analysis, in silico expression patterns of five developmental stages, the protein–protein interaction analysis between DkbHLH and DkMYB proteins showed that the bHLH_Cluster-15548.1 protein sequence was identified to be highly similar to the AtGL3 (AT5G41315.1) protein, which is associated with flavonoid and proanthocyanidin (PA) biosynthesis. This study presents the systematic analysis of bHLH genes from D. kaki and provides valuable information for further research on the involvement of bHLH protein in anthocyanin biosynthesis.

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.

To explore the mechanisms of the color formation of different colored persimmon fruits, we used two different colored persimmon cultivars (yellow-peeled persimmon fruit ‘Zhongshi No.6’ and red-peeled persimmon fruit ‘Hongdenglong’) as materials to study the synthesis and accumulation of carotenoids in three stages (full green, color transition, and full yellow or red) using targeted metabolomic and transcriptomic methods. A total of 14 carotenoids and 30 carotenoid lipids were identified in the peel of the two persimmon cultivars. After analysis, it was found that the total carotenoid content of the red persimmon cultivar was higher than that of the yellow persimmon cultivar. The contents of lycopene, α-carotenoid, β-carotenoid, (E/Z)-phytoene, and β-cryptoxanthin are the main reasons for the difference in total carotenoid content between the two persimmon cultivars, especially lycopene. Twelve structural genes involved in the metabolism of carotenoids were also found in this study. In comprehensive metabolome and transcriptome analysis, we found that, between the two persimmon cultivars, lycopene was the key metabolite responsible for the color difference, and PSY, LCYE, and ZDS were the key genes that regulated the differences in lycopene accumulation. The results of this study provide us with new information regarding persimmon fruit synthesis and accumulation. In addition, they also provide a theoretical foundation for improving persimmon fruit germplasm.

Transcription factors are crucial for plant growth and development. MADS-box, WRKY, NAC, and SBP-box, some of the most vital transcription factors, are not well identified in Diospyros spp., and their floral development and sex differentiation related functions remain unknown. We identified and analysed 53 MADS-box, 66 WRKY, 83 NAC, and 17 SBP-box transcription factors using the chromosomal D. oleifera genome. There were six DolSBPs identified as miR156 and miR157 targets. According to the multiple sequence alignments of Arabidopsis and D. oleifera proteins and their conserved domains and motifs, DolMADSs were divided into 23 type I and 30 type II. The DolWRKYs, DolNACs, and DolSBPs were divided into 7, 16, and 6 subgroups, respectively. It was found that one DolMADS, five DolWRKYs, one DolNAC, and four DolSBPs may promote unisexual female flowers development, while three DolMADSs, four DolWRKYs, and one DolNAC may enhance unisexual male flowers development. The functions among the different members of the same family may, thus, vary widely. The results will help to elucidate the evolution of the MADS-box, WRKY, NAC, and SBP-box genes families in D. oleifera and to determine their functions in flower development for Diospyros spp.

The complete chloroplast genome sequence of Hippophae rahmnoides subsp. sinensis was characterized from Illumina pair-end sequencing. The chloroplast genome of H. rahmnoides subsp. sinensis was 156,355 bp in length, containing a large single-copy region (LSC) of 84,002 bp, a small single-copy region (SSC) of 19,055 bp, and two inverted repeat (IR) regions of 26,649 bp. The overall GC content is 36.6%, while the corresponding values of the LSC, SSC, and IR regions are 64.5%, 69.2%, and 60.1%, respectively. The genome contains 131 complete genes, including 88 protein-coding genes, 38 tRNA genes (29 tRNA species), and 8 rRNA genes (4 rRNA species). The neighbour-joining phylogenetic analysis showed that H. rahmnoides subsp. sinensis and H. rahmnoides clustered together as sisters to other H. rahmnoides species.

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.

Most varieties of persimmon (Diospyros kaki Thunb.) are gynoecious, while just a few are either monoecious, androgynomonoecious, or androecious. Persimmon flowers initially contain the original androecium and gynoecium followed by arrest of either pistil or stamen primordia before maturity. Abortion of inappropriate primordium in persimmon may be related to programmed cell death (PCD). To test this hypothesis, hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cyt-c immunohistochemistry (IHC) assay, transmission electron microscopy (TEM) observation, and real-time quantitative polymerase chain reaction (RT-qPCR) were used to clarify the occurrence and molecular regulatory mechanism of PCD in male and female floral buds during the 14 days prior to the second crucial morphological stage when inappropriate sexual primordia were arrested to form unisexual flowers. Accordingly, dead cells in inappropriate sex organs were largely accumulated during the microsporocyte and macrosporocyte period of male and female floral buds, respectively. This may explain the abortion of inappropriate sex organs, leading to unisexual flowers. PCD is necessary for normal growth and development in persimmons, as dead cells could also be observed in the normal flower organs. High levels of a gene homologous to AMC9 may have accelerated the arrest of the pistil primordium during differentiation, leading to male unisexual flowers, and high levels of genes homologous to MeGI, BAG5, AifA, and HSP70 in female floral buds were positively correlated with the arrest of stamen primordium. Future studies may try to transform unisexual flowers into hermaphroditic flowers by the regulation of PCD artificially, which will be helpful to the controlled pollination experiments.