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   D. kaki genome (unpublished).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.

   Subject terms: Plant sciences, Plant breeding, Plant cell biology,
   Plant physiology, Secondary metabolism

Introduction

   Diospyros kaki Thunb. is a member of the family Ebenaceae and is among
   the important fruit trees cultivated and harvested worldwide^[40]1.
   Based on genetic traits and astringency removal, persimmon varieties
   can be classified into pollination-constant non-astringent (PCNA),
   pollination-variant non-astringent (PVNA), pollination-variant
   astringent (PVA), and pollination-constant astringent (PCA)
   types^[41]2. The Chinese PCNA (C-PCNA) and Japanese PCNA (J-PCNA)
   persimmon can lose astringency naturally on the tree without any
   postharvest treatment but have different proanthocyanidin (PA)
   accumulation mechanisms^[42]3,[43]4. The characteristic of losing
   astringency in J-PCNA and C-PCNA persimmon is genetically controlled by
   a recessive and a dominant allele, respectively^[44]5. Because of
   dominant inheritance, the C-PCNA genotype is often selected as the
   superior parent for persimmon breeding programs.

   Both the “dilution effect” and “coagulation effect” contribute to the
   deastringency of C-PCNA persimmon fruit at the late stage^[45]6. The
   “dilution effect” means that PA biosynthesis is reduced with the fruit
   grows larger, resulting in a decrease in the proportion of soluble PA
   content in persimmon fruit^[46]7,[47]8. PAs are synthesized via the
   chorismic acid pathway, phenylpropane metabolic pathway, flavonoid
   synthesis and proanthocyanidin pathway^[48]9. The structural genes
   encoding the enzymes have been identified in the three pathways,
   including chalcone isomerase (CHI), chalcone synthase (CHS),
   leucoanthocyanidin reductase (LAR), flavonoid 3′-hydroxylase (F3′H),
   and anthocyanidin reductase (ANR)^[49]10. The “coagulation effect”
   refers to losing astringency by PA insolubilization by converting
   soluble PAs into insoluble PAs with acetaldehyde^[50]6. The
   acetaldehyde metabolic pathway plays an important role in the
   astringency loss of PCNA persimmon^[51]11,[52]12 and is catalyzed by
   pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and
   acetaldehyde dehydrogenase (ALDH)^[53]13,[54]14.

   Transcription factors involved in PA biosynthesis have been identified
   previously. The R2R3MYB, bHLH, and WD40 (MBW) complex regulates the
   expression of the structural genes DFR, ANS, and ANR of late
   flavonoid/phenylpropanoid metabolism^[55]15. The expression of PA
   biosynthesis genes is specifically induced by AtTT2 (AtMYB123), AtTT8
   (AtbHLH042), and AtTTG1 (WD40-repeat protein) in Arabidopsis
   thaliana^[56]16. In strawberries, FaTTG1 can form a complex with
   FaMYB9/FaMYB11 and FabHLH3, which increases the accumulation of PA by
   upregulating the expression of ANS and LAR^[57]17. In persimmon,
   reduced expression of DkMYB4 in J-PCNA leads to the downregulation of
   PA biosynthesis during the early stages^[58]18, and in C-PCNA, reduced
   expression of DkMYB14 results in the nonastringency, which directly
   represses PA biosynthesis and promotes its insolubilization, leading to
   nonastringency^[59]19. A bHLH gene (DkMYC1) involved in PA synthesis
   regulation was isolated from ‘Luotian-tianshi’, and the expression
   pattern of DkMYC1 was correlated with PA accumulation and the
   expression patterns of DkANR and DkF3′5′H^[60]20.

   Several studies have tried to elucidate the underlying molecular basis
   for C-PCNA persimmon deastringency, either by ethanol or by 40 °C water
   treatments via transcriptome sequencing. These procedures have no doubt
   provided crucial insight into the deastringency process^[61]21,[62]22;
   however, C-PCNA persimmon can lose its astringency naturally. The PA
   biosynthesis and insolubilization in C-PCNA persimmon have not been
   adequately explained in the natural state, especially at the late
   developmental stages of fruit. In this study, fruits from the C-PCNA
   genotype ‘Xiaoguo-tianshi’ persimmon were specifically collected at
   five later stages, as natural deastringency occurs late on the C-PCNA
   persimmon tree. In addition, the completion of the C-PCNA D. kaki
   reference genome (unpublished) has facilitated the genome-wide analysis
   of dynamic gene expression during fruit development; thus, the PA
   content combined with RNA-seq analysis at key late development stages
   can provide basic support for C-PCNA persimmon natural astringency
   loss.

Material and methods

Plant material

   Fruits of C-PCNA persimmon (D.kaki, variety ‘Xiaoguo-tianshi’) at five
   developmental stages (T1 = 70, T2 = 100, T3 = 120, T4 = 140, T5 = 160
   DAF) were harvested from Yuanyang County, Henan Province, China (34°
   55′ 18″–34° 56′ 27″ N, 113° 46′ 14″–113° 47′ 35″ E). The collection and
   conservation of plant material in this study complied with relevant
   institutional, national, and international guidelines and legislation.
   Fresh material was immediately frozen in liquid nitrogen and stored
   at − 80 °C until the PA content and RNA extraction were analyzed.

Extraction and determination of PAs

   Approximately 2.5 g (precise weight) of the persimmon fruit powder was
   extracted with 80% (v/v) ethanol in an ultrasonic bath for 30 min, and
   the precipitates were suspended in 1% HCl-MeOH for insoluble PA
   extraction. Soluble and insoluble PA contents were detected using the
   Folin-Ciocalteu method as described by Oshida et al.^[63]23.

RNA extraction, transcriptome sequencing, and data analysis

   Total RNA was extracted using the UNlQ-10 Column TRIzol Total RNA
   Isolation Kit (B511321; Sangon, Shanghai, China). The quality and
   quantity of the RNA were tested on a 1% agarose gel and then assessed
   on a Merinton SMA4000 spectrophotometer (Merinton Inc., Beijing,
   China). The paired-end cDNA sequencing libraries of fruits at five
   stages were prepared with three biological replicates and then
   sequenced on an Illumina NovaSeq platform (Illumina, San Diego, CA,
   USA). The files of raw reads were cleaned by removing adapter sequences
   and then the clean reads were mapped to the hexaploid C-PCNA persimmon
   genome (variety ‘Xiaoguo-tianshi’, unpublished).

   DEseq2 was used to detect the DEGs^[64]24, with a padj ≤ 0.05 and
   |log2-fold change|≥ 1. Gene Ontology (GO)
   ([65]www.geneontology.org)^[66]25 and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) ([67]www.kegg.jp/kegg/kegg1.html)^[68]26 databases were
   used for functional enrichment analysis by ClusterProfiler
   (3.8.1)^[69]27. The R packages Cluster, Biobase, and Q-value were used
   to display the expression profiles of the DEGs in the five stages using
   K-means clustering. Heatmaps were prepared with the online website
   Hiplot. Pearson’s correlation analysis was performed using SPSS
   statistical software (v 24.0; SPSS Inc., Chicago, IL, USA). The
   coexpression networks were constructed using DEGs and were analyzed
   using the R package WGCNA^[70]28 and visualized using Cytoscape
   (v3.9.1) and its plugin cytoHubba^[71]29.

Quantitative RT-PCR analysis

   Total RNA for the RNA-seq analysis was reverse-transcribed into cDNA
   with the TRUE-script First-Strand cDNA Synthesis Kit (Kemix, Beijing,
   China). Reactions were run on a LightCycler 480 II (Roche) with a
   96-well plate. The reaction conditions for each gene were 95 °C for
   3 min, followed by 45 cycles of 5 s at 95 °C and 30 s at 55–60 °C.
   Three technical replicates were performed for each gene. GAPDH was used
   as the reference gene^[72]30. The RT-qPCR primers are listed in Table
   [73]S1.

Identification of MYB-bHLH-WD40 complex members in C-PCNA persimmon

   To identify MYB-bHLH-WD40 Complex Members in the persimmon genome,
   local tBLASTp ([74]https://blast.ncbi.nlm.nih.gov/) (E-value ≤ 1e^−5)
   was performed using the sequences of homologous MBW complex proteins in
   Arabidopsis thaliana^[75]16,[76]31, Malus × domestica^[77]32, Vitis
   vinifera^[78]33–[79]35, and Fragaria × ananassa^[80]17 (Table [81]S2).
   The online website Pfam ([82]https://pfam.xfam.org/) was used to remove
   sequences without conserved MYB_DNA-binding (PF00249.30, PF13921.5),
   bHLH-MYC_N (PF14215.5), or WD40 (PF00400.31) domains (Table [83]S2).
   Multiple sequence alignments and phylogenetic analysis were implemented
   using Clustal X^[84]36 and MEGA 5^[85]37 software, respectively.

Result

Phenotype and PA content of C-PCNA persimmon fruit

   To evaluate the C-PCNA persimmon deastringency process, the fruit’s
   late developmental stages were grouped into 5 time points (T1–T5) and
   the fruits were harvested at 70, 100, 120, 140, and 160 days after
   flower (DAF) (Fig. [86]1a). Morphological differences were measured
   based on the changes in fruit size and weight. Persimmon fruit
   experienced the fastest growth stage from 120 to 140 DAF, with a
   39.67–59.27 g weight, and then grew slowly until deastringency
   (Fig. [87]1b). During the ‘expansion’ phase, the soluble and insoluble
   PA contents decreased, followed by a continuous decrease in the soluble
   PA content in the T4-T5 stages and a significant increase in the
   insoluble PA content. This resulted in the astringency loss of
   ‘Xiaoguo-tianshi’ persimmon fruit at stage T5 (Fig. [88]1c).

Figure 1.

   [89]Figure 1
   [90]Open in a new tab

   Five developmental stages of C-PCNA persimmon ‘Xiaoguo-tianshi’. (a)
   Fruit morphology. (b) Mean fruit weight and diameter. (c) Soluble PAs,
   insoluble PAs, and total PA content. Values are the means of four
   replicates. **Indicates a significant difference at P < 0.01. *
   indicates a significant difference at P < 0.05.

RNA-Seq of C-PCNA persimmon developing fruit

   After the low-quality reads, the transcriptome sequencing of the
   ‘Xiaoguo-tianshi’ fruit at five developmental stages yielded 99.24 GB
   of data. Each filtered sample consisted of 6.79 GB of high-quality data
   with a Q30 base percentage of 95.28% on average, approximately 87.61%
   of the reads could be with the reference D. kaki genome, and 3,472
   novel genes were also identified. These results indicated that
   sequencing data accuracy was sufficient for further analysis. A
   principal component analysis (PCA) based on Fragments Per Kilobase of
   transcript per Million mapped reads (FPKM) values separated the samples
   into five distinct groups, with each sample making a separate group
   with its replicates, indicating that there were good correlations and
   differences among the samples (Fig. [91]2a). To analyze the expression
   variabilities associated with C-PCNA persimmon fruit PA biosynthesis
   and insolubilization at different developmental stages, genes in four
   pairwise transcriptome groups (T2 vs T1, T3 vs T2, T4 vs T3, and T5 vs
   T4) were compared. In all, 7,102 genes were significantly
   differentiated in the four groups, including 2,851 DEGs in T2 vs T1,
   2,428 DEGs in T3 vs T2, 2,996 DEGs in T4 vs T3, and 1,914 DEGs in T4 vs
   T3. The Venn diagram shows that 1,484, 1039, 1,384, and 793 DEGs were
   differentially expressed in the four respective groups (Fig. [92]2b–e).
   Most of the DEGs were downregulated in the control groups. The
   reliability of the transcriptomic results was confirmed by the
   consistent gene expression trend of 9 DEGs, which was observed using
   real-time quantitative reverse transcription polymerase chain reaction
   (RT-qPCR) analysis (Fig. [93]S1) compared with the transcriptome FPKM
   data.

Figure 2.

   [94]Figure 2
   [95]Open in a new tab

   Overview of the C-PCNA Persimmon Developing Fruit transcriptome. (a)
   Principal component analysis. (b) Summary of DEGs in all combinations
   of developmental stage comparisons. The number of total DEGs (c),
   upregulated DEGs (d), and downregulated DEGs (e) are presented by Venn
   diagrams (|log2-fold change|> 1 and padj < 0.05).

Functional enrichment analysis of DEGs

   The GO and KEGG databases were used to further analyze DEGs in four
   comparison groups (T2 vs T1, T3 vs T2, T4 vs T3, and T5 vs T4) using
   padj < 0.05 to indicate a significant difference. The results of GO
   functional enrichment analysis revealed significant enrichment in four
   terms associated with PA transport: UDP-glycosyltransferase,
   glucosyltransferase, drug transmembrane transporter, and transferase
   activities. GO terms of glucosyltransferase activity were observed in
   T2 vs T1, T4 vs T3, and T5 vs T4, and the other three terms were only
   shared in T5 vs T4. KEGG pathway enrichment analysis revealed that T4
   vs T3 had enriched phenylpropanoid biosynthesis. The significantly
   enriched KEGG pathway term flavonoid biosynthesis was shared in T4 vs
   T3 and T5 vs T4. The two candidate KEGG pathways were considered to be
   heavily involved in PA accumulation. Our results suggest that the T3–T4
   stages may be a key developmental period for the persimmon PA
   biosynthesis process phenylpropane metabolic pathway and flavonoid
   synthesis proanthocyanidin pathway. Stages T4-T5 may be involved in
   persimmon PA transport and the subsequent process (Fig. [96]S2).

Gene expression trends during C-PCNA persimmon fruit development

   Six gene profiles based on their expression patterns were clustered
   together, as shown in Fig. [97]3a. The genes within these 6 expression
   profiles were analyzed by KEGG annotation (Fig. [98]3b). Cluster 2
   comprised 1,090 DEGs and their expression showed a downregulating trend
   from T1 to T5, and these genes rapidly decreased between T2 and T4.
   Cluster 5 contained 1,332 DEGs, and their expression increased at late
   time points. The expression pattern of cluster 2 was positively
   consistent with soluble PA and total PA content (Fig. [99]1b), whereas
   5 was positively aligned with insoluble PA content. DEGs in cluster 2
   were significantly enriched in flavonoid biosynthesis and
   photosynthesis, where flavonoid biosynthesis had an enrichment factor
   of nearly 0.4, indicating greater intensity. The plant hormone signal
   transduction and MAPK signaling pathways were significantly enriched in
   cluster 5. Among those, some of the final metabolites of flavonoid
   biosynthesis are proanthocyanins. The results of the functional
   classification analysis of the common expression patterns of DEGs
   combined with KEGG showed that genes in cluster 2 and cluster 5 might
   be associated with PA accumulation and were selected for subsequent
   analysis.

Figure 3.

   [100]Figure 3
   [101]Open in a new tab

   Gene expression profiles and KEGG enrichment analysis of the DEGs in
   the six common expression clusters composing the fruit transcriptome.
   (a) Heatmap of the overall common expression pattern. Heat maps depict
   the normalized gene expression values, which represent the mean value
   of three biological replicates. (b) Expression patterns and KEGG
   pathway annotations of 6 clusters. Each line represents the normalized
   gene expression values for an individual transcript. The y-axis of each
   cluster is the pathway name, while the x-axis represents the rich
   factor. Significantly overrepresented KEGG categories are represented
   by red dots.

DEGs related to PA biosynthesis and insolubilization

   PA accumulation gene expression levels across the five developmental
   stages were investigated by generating heat maps (Fig. [102]4). We
   focused on the 43 DEGs involved in the PA biosynthesis and
   insolubilization pathway, which included 18 genes from cluster 2, 8
   from cluster 5, 7 from cluster 6, 5 from cluster 4, 3 from cluster 1,
   and 2 from cluster 3. Since the aim was to identify genes associated
   with PA content variation in the C-PCNA persimmon, our analysis was
   focused on DEGs in clusters 2 and 5. Nearly all DEGs from cluster 2
   were downregulated in T4 vs T3 and T5 vs T4. Most of the cluster 2
   genes were involved in PA biosynthesis, such as DAHPS
   (evm.TU.contig8955.26) and DHD/SDH (evm.TU.contig8908.198), the
   precursors of proanthocyanin biosynthesis that participate in the
   chorismic acid pathway. PAL (evm. TU.contig9504.51,
   evm.TU.contig3686.428), C4H (evm.TU.contig22.251), and 4CL
   (evm.TU.contig7272.385) are phenylpropanoid genes. ANR
   (evm.TU.contig4466.754), ANS (evm.TU.contig5828.5), CHS
   (evm.TU.contig4175.257), F3’H (evm.TU.contig7272.244), F3′5'H
   (evm.TU.contig31.16), and F3H (evm.TU.contig4466.49) are involved in
   flavonoid synthesis and the proanthocyanidin pathway. Cluster 5 DEGs,
   however, were upregulated in T5 vs T4, and most of these genes were
   involved in PA insolubilization, such as PDC (evm.TU.contig3684.117,
   evm.TU.contig4466.583, evm.TU.contig8029.113, evm.TU.contig9507.111),
   ADH (evm.TU.contig2067.296, evm.TU.contig4397.68), and ALDH
   (evm.TU.contig7272.653), which take part in acetaldehyde metabolism.
   However, the MATE gene (evm.TU.contig2065.66) was significantly
   downregulated at T4 vs T3, which might be involved in transporting PA
   to the vacuole. This study revealed that PA biosynthesis-associated
   genes were highly downregulated in T4 vs T3 and T5 vs T4, whereas PA
   insolubilization genes were highly upregulated in T5 vs T4, providing a
   reasonable explanation for the variation in PA content at stages T4–T3
   and T5–T4.

Figure 4.

   [103]Figure 4
   [104]Open in a new tab

   Expression patterns of 43 DEGs involved in PA biosynthesis and
   insolubilization. PA biosynthesis pathways include the chorismic acid
   pathway, phenylpropane pathway, flavonoid synthesis and
   proanthocyanidin-specific pathway. PA insolubilization refers to
   converting soluble PA into insoluble PA with acetaldehyde metabolism.
   Heatmaps depict the normalized gene expression values, which represent
   the mean value of three biological replicates.

Phylogenetic analysis and expression patterns of the persimmon MYB-bHLH-WD40
complex

   It has been reported that an MBW complex consisting of MYB, bHLH, and
   WD40 is a critical factor for PA biosynthesis. Our study discovered a
   total of 14 R2R3-MYB, 13 bHLH, and 13 WD40 transcription factors from
   the D.kaki genome. These transcription factors clustered with
   homologous MYB-bHLH-WD40 complex genes of A. thaliana, V. vinifera, and
   F. ananassa (Fig. [105]5a–c). Among these transcription factors, only 6
   R2R3-MYB, 5 bHLH, and 2 WD40 genes were differentially expressed during
   C-PCNA persimmon fruit development, so these were used for further
   analysis. Of the 6 R2R3-MYB family genes, 5 showed significantly
   decreased expression at T4 vs T3. The evm.TU.contig8910.486 and
   evm.TU.contig7396.13 gene expression showed a remarkably positive
   correlation with total PA content (P < 0.05), where
   evm.TU.contig8910.486 was from cluster 2. All 5 bHLH family genes were
   downregulated at T4 vs T3, and of these 5 genes, the expression of
   evm.TU.contig1398.203 and evm.TU.contig8910.303 cluster 2 genes
   presented a positive correlation with total PA content (P < 0.05). Only
   1 WD40 gene, evm.TU.contig1.155, from cluster 5 exhibited a
   significantly negative correlation with soluble PA content (P < 0.01).

Figure 5.

   [106]Figure 5
   [107]Open in a new tab

   Phylogenetic trees of the MYB-bHLH-WD40 complex members in C-PCNA
   persimmon. (a) Phylogenetic tree of R2R3MYB, (b) phylogenetic tree of
   bHLH, and (c) phylogenetic tree of WD40. Heat maps depict the
   normalized gene expression values, which represent the mean value of
   three biological replicates.

Weighted gene coexpression network analysis

   To construct a potential regulatory network of PA accumulation in the
   C-PCNA persimmon, weighted gene coexpression network analysis (WGCNA)
   was performed using 7,102 DEGs, and 15 merged coexpression gene modules
   were identified. Among these modules, the brown module contained 904
   genes, which had a significant and positive correlation with soluble PA
   (r = 0.76, P = 0.001) and total PA content (r = 0.94, P = 2e^−07). The
   yellow module (864 DEGs) had a significant negative correlation with
   insoluble PA content (r = 0.75, P = 0.001) (Fig. [108]6a). This
   indicates that the brown and yellow modules may have a closer
   association and more significant correlations with PA biosynthesis and
   PA insolubilization, respectively.

Figure 6.

   [109]Figure 6
   [110]Open in a new tab

   Weighted gene coexpression network analysis. (a) Gene modules defined
   using WGCNA and the association with soluble PA, insoluble PA, and
   total PA content. The numbers in the heatmap show the P-value (lower)
   and the correlation (upper). (b) The linkages of PA biosynthesis and
   insolubilization DEGs in yellow and brown modules, and the circles are
   sized by the gene connectivity.

   Strikingly, the brown module genes included the structural genes DAHPS
   (evm.TU.contig8955.26), PAL (evm.TU.contig9504.51), C4H
   (evm.TU.contig22.251), 4CL (evm.TU.contig7272.385), ANS
   (evm.TU.contig5828.5), F3'H (evm.TU.contig7272.244), F3′5'H
   (evm.TU.contig31.16), and F3H (evm.TU.contig4466.49), which have been
   linked to PA biosynthesis. Yellow module genes included the structural
   genes PDC (evm.TU.contig4466.583, evm.TU.contig9507.111,
   evm.TU.contig3684.117) and ADH (evm.TU.contig4397.68,
   evm.TU.contig2067.296), which have been linked to PA insolubilization.
   The evm.TU.contig1407.18 (probable aminotransferase ACS10) and
   evm.TU.contig1077.85 (AHA10) had high interconnections in the brown
   module and the yellow module, respectively. In addition, the critical
   transcription factors MYB (evm.TU.contig8910.486), bHLH
   (evm.TU.contig1398.203), and WD40 (evm.TU.contig1.155) were identified.
   Among them, the WD40 transcription factor in the yellow module was
   connected with the PA insolubilization genes ADH and PDC and presented
   a significant negative correlation with soluble PA content. Combined
   with the MYB-bHLH-WD40 complex analysis above, WD40 probably interacts
   with MYB (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203) in
   the brown module, connecting and coregulating PA biosynthesis and
   insolubilization in C-PCNA persimmon (Fig. [111]6b).

Discussion

   An overview of genes and transcription factors associated with PA
   content variation in C-PCNA persimmon fruits at five late stages was
   obtained using the transcriptome based on a well-annotated C-PCNA
   persimmon genome. According to previous studies, research on the
   natural deastringency of the C-PCNA genotype has always been more
   inclined towards fruit that underwent 40 °C water deastringent
   treatment^[112]21 or the whole development stages as materials^[113]38,
   rather than using fruit at late developmental stages, which is more
   critical for natural C-PCNA deastringency. In the present study, we
   analyzed PA content and constructed C-PCNA genotype libraries
   representing five late development stages and used the transcriptome to
   evaluate differentially expressed genes and TFs associated with PA
   variation to identify potential PA biosynthesis-related and
   insolubilization-related genes.

   Comprehensive analysis of phenotype (Fig. [114]1a), PA content
   (Fig. [115]1b), and DEG enrichment (Fig. [116]S2) at five late
   developmental stages of the C-PCNA persimmon ‘Xiaoguo-tianshi’ helped
   us pinpoint the critical phase for PA variation. The C-PCNA persimmon
   fruit experiences the fastest growth stage from 120 to 140 DAF, where
   the soluble, insoluble, and total PA content was decreased. Moreover,
   the enrichment of “flavonoid biosynthesis” at this time point was
   related to PA biosynthesis^[117]39. These results indicated that
   120–140 DAF could be the critical phase for the “dilution effect”.
   Soluble PA decreased while insoluble PA levels for C-PCNA persimmon
   increased rapidly from 140 to 160 DAF. Meanwhile, GO terms such as
   “drug transmembrane transporter activity” relating to PA
   transport^[118]40 and KEGG term “flavonoid biosynthesis” were
   significantly enriched. The results showed that 140–160 DAF could be
   the critical phase for PA transport and the “coagulation effect”. These
   results are consistent with previous research showing that C-PCNA
   persimmon undergoes deastringency at the late stage^[119]6.

   Chorismate acid links primary and secondary metabolism in the shikimate
   pathway, which refers to the synthesis of phenylalanine from erythrose
   4-phosphate and phosphoenolpyruvate in several steps^[120]41. It has
   been reported previously that inhibiting the DAHPS or DHD/SDH of the
   shikimate pathway results in the reduction of some secondary
   metabolites, such as chlorogenate and lignin, in tobacco and potato
   plants^[121]42,[122]43. In our study, it was revealed that 1 DAHPS and
   1 DHD/SDH were aggregated in cluster 2 and were downregulated between
   T4 vs T3 and T5 vs T4, which matched the proanthocyanin content.
   Downregulation of DHD/SDH has been reported in J-PCNA^[123]9,
   C-PCNA^[124]5, and artificially treated PCA persimmon^[125]44. However,
   DAHPS downregulation was observed in C-PCNA and C-PCNA persimmon that
   underwent artificial 40 °C water treatment^[126]21. These results
   showed that termination of PA accumulation accompanies downregulation
   of DAHPS and DHD/SDH in persimmon fruit.

   Biosynthesis of the PA precursor flavan-3-ols shares the phenylpropane
   metabolic pathway, flavonoid synthesis and proanthocyanidin
   pathway^[127]9,[128]45. LAR and ANR encode key enzymes involved in PA
   biosynthesis, which act in the production of 2,3-trans-flavan-3-ols
   [(+)-catechin (CA), and (+)-gallocatechin (GC)], 2,3-cis-flavan-3-ols
   [(−)-epicatechin (EC), (−)-epigallocatechin (ECG), and
   (−)-epi-gallocatechin 3-O-gallate (EGCG)], respectively^[129]44. We
   found that the expression of the phenylpropane and flavonoid structural
   genes PAL, C4H, 4CL, ANR, ANS, CHS, F3’H, F3′5'H, and F3H decreased at
   T4 compared to T3, which was consistent with the decreased PA level.
   However, the LAR gene showed no differential expression in any
   developmental stage, which caused a reduction in epigallocatechin (EGC)
   and EGCG. This was one of the main reasons for PA reduction in the PCNA
   genotype^[130]9. Moreover, overexpression of Chinese Bayberry MrANR and
   MrLAR in tobacco indicated that MrANR increased PA accumulation, while
   MrLAR was unable to affect the total PA contents^[131]46. These results
   revealed that ANR was highly downregulated at the late stage of
   persimmon, which might be associated with the decrease in PA content by
   inhibiting PA precursor biosynthesis.

   Previous studies suggest that acetaldehyde metabolism plays an
   important role in the astringency loss in both C-PCNA genotype^[132]21
   and PCA persimmon with artificial deastringency treatment^[133]47. In
   plants, pyruvate is converted to acetaldehyde by PDC, then to ethanol
   by ADH, and acetate by mitochondrial ALDH2a and ALDH2b during the
   acetaldehyde biosynthesis process^[134]48,[135]49. We found that 3 ADH
   and 2 ALDH genes that grouped in cluster 2 were downregulated, and 2
   ADH and 1 ALDH that grouped in cluster 5 were upregulated. Three PDC
   genes were found to aggregate in cluster 5 and were upregulated between
   T5 and T4, which matched the insoluble PA content. The ADH-like and
   ALDH2 genes were downregulated in the C-PCNA genotype natural
   deastringency process, and PDC2 was specifically upregulated^[136]21.
   However, the expression patterns of the DkADH1,3 and DkPDC1,2 genes
   were upregulated in PCA persimmon during artificial ethylene
   deastringency treatment^[137]47, and 1 ADH-like gene showed high
   expression in 40 °C water treatment^[138]21. The upregulation of ADH
   and PDC genes to ethylene was also reported in other fruits, such as
   apricot^[139]50 and melon^[140]51, implying that the decrease in ALDH
   genes and the increase in ADH and PDC genes might result in
   acetaldehyde accumulation, causing C-PCNA deastringency naturally via
   PA insolubilization.

   PA and anthocyanin biosynthesis pathway genes are mainly regulated by
   the MBW complex in plants, such as Medicago truncatula^[141]52 and
   Anthurium andraeanum^[142]53. MYB transcription factors are core
   members of the MBW complex. MYB182 overexpression results in the
   reduction of PA and anthocyanin levels in poplar by reducing the
   expression of flavonoid biosynthesis structural genes^[143]54. DkMYB14
   in persimmon acts as both a repressor in PA biosynthesis and an
   activator in acetaldehyde biosynthesis^[144]19. MYB and bHLH
   autonomously mediate the expression of genes involved in the middle
   steps of the phenylpropanoid pathway^[145]16. In the ‘Red Delicious’,
   only a few MYB and bHLH members in the fruit skin were significantly
   correlated with anthocyanin content and suggested to regulate
   anthocyanin accumulation^[146]55. WD40 proteins in the MBW complex are
   thought to confer a docking platform for the MYB–bHLH
   interaction^[147]56. A member of the MBW protein complex has been
   identified in persimmon through homologous cloning^[148]20 and
   transcriptome sequencing^[149]21. DkMYB2, DkMYB4, and DkMYC1 (MBW)
   cooperatively increase the expression of a persimmon ANR gene involved
   in the biosynthesis of PA precursor cis-flavan-3-ols, which not only
   echoes the above analysis of PA biosynthesis structural genes, but also
   supports the presence of MBW complexes in persimmon^[150]57. We also
   identified R2R3MYB, bHLH, and WD40 members based on the unpublished
   C-PCNA persimmon genome and indicated that the expression of the
   transcription factors R2R3-MYB (evm.TU.contig8910.486 and
   evm.TU.contig7396.13) and bHLH (evm.TU.contig1398.203 and
   evm.TU.contig8910.303) positively correlated with the total PA content
   and WD40 (evm.TU.contig1.155) negatively correlated with the soluble PA
   content. These data provide the basis for targeted gene resources of
   transcription factors involved in PA accumulation in C-PCNA persimmon.

   To reveal the potential regulatory network underlying PA content
   variation in C-PCNA persimmon, we performed WGCNA to identify modules
   of highly correlated genes associated with soluble PA, insoluble PA,
   and total PA content. We found that most PA biosynthesis and
   insolubilization DEGs were included in the brown and yellow modules,
   which were highly correlated with the soluble, insoluble, and total PA
   contents of the C-PCNA persimmon. A network of genes that were highly
   coexpressed with these PA biosynthesis-related and
   insolubilization-related DEGs was constructed. Among these genes, an
   MBW complex homologous WD40 gene (evm.TU.contig1.155) showed a high
   correlation with the PA insolubilization genes ADH and PDC in WGCNA.
   This WD40 gene also presented a highly negative correlation with
   soluble PA content. Previous studies have reported that the expression
   pattern of DkWD40 was consistent with that of PA insolubilization genes
   such as ADH, and it is also consistent with the transformation of
   soluble PA to insoluble PA in the “Nishimura-wase” persimmon^[151]58.
   Combining WGCNA and phylogenetic analysis of the MBW complex, we found
   that the WD40 gene (evm.TU.contig1.155), MYB (evm.TU.contig8910.486)
   and bHLH (evm.TU.contig1398.203) might be linked between PA
   biosynthesis and PA insolubilization. In this study,
   evm.TU.contig1407.18 (probable aminotransferase ACS10) and
   evm.TU.contig1077.85 (AHA10) were coexpressed with the WD40 gene
   (evm.TU.contig1.155), MYB (evm.TU.contig8910.486) and bHLH
   (evm.TU.contig1398.203) and harbored the highest connections in the
   brown and yellow modules, respectively. ACS is the key enzyme in the
   ethylene biosynthetic pathway in plants^[152]59. In Arabidopsis,
   AtACS10 is not ACC synthase but aminotransferase, it was clustered with
   alanine AT and aspartate AT, which speculated that it might encode
   aminotransferase^[153]60. Complementation experiments showed that
   AtACS10 and AtACS12 were ATases with broad specificity for aspartic
   acid and aromatic amino acids such as tyrosine and
   phenylalanine^[154]60. Besides, aspartate AT, the homologous genes of
   AtACS10, catalyze the synthesis of compounds such as phenylalanine via
   a transamination reaction^[155]61. Phenylalanine is the precursor of
   many flavonoid compounds such as PA, anthocyanins, and
   isoflavonoids^[156]62. The conversion of phenylalanine to PA and
   anthocyanins was catalyzed by enzymes such as PAL, C4H, and
   4CL^[157]63, which is consistent with our results that ACS10 and the
   genes encoding these enzymes were coexpressed. AHA10 that have been
   associated with PA accumulation with the disturbance of vacuolar
   acidification activates TT12 to transport PA into vacuoles^[158]64.
   Thus, evm.TU.contig1407.18 (probable aminotransferase ACS10) and
   evm.TU.contig1077.85 (AHA10) might play important roles in the
   regulation of PA accumulation. The overall results suggest that the MBW
   complex might play important roles in regulating PA biosynthesis and PA
   insolubilization; however, the mechanism remains unclear and requires
   further investigation.

Conclusion

   Transcriptome data of C-PCNA persimmon fruit at five developmental
   stages have provided the specific processes that lead to PA
   biosynthesis and PA insolubilization. Altogether, the physiological, PA
   content, and transcriptome data revealed that 120 to 140 DAF is the
   critical phase for PA biosynthesis, while 140 to 160 DAF is the
   critical phase for PA transport and PA insolubilization. The results
   indicate that the downregulation of the ANR gene at T5 vs T4 may be
   associated with a reduction in PA biosynthesis by inhibiting its
   precursor cis-flavan-3-ols. The decrease in ALDH and an increase in ADH
   and PDC genes might result in C-PCNA persimmon’s PA insolubilization.
   MBW complex homologous R2R3-MYB (evm.TU.contig8910.486 and
   evm.TU.contig7396.13), bHLH (evm.TU.contig1398.203 and
   evm.TU.contig8910.303), and WD40 (evm.TU.contig1.155) were isolated
   from the C-PCNA D.kaki genome (unpublished) and were highly correlated
   with PA content. In addition, the WD40 (evm.TU.contig1.155), MYB
   (evm.TU.contig8910.486) and bHLH (evm.TU.contig1398.203) genes might
   link and coregulate both PA biosynthesis and insolubilization via
   WGCNA. These genes might play important roles in PA content variation.
   This study laid an empirical foundation for ongoing investigations of
   PA biosynthesis and PA insolubilization in C-PCNA persimmon.

Supplementary Information

   [159]Supplementary Information.^ (507.4KB, docx)

Acknowledgements