Multi-omics provide insights into the regulation of DNA methylation in pear fruit metabolism.

BACKGROUND: Extensive research has been conducted on fruit development in crops, but the metabolic regulatory networks underlying perennial fruit trees remain poorly understood. To address this knowledge gap, we conduct a comprehensive analysis of the metabolome, proteome, transcriptome, DNA methylome, and small RNAome profiles of pear fruit flesh at 11 developing stages, spanning from fruitlet to ripening. Here, we systematically investigate the metabolic landscape and regulatory network involved. RESULTS: We generate an association database consisting of 439 metabolites and 14,399 genes to elucidate the gene regulatory network of pear flesh metabolism. Interestingly, we detect increased DNA methylation in the promoters of most genes within the database during pear flesh development. Application of a DNA methylation inhibitor to the developing fruit represses chlorophyll degradation in the pericarp and promotes xanthophyll, ß-carotene, and abscisic acid (ABA) accumulation in the flesh. We find the gradual increase in ABA production during pear flesh development is correlated with the expression of several carotenoid pathway genes and multiple transcription factors. Of these transcription factors, the zinc finger protein PbZFP1 is identified as a positive mediator of ABA biosynthesis in pear flesh. Most ABA pathway genes and transcription factors are modified by DNA methylation in the promoters, although some are induced by the DNA methylation inhibitor. These results suggest that DNA methylation inhibits ABA accumulation, which may delay fruit ripening. CONCLUSION: Our findings provide insights into epigenetic regulation of metabolic regulatory networks during pear flesh development, particularly with regard to DNA methylation.

Comparative microbiome analysis reveals the variation in microbial communities between 'Kyoho' grape and its bud mutant variety.

Microbes are an important part of the vineyard ecosystem, which significantly influence the quality of grapes. Previously, we identified a bud mutant variety (named 'Fengzao') from 'Kyoho' grapes. The variation of microbial communities in grape and its bud mutant variety has not been studied yet. So, in this study, with the samples of both 'Fengzao' and 'Kyoho', we conducted high-throughput microbiome sequencing and investigated their microbial communities in different tissues. Obvious differences were observed in the microbial communities between 'Fengzao' and 'Kyoho'. The fruit and the stem are the tissues with relatively higher abundance of microbes, while the leaves contained less microbes. The fruit and the stem of 'Kyoho' and the stem of 'Fengzao' had relatively higher species diversity based on the alpha diversity analysis. Proteobacteria, Enterobacteriaceae and Rhodobacteraceae had significantly high abundance in 'Fengzao'. Firmicutes and Pseudomonas were highly abundant in the stems of 'Kyoho', and family of Spirochaetaceae, Anaplasmataceae, Chlorobiaceae, and Sphingomonadaceae, and genera of Spirochaeta, Sphingomonas, Chlorobaculum and Wolbachia were abundant in the fruits of 'Kyoho'. These identified microbes are main components of the microbial communities, and could be important regulators of grapevine growth and development. This study revealed the differences in the microbial compositions between 'Kyoho' and its bud mutant, and these identified microbes will be significant resources for the future researches on the quality regulation and disease control of grapevines.

Identification of watermelon H3K4 and H3K27 genes and their expression profiles during watermelon fruit development.

BACKGROUND: The ClaH3K4s and ClaH3K27s gene families are subfamilies of the SET family, each with a highly conserved SET structure domain and a PHD structural domain. Both participate in histone protein methylation, which affects the chromosome structure and gene expression, and is essential for fruit growth and development. METHODS AND RESULTS: In order to demonstrate the structure and expression characteristics of ClaH3K4s and ClaH3K27s in watermelon, members of the watermelon H3K4 and H3K27 gene families were identified, and their chromosomal localization, gene structure, and protein structural domains were analyzed. The phylogeny and covariance of the gene families with other species were subsequently determined, and the expression profiles were obtained by performing RNA-Seq and qRT-PCR. The watermelon genome had five H3K4 genes with 3207-8043 bp nucleotide sequence lengths and four H3K27 genes with a 1107-5499 bp nucleotide sequence. Synteny analysis revealed the close relationship between watermelon and cucumber, with the majority of members displaying a one-to-one covariance. Approximately half of the 'Hua-Jing 13 watermelon' ClaH3K4s and ClaH3K27s genes were expressed more in the late fruit development stages, while the changes were minimal for the remaining half. H3K4-2 expression was observed to be slightly greater on day 21 compared to other periods. Moreover, ClaH3K27-1 and ClaH3K27-2 were hardly expressed throughout the developing period, and ClaH3K27-4 exhibited the highest expression. CONCLUSION: These results serve as a basis for further functional characterization of the H3K4 and H3K27 genes in the fruit development of watermelon.

Proteome-Wide Identification of Non-histone Lysine Methylation during Grape Berry Ripening.

To gain a comprehensive understanding of non-histone methylation during berry ripening in grape (Vitis vinifera L.), the methylation of non-histone lysine residues was studied using a 4D label-free quantitative proteomics approach. In total, 822 methylation sites in 416 methylated proteins were identified, with xxExxx_K_xxxxxx as the conserved motif. Functional annotation of non-histone proteins with methylated lysine residues indicated that these proteins were mostly associated with "ripening and senescence", "energy metabolism", "oxidation-reduction process", and "stimulus response". Most of the genes encoding proteins subjected to methylation during grape berry ripening showed a significant increase in expression during maturation at least at one developmental stage. The correlation of methylated proteins with QTLs, SNPs, and selective regions associated with fruit quality and development was also investigated. This study reports the first proteomic analysis of non-histone lysine methylation in grape berry and indicates that non-histone methylation plays an important role in grape berry ripening.

Genome-wide characterization of long terminal repeat retrotransposons provides insights into trait evolution of four cucurbit species.

Cucurbits are a diverse plant family that includes economically important crops, such as cucumber, watermelon, melon, and pumpkin. Knowledge of the roles that long terminal repeat retrotransposons (LTR-RTs) have played in diversification of cucurbit species is limited; to add to understanding of the roles of LTR-RTs, we assessed their distributions in four cucurbit species. We identified 381, 578, 1086, and 623 intact LTR-RTs in cucumber (Cucumis sativus L. var. sativus cv. Chinese Long), watermelon (Citrullus lanatus subsp. vulgaris cv. 97103), melon (Cucumis melo cv. DHL92), and Cucurbita (Cucurbita moschata var. Rifu), respectively. Among these LTR-RTs, the Ale clade of the Copia superfamily was the most abundant in all the four cucurbit species. Insertion time and copy number analysis revealed that an LTR-RT burst occurred approximately 2 million years ago in cucumber, watermelon, melon, and Cucurbita, and may have contributed to their genome size variation. Phylogenetic and nucleotide polymorphism analyses suggested that most LTR-RTs were formed after species diversification. Analysis of gene insertions by LTR-RTs revealed that the most frequent insertions were of Ale and Tekay and that genes related to dietary fiber synthesis were the most commonly affected by LTR-RTs in Cucurbita. These results increase our understanding of LTR-RTs and their roles in genome evolution and trait characterization in cucurbits.

Dissection of the Pearl of Csaba pedigree identifies key genomic segments related to early ripening in grape.

Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of ∼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs), and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANOVA, P < 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of "Kyoho". This work uncovered core haplotypes and genomic segments related to the early ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.

DNA methylation reprogramming provides insights into light-induced anthocyanin biosynthesis in red pear.

DNA methylation, an epigenetic mark, is proposed to regulate plant anthocyanin biosynthesis. It well known that light induces anthocyanin accumulation, with bagging treatments commonly used to investigate light-controlled anthocyanin biosynthesis. We studied the DNA methylome landscape during pear skin coloration under various conditions (fruits re-exposed to sunlight after bag removal). The DNA methylation level in gene body/TE and its flanking sequence was generally similar between debagged and bagged treatments, however differentially methylated regions (DMRs) were re-modelled after light-exposure. Both DNA demethylase homologs and the RNA-directed DNA methylation (RdDM) pathways contributed to this re-distribution. A total of 310 DEGs were DMR-associated during light-induced anthocyanin biosynthesis between debagged and bagged treatments. The hypomethylated mCHH context was seen within the promoter of PyUFGT, together with other anthocyanin biosynthesis genes (PyPAL, PyDFR and PyANS). This enhanced transcriptional activation and promoted anthocyanin accumulation after light re-exposure. Unlike previous reports on bud sports, we did not detect DMRs within the MYB10 promoter. Instead, we observed the genome-wide re-distribution of methylation patterns, suggesting different mechanisms underlying methylation patterns of differentially accumulated anthocyanins caused by either bud mutation or environment change. We investigate the dynamic landscape of genome-scale DNA methylation, which is the combined effect of DNA demethylation and RdDM pathway, in the process of light-induced fruit colour formation in pear. This process is regulated by methylation changes on promoter regions of several DEGs. These results provide a DMR-associated DEGs set and new insight into the mechanism of DNA methylation involved in light-induced anthocyanin biosynthesis.

A simple and efficient protocol for transient transformation of sliced grape berries.

Grape is an economically important crop but recalcitrant to Agrobacterium-mediated genetic transformation and in vitro regeneration. Here, we have developed a protocol for transient transformation of grapes by investigating the effects of explant pre-culture and duration of vacuum infiltration on transformation efficiency. Using sliced grape berries of "Shine-Muscat" (Vitis labrusca × Vitis vinifera) between the end of fruit expansion phase and the mature stage as explants, we firstly compared the effect of pre-culture explants into a susceptible state (incubation on Murashige and Skoog (MS) agar plate in the dark at 25 ± 1 °C for 48 h) with no pre-culture and then tested different vacuum infiltration times on transformation efficiency using ß-glucuronidase (GUS) reporter system. Pre-culture increased the susceptibility of explants to the agrobacteria infection and increased transient transformation efficiency as assessed by histochemical GUS activity, with intense blue coloration compared with the faint staining observed in the non-susceptible explants. Using a Circulating Water Vacuum Pump system to facilitate agrobacteria entry into berry cells, we tested vacuum durations of 5, 10, and 15 min and observed that transformation efficiency increased with vacuum duration of infiltration. These results were confirmed by relative gene expression of GUS transgene as assessed by RT-qPCR and GUS activity assay. To further confirm the usefulness of our protocol, we transiently transformed grape berries with the hydrogen peroxide sensor gene VvHPCA3, and this was confirmed by gene expression analysis as well as increased sensitivity of the explants to hydrogen peroxide treatment. Overall, this study has resulted in a simple but efficient transient transformation protocol for grape berries and would be a valuable tool for the rapid testing of gene function and the study of key regulatory networks in this important crop.

Large-scale discovery of non-conventional peptides in grape (Vitis vinifera L.) through peptidogenomics.

Non-conventional peptides (NCPs), which are peptides derived from previously unannotated coding sequences, play important biological roles in plants. In this study, we used peptidogenomic methods that integrated mass spectrometry (MS) peptidomics and a six-frame translation database to extensively identify NCPs in grape. In total, 188 and 2021 non-redundant peptides from the Arabidopsis thaliana and Vitis vinifera L. protein database at Ensembl/URGI and an individualized peptidogenomic database were identified. Unlike conventional peptides, these NCPs derived mainly from intergenic, intronic, upstream ORF, 5'UTR, 3'UTR, and downstream ORF regions. These results show that unannotated regions are translated more broadly than we thought. We also found that most NCPs were derived from regions related to phenotypic variations, LTR retrotransposons, and domestication selection, indicating that the NCPs have an important function in complex biological processes. We also found that the NCPs were developmentally specific and had transient and specific functions in grape berry development. In summary, our study is the first to extensively identify NCPs in grape. It demonstrated that there was a large amount of translation in the genome. These results lay a foundation for studying the functions of NCPs and also provide a reference for the discovery of new functional genes in grape.

Overexpression of VvPPR1, a DYW-type PPR protein in grape, affects the phenotype of Arabidopsis thaliana leaves.

Pentatricopeptide repeat (PPR) proteins play important roles in plant growth and development. However, little is known about their functions in the leaf morphogenesis of Jingxiu grape (Vitis vinifera L.). Here, we explored the function of VvPPR1, which encodes a DYW-type PPR protein in grape. We showed that VvPPR1 is involved in the regulation of leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis thaliana. Analysis of structural characteristics showed that VvPPR1 is a DYW-type PPR gene in the PLS subfamily consisting of 15 PPR motifs. The N-terminal had a targeted chloroplast site, and the C-terminal had a DYW domain. Quantitative PCR analysis revealed that the expression level of VvPPR1 was highest in grape leaves. Subcellular localization revealed that VvPPR1 is localized in the cytoplasm and chloroplast. VvPPR1-overexpressing plants had rolled leaves, high degrees of anthocyanin accumulation, and longer trichomes. The expression levels of genes related to these phenotypes were either significantly up-regulated or down-regulated. These results demonstrate that VvPPR1 is involved in leaf rolling, anthocyanin accumulation, and trichome formation in Arabidopsis; more generally, our findings indicate that VvPPR1 could be a target for improving the cultivation of horticultural crops.

The PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10 module regulates light-induced anthocyanin biosynthesis in red pear.

Some cultivars of pear (Pyrus L.) show attractive red fruit skin due to anthocyanin accumulation. This pigmentation can be affected by environmental conditions, especially light. To explore the light-induced regulation network for anthocyanin biosynthesis and fruit coloration in pear, small RNA libraries and mRNA libraries from fruit skins of 'Yunhongyihao' pear were constructed to compare the difference between bagging and debagging treatments. Analysis of RNA-seq of fruit skins with limited light (bagged) and exposed to light (debagged), showed that PyPIF5 was down-regulated after bag removal. PymiR156a was also differentially expressed between bagged and debagged fruit skins. We found that PyPIF5 negatively regulated PymiR156a expression in bagged fruits by directly binding to the G-box motif in its promoter. In addition, PymiR156a overexpression promoted anthocyanin accumulation in both pear skin and apple calli. We confirmed that PymiR156a mediated the cleavage of PySPL9, and that the target PySPL9 protein could form heterodimers with two key anthocyanin regulators (PyMYB114/PyMYB10). We proposed a new module of PyPIF5-PymiR156a-PySPL9-PyMYB114/MYB10. When the bagged fruits were re-exposed to light, PyPIF5 was down-regulated and its inhibitory effect on PymiR156a was weakened, which leads to degradation of the target PySPL, thus eliminating the blocking effect of PySPL on the formation of the regulatory MYB complexes. Ultimately, this promotes anthocyanin biosynthesis in pear skin.

Transcriptome analysis reveals mechanism of early ripening in Kyoho grape with hydrogen peroxide treatment.

BACKGROUND: In a previous study, the early ripening of Kyoho grape following H2O2 treatment was explored at the physiological level, but the mechanism by which H2O2 promotes ripening at the molecular level is unclear. To reveal the molecular mechanism, RNA-sequencing analysis was conducted on the different developmental stages of Kyoho berry treated with H2O2. RESULTS: In the comparison of treatment and control groups, 406 genes were up-regulated and 683 were down-regulated. Time course sequencing (TCseq) analysis showed that the expression patterns of most of the genes were similar between the treatment and control, except for some genes related to chlorophyll binding and photosynthesis. Differential expression analysis and the weighted gene co-expression network were used to screen significantly differentially expressed genes and hub genes associated with oxidative stress (heat shock protein, HSP), cell wall deacetylation (GDSL esterase/lipase, GDSL), cell wall degradation (xyloglucan endotransglucosylase/ hydrolase, XTH), and photosynthesis (chlorophyll a-b binding protein, CAB1). Gene expression was verified with RT-qPCR, and the results were largely consistent with those of RNA sequencing. CONCLUSIONS: The RNA-sequencing analysis indicated that H2O2 treatment promoted the early ripening of Kyoho berry by affecting the expression levels of HSP, GDSL, XTH, and CAB1 and- photosynthesis- pathways.

Comparative transcriptome analyses of fruit development among pears, peaches, and strawberries provide new insights into single sigmoid patterns.

BACKGROUND: Pear fruit exhibit a single sigmoid pattern during development, while peach and strawberry fruits exhibit a double sigmoid pattern. However, little is known about the differences between these two patterns. RESULTS: In this study, fruit weights were measured and paraffin sections were made from fruitlet to maturated pear, peach, and strawberry samples. Results revealed that both single and double sigmoid patterns resulted from cell expansion, but not cell division. Comparative transcriptome analyses were conducted among pear, peach, and strawberry fruits at five fruit enlargement stages. Comparing the genes involved in these intervals among peaches and strawberries, 836 genes were found to be associated with all three fruit enlargement stages in pears (Model I). Of these genes, 25 were located within the quantitative trait locus (QTL) regions related to fruit weight and 90 were involved in cell development. Moreover, 649 genes were associated with the middle enlargement stage, but not early or late enlargement in pears (Model II). Additionally, 22 genes were located within the QTL regions related to fruit weight and 63 were involved in cell development. Lastly, dual-luciferase assays revealed that the screened bHLH transcription factors induced the expression of cell expansion-related genes, suggesting that the two models explain the single sigmoid pattern. CONCLUSIONS: Single sigmoid patterns are coordinately mediated by Models I and II, thus, a potential gene regulation network for the single sigmoid pattern was proposed. These results enhance our understanding of the molecular regulation of fruit size in Rosaceae.

Studying on the strictly self-compatibility mechanism of 'Liuyefeitao' peach (Prunus persica L.).

Peach (Prunus persica L.) generally exhibits self-pollination, however, they can also be pollinated by other varieties of pollen. Here we found two varieties that are different from other peaches: 'Daifei' and 'Liuyefeitao'. 'Daifei' produces less pollen, which needs artificial pollination, honeybee pollination, and the fruit setting depends on other varieties of peach pollen. 'Liuyefeitao' exhibits strictly self-pollination, hence pollen from other species is rejected. To explore the mechanism of this phenomenon, we performed a high-throughput sequencing of the stigma (including style) of 'Daifei' and 'Liuyefeitao' to explain the rejection mechanism of other varieties of pollen of 'Liuyefeitao' peach. In our study, we found one S gene, and lots of non-S-locus factors such as: F-box proteins, Ub/26S, MAPKs, RLK, and transcription factor were differential expressed between 'Daifei' and 'Liuyefeitao'. We supposed that the strictly self-compatible of 'Liuyefeitao' may result from the synthesis of these factors.

Identification and expression analysis of genes related to calyx persistence in Korla fragrant pear.

BACKGROUND: The objective of this study was to increase understanding about genetic mechanisms affecting calyx persistence in Korla fragrant pear (Pyrus brestschneideri Rehd). Flowers were collected at early bloom, full bloom, and late bloom. The RNA was extracted from the flowers and then combined according to calyx type. Transcriptome and digital gene expression (DGE) profiles of flowers, ovaries, and sepals with persistent calyx (SC_hua, SC_ep, and SC_zf, respectively) were compared with those of flowers, ovaries, and sepals with deciduous calyx (TL_hua, TL_ep, and TL_zf, respectively). Temporal changes in the expression of selected genes in floral organs with either persistent or deciduous calyx were compared using real-time quantitative PCR (qRT-PCR). RESULTS: Comparison of the transcriptome sequences for SC_hua and TL_hua indicated 26 differentially expressed genes (DEGs) with known relationship to abscission and 10 DEGs with unknown function. We identified 98 MYB and 21 SPL genes from the assembled unigenes. From SC_zf vs TL_zf, we identified 21 DEGs with known relationship to abscission and 18 DEGs with unknown function. From SC_ep vs TL_ep, 12 DEGs with known relationship to abscission were identified along with 11 DEGs with unknown function. Ten DEGs were identified by both transcriptome sequencing and DGE sequencing. CONCLUSIONS: More than 50 DEGs were observed that were related to calyx persistence in Korla fragrant pear. Some of the genes were related to cell wall degradation, plant hormone signal transduction, and stress response. Other DEGs were identified as zinc finger protein genes and lipid transfer protein genes. Further analysis showed that calyx persistence in Korla fragment pear was a metabolic process regulated by many genes related to cell wall degradation and plant hormones.