Transcriptome profiling of ‘Kyoho’ grape at different stages of berry development following 5-azaC treatment
5-Azacytidine (5-azaC) promotes the development of ‘Kyoho’ grape berry but the associated changes in gene expression have not been reported. In this study, we performed transcriptome analysis of grape berry at five developmental stages after 5-azaC treatment to elucidate the gene expression networks controlling berry ripening.
The expression patterns of most genes across the time series were similar between the 5-azaC treatment and control groups. The number of differentially expressed genes (DEGs) at a given developmental stage ranged from 9 (A3_C3) to 690 (A5_C5). The results indicated that 5-azaC treatment had not very great influences on the expressions of most genes. Functional annotation of the DEGs revealed that they were mainly related to fruit softening, photosynthesis, protein phosphorylation, and heat stress. Eight modules showed high correlation with specific developmental stages and hub genes such as PEROXIDASE 4, CAFFEIC ACID 3-O-METHYLTRANSFERASE 1, and HISTONE-LYSINE N-METHYLTRANSFERASE EZA1 were identified by weighted gene correlation network analysis.
5-AzaC treatment alters the transcriptional profile of grape berry at different stages of development, which may involve changes in DNA methylation.
KeywordsKyoho Grape Ripening Transcriptome 5-azaC DEG
Histone-lysine N-methyltransferase EZA1
Differentially expressed genes
Days post anthesis
Fragments per kilobase per million mapped reads
Probable galacturonosyltransferase-like 1
Heat shock protein
Kyoto Encyclopedia of Genes and Genomes
Non-specific lipid-transfer protein A
Caffeic acid 3-O-methyltransferase
Purple acid phosphatase 15
Real-Time Quantitative PCR
Weighted Gene Co-Expression Network Analysis
Probable xyloglucan endotransglucosylase/hydrolase protein 23
Grape (Vitis vinifera L.) is one of the most important perennial woody fruit crops in the world. The grape berry is consumed whole or in the form of raisins or wine and has high nutritional, medicinal, and economic value , making it one of the most popular fruits. Grape berry exhibits change in pigmentation, sugar and organic acid contents, and other quality components during development and ripening  and is a useful model for studying fruit development.
Transcriptome sequencing is the main technology for investigating genome-wide changes in gene expression patterns, and has been used to study metabolic pathways and gene expression during fruit development in many plants. Most of the research has focused on climacteric fruits such as bayberry , pear [4, 5], kiwifruit , peach , tomato , and apricot , although recent studies have also investigated non-climacteric fruits such as sweet orange  and strawberry . For example, cell wall biosynthesis, carbohydrate metabolism, the tricarboxylic acid cycle, and carotenoid biosynthesis were shown to be differentially regulated during fruit development and ripening of the sweet orange variety ‘Anliu’ and its red-fleshed mutant ‘Hong Anliu’ . Metabolic shifts occurred in the green-white-red stages of strawberry that were associated with differential gene expression, and it was found that oxidative phosphorylation plays an important role in the regulation of fruit maturation .
Whole-genome sequencing of the PN40024 genotype of grapevine, originally derived from Pinot Noir, was completed in 2007 and has provided a useful resource for functional genomic studies . A transcriptome analysis revealed that reduced biosynthesis, photosynthesis, and transport was the main reason for delayed senescence of the peel . Most genes showed comparable expression levels between ‘Kyoho’ berry and its early-ripening mutant ‘Fengzao’ , and an analysis of differentially expressed genes (DEGs) revealed that those related to oxidative stress genes likely promote the early ripening of ‘Fengzao’ grape berry. Genes involved in carbohydrate metabolism and regulation of flavonoid metabolism and those of the solute carrier family showed the most marked changes in expression in ‘Kyoho’ and transgenic berry peels , and it was later reported that V. vinifera VACUOLAR H+-PPASE 1 was activated by the MYB transcription factor MYBA1 and that hexokinase-mediated glucose signaling increased the expression of anthocyanin biosynthesis and transport-related genes to promote anthocyanin accumulation in grape peel. In addition, differences in the levels of microRNAs (miR169-NF-Y subunit, miR398-CSD, miR3626-RNA helicase, miR399-phosphate transporter, and miR477-GRAS transcription factor) and their targets have been observed in ‘Kyoho’ and ‘Fengzao’ during berry development and ripening .
DNA methylation is a mitotically reversible and meiotically heritable epigenetic modification  that is important in plant growth and development [18, 19, 20]. Recent studies have shown that DNA methylation is associated with fruit development and ripening [21, 22, 23, 24, 25, 26]. Treatment with 5-azacytidine (5-azaC), a methyltransferase inhibitor, was shown to affect the development of tomato , strawberry , and Acca sellowiana  fruit by decreasing DNA methylation levels, resulting in an early ripening phenotype. Although 5-azaC treatment delayed fruit ripening in sweet orange , it also had a genome-wide demethylating effect . 5-AzaC promoted the early ripening of grape berry and reduced global methylation level at a concentration of 100 μΜ in our previous study . However, the mechanism by which 5-azaC alters gene expressions to accelerate berry ripening remains unknown.
To answer this question, in this study we carried out RNA-sequencing (RNA-seq) analysis of ‘Kyoho’ grape berry at five different stages of fruit development after 5-azaC treatment. The results provide novel insight into the molecular basis of grape berry ripening and a basis for future molecular studies.
Analysis of RNA-seq libraries
To identify the genes involved in grape berry development, we performed transcriptome sequencing of ‘Kyoho’ grape berry with or without 5-azaC treatment at different developmental stages. The RNA-seq data have been uploaded to the National Center for Biotechnology Information Sequence Read Archive under the accession number PRJNA542248. A total of 30 cDNA libraries were constructed comprising 1.37 billion raw reads; 1.33 billion clean reads (accounting for 96.74% of raw reads) were recorded after removing adapter sequences and reads of low quality and those with more than 5% N bases. The average number of clean reads per sample was about 45.76 million and the clean Q30 (sequencing error rate < 0.1%) base rate was > 93.6% for each sample. Ultimately, 1.21 billion high-quality reads (accounting for 91.32% of clean reads) were mapped to the grape reference genome; 29.56 million of these were mapped to multiple locations in the genome at a ratio of 2.23% (Additional file 1).
In the 5-azaC-treated and untreated control samples, more genes were expressed at the A3 (23883) stage than at the C3 (22710) stage, whereas fewer genes were expressed at the other four stages. We also analyzed the number of genes expressed at different levels (fragments per kilobase million [FPKM] ≥ 50, 50 > FPKM ≥10, 10 > FPKM ≥2, 2 > FPKM ≥0.1, FPKM < 0.1) and found that the number of genes with FPKM ≥10 was higher in berries at A2 and A3 stages than in berries at stages C2 and C3; the number of genes with different expression levels was greater at C2 than at A2 (Additional file 2).
Gene expression profile following 5-azaC treatment
Comparison of overall expression patterns by time course sequencing (TCseq) analysis
Analysis of differentially expressed genes (DEGs)
Numbers of DEGs in each developmental stage or in two adjacent stages of ‘Kyoho’ grape berry for control and 5-azaC treatment
DEGs between the treatment and the control at the same developmental stage. (q < 0.05)
Purple acid phosphatase 15
Probable xyloglucan endotransglucosylase
xyloglucan: xyloglucosyl transferase
/hydrolase protein 23
NDR1/HIN1-like protein 12
Non-specific lipid-transfer protein A
Basic 7S globulin
Late embryogenesis abundant protein Dc3
Probable pectinesterase/pectinesterase inhibitor 36
Probable galacturonosyltransferase-like 1
Probable sarcosine oxidase
PIPOX; sarcosine oxidase/L- pipecolate oxidase oxidase1111111pipecolapipecolate oxidase
Histidine kinase CKl1
Pleiotropic drug resistance protein 2
Protein ROOT INITIATION DEFECTIVE 3
Probable potassium transporter 13
Protein DETOXIFICATION 48
TC.MATE, SLC47A, norM, mdtK, dinF; multidrug resistance protein, MATE family
resistance protein, MATE family
GDSL esterase/lipase 1
Cationic peroxidase 1
Non-specific lipid-transfer protein 2
Probable sucrose-phosphate synthase 1
Exocyst complex component EXO70A1
EXOC7, EXO70; exocyst complex component 7
Arogenate dehydrogenase 2, chloroplastic
TYRAAT; arogenate dehydrogenase (NADP+), plant
Probable flavin-containing monooxygenase 1
FMO; dimethylaniline monooxygenase (N-oxide forming) forming)
23.6 kDa heat shock protein, mitochondrial
HSP20; HSP20 family protein
Nine DEGs in A3_C3 were annotated; two of these—encoding Probable pectinesterase/pectinesterase inhibitor 36 (VIT_15s0048g00500, PE) and Endonuclease 1 (VIT_00s0301g00100)—were downregulated after 5-azaC treatment whereas seven genes encoding UDP-glycosyltransferase 89B2 (VIT_17s0000g04750, UGT89B2), Probable galacturonosyltransferase-like 1 (VIT_18s0001g11860, GTL), Histidine kinase CKl1 (VIT_07s0005g01380), Beta-glucosidase 13 (VIT_13s0064g01760, BGLU13), and Probable sarcosine oxidase (VIT_04s0069g00860) were upregulated. BGLU13 and VIT_04s0069g00860 are involved in the β-glucosidase and sarcosine oxidase/l-pipecolate oxidase pathways, respectively (Table 2).
A total of 19 DEGs were identified in A4_C4: Probable sucrose-phosphate synthase 1 (VIT_04s0008g05730), polygalacturonase (PG) QRT3 (VIT_01s0011g01300, QRT3), and Probable flavin-containing monooxygenase 1 (VIT_18s0122g01430) were upregulated; VIT_18s0122g01430, is involved in the flavin monooxygenase and dimethylaniline monooxygenase (NO-forming) pathways. Downregulated genes were Non-specific lipid-transfer protein 2 (VIT_14s0006g02570), Acidic endochitinase (VIT_15s0046g01570), Pleiotropic drug resistance protein 2 (VIT_13s0074g00700), GDSL esterase/lipase 1 (VIT_09s0002g00550), Cationic peroxidase 1 (VIT_18s0001g06840), Glutathione S-transferase (VIT_07s0005g00030), and 23.6-kDa heat shock protein (VIT_16s0022g00510, HSP 23.6). The HSP23.6 gene belongs to the HSP20 family (Table 2). A5_C5 had the most DEGs. The expression levels of 28 DEGs encoding heat shock proteins and belonging to HSP20, HSP70, HSP90, and HSF_DNA-binding gene families were downregulated after 5-azaC treatment, as were all 28 DEGs related to photosynthesis and some methyltransferase genes including VIT_04s0023g02290, VIT_05s0049g01650, VIT_12s0028g02370, and VIT_08s0007g08470 (Additional file 5).
We performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs in the treatment and control groups at the same developmental stage and found that only DEGs in A5_C5 were significantly enriched in KEGG pathways—namely, Protein processing in endoplasmic reticulum, Photosynthesis, Photosynthesis antenna proteins, Galactose metabolism, Flavone and flavanol biosynthesis, Diterpenoid biosynthesis, and ABC transporters; most genes were involved in Protein processing in endoplasmic reticulum (Additional file 6) and DEGs related to Photosynthesis were downregulated. The expression patterns and details of representative genes in key pathways are shown in Additional file 5.
Weighted gene correlation network analysis (WGCNA)
Validation by quantitative real-time (qRT-)PCR
Tomatoes treated with 5-azaC ripen prematurely, and whole-genome bisulfite sequencing of the fruit at four stages of development has revealed many differentially methylated regions, suggesting that DNA methylation is an important regulator of fleshy fruit ripening . As a DNA methyltransferase inhibitor, 5-azaC has been used to reduce methylation levels in other plants [28, 30, 33]. We previously showed that 5-azaC decreased methylation in developing ‘Kyoho’ grape berry . 5-AzaC not only affects multiple physiological processes during plant development such as enhancing resistance and oxidation, inducing flowering, and influencing phenotype, but also affects gene expression during fruit maturation. RNA-seq is a useful tool for whole-genome expression profiling and identification of candidate genes related to this process. The TCseq results showed that the expression patterns of most genes were similar between the treatment and control groups (Fig. 4). No DEGs were identified in A1_C1 and only a few were detected in A2_C2, A3_C3, and A4_C4, confirming that 5-azaC treatment had little effect on the expression of most genes. However, there were 690 DEGs in A5_C5, which was more than the number in earlier developmental stages. Based on physiological indices and developmental state , 5-azaC was found to promote the early ripening of grape berry. Methylation normally leads to gene silencing but demethylation activates gene expression. 5-AzaC inhibited methylation but the DEGs expressed at A2 were downregulated, indicating that 5-azaC can also inhibit gene expression as previously reported . Thus, 5-azaC treatment has pleiotropic effects on gene expression and related regulatory networks.
Fruit softening involves the dissolution of the cell wall due to polymer depolymerization . The degradation of cell wall starch was shown to be the main cause of banana fruit softening/ripening [35, 36]. ‘Kyoho’ grape berry has a very hard texture at the very early stage of development, reaching peak hardness at 45 dpa before softening, which reflects the breakdown of the cell wall . Our transcriptome analysis revealed that genes associated with cell wall softening such as UGT89B2, GTL, and BGLU13 were significantly upregulated at 45 dpa after 5-azaC treatment (Table 1). Similarly, QRT3 expression was increased at 55 dpa when the berries were at the softening stage, suggesting that it regulates berry ripening. Tomato genome sequencing revealed the expression of over 50 structural genes encoding known or putative cell wall-modifying proteins during fruit development and ripening . Inhibiting PG activity was found to slow the softening of bayberry . Phospholipase (PL) activity degrades cross-linked pectin polymers, and pectin polysaccharide in the cell wall is further degraded by PG, resulting in fruit softening; silencing PL expression slows the degree of fruit softening in strawberry . As a PG, QRT3 was upregulated after 5-azaC treatment in early-ripening berries in this study. BG participates in the synthesis of abscisic acid, which is associated with grape ripening; VvBG1 overexpression in strawberry increases BG activity and promotes fruit ripening . Interestingly, we found that BGLU13 was upregulated by 5-azaC treatment.
HSPs are responsible for protein folding, assembly, translocation, and degradation during normal cell growth and development . HSP70, HSP90, and their co-chaperones are involved in signal transduction and protein targeting and degradation . Small HSP is implicated in seed, pollen, and fruit development [43, 44]. MiHSP17.6 expression, which regulates the development of mango fruit, declined during the early stage of fruit development, increased during the middle stage, and then decreased at the late stage; the highest expression level was at 60 dpa . Class II sHSP 17.4 mRNA was detected at all stages of the ripening process of tomato fruit and reached a maximal level at the later stage, whereas Class II HSP 17.6 and 17.7 mRNA had the highest expression at the turning and pink stages, respectively . FaHSP17.4 expression was increased during cell division in strawberry fruit development, but gradually decreased during maturation after cell division ceased . In this study, the HSP 23.6 gene belonging to the HSP20 family was downregulated at 55 dpa following 5-azaC treatment, with other HSPs (e.g., HSP20, HSP70, and HSP90) showing a similar trend at 65 dpa. These results indicate that 5-azaC inhibits the expression of HSP genes although the underlying mechanism remains to be determined.
Protein phosphorylation plays an important role in the regulation of growth and development of strawberry fruit. Phosphorylated proteins not only participate in transcriptional regulation and cell division but also modulate the response to plant hormones and sugar metabolism, which are related to fruit ripening and softening . In this study, GO enrichment analysis of genes at different developmental stages revealed factors that control berry development; their expression was stable and was not readily perturbed by exogenous substances. Moreover, they had related functions during berry development such as nuclease, isomerase, and endonuclease activities. However, the expression of some genes was altered at A4 by 5-azaC treatment including those encoding pyrophosphatase, nucleoside triphosphatase, and hydrolase, possibly as a result of changes in their methylation status. Meanwhile, the results of the TCseq analysis showed that most of the genes showed the same or similar expression patterns between treatment and control groups at different developmental stages, although gene expression patterns in cluster 1 were altered at 25 dpa. The GO enrichment analysis of cluster 1 genes also revealed that many were related to pyrophosphatase, nucleoside triphosphatase, and hydrolase activities.
PAP is produced by plant secretion under low phosphorus conditions and can hydrolyze phosphate groups on organophosphorus substrates and produce phosphorus for absorption and utilization by plants . The PAP-related gene MdPAP10 cloned from apple was found to be expressed in root, stem, leaf, flower and fruit, and its overexpression induced phosphorus-related gene expression . In this study, 5-azaC treatment reduced the level of PAP15 at 35 dpa, suggesting that this gene is related to grape berry development. However, the specific functions of PAP remain to be determined. In summary, our results suggest that grape berry ripening involves changes in protein phosphorylation state.
DNA methylation is generally considered to be a marker of transcriptional repression. However, decreased DNA methylation levels are also associated with the repression of genes, for instance during grape berry ripening; this is especially true in the case of genes related to photosynthesis, which are not required by ripened berries. Photosynthesis is important for the rapid growth of young fruit and starch production. With fruit ripening, the chloroplast differentiates into pigment cells and promotes coloration. Hypomethylation of DEGs is increased during chlorophyll biosynthesis. Accordingly, many photosynthesis-related genes were downregulated at A5_C5 (65 dpa). Whereas the berries of control plants were still green at this stage, those in the 5-azaC treatment group had changed color. Thus, 5-azaC may inhibit the expression of photosynthesis-related genes. However, additional studies are needed to clarify the molecular link between DNA methylation and gene expression changes during grape berry development and ripening.
Transcriptome analysis of grape berry was performed at five developmental stages to elucidate the gene expression networks controlling ripening of ‘Kyoho’ grape berry following 5-azaC treatment. Most genes were detected at similar levels between the treatment and control groups in the time series analysis, indicating that 5-azaC treatment does not significantly influence the expression of most genes. Functional annotation of DEGs revealed that they were mainly related to fruit softening, photosynthesis, protein phosphorylation, and heat stress. The results provide insight into the mechanisms that regulate grape berry development, which is useful for establishing grape varieties with specific favorable characteristics.
Plant material and 5-azaC treatment
Sampling time points of ‘Kyoho’ grape berries for 5-azaC treatment and the control
Sampling date (dpa)
Library preparation and transcriptome sequencing
cDNA library preparation and transcriptome sequencing was performed as previously described . Briefly, mRNA was enriched with oligo (dT) magnetic beads from total RNA. First-strand cDNA was synthesized using random hexamers with the fragmented mRNAs as templates; second strand buffer, dNTPs, RNaseH, and DNA polymerase I were then added to the reaction. The QIAQuick PCR kit and EB buffer were used for purification and elution, and end repair of the double-stranded cDNA was performed using A bases and an adapter. Fragments of the appropriate size were recovered by agarose gel electrophoresis and PCR amplification was performed to complete whole library preparation. RNA-seq libraries for the control and treatment groups were labeled as C and A, respectively. Three biological replicates of each library were sequenced on a HiSeq 2500 platform (Illumina, San Diego, CA, USA) with 150-bp paired-ends by Annoroad Gene Technology Co. (Beijing, China). RNA-seq data were uploaded to the Sequence Read Archive of the National Center for Biotechnology Information (accession number: PRJNA542248).
RNA-seq data analysis
RNA-seq data analysis was performed according to previously published protocols . To ensure data quality, clean reads were obtained by removing reads contaminated with adapters, those of low quality, and those in which N bases constituted > 5% of total bases. The Q30 of clean reads was calculated.
Bowtie2 v.2.2.3 was used to build a genome index and clean reads were aligned to the grape reference genome  using HISAT2 v.2.1.0  with the BWT algorithm . The expression levels of genes in each sample were calculated using HTSeq 0.6.0 and normalized, and are expressed as FPKM values to facilitate comparisons between different genes .
Differential expression analysis
Linear regression was used to estimate gene expression levels in each sample and the probability value (P value) of each gene that was differentially expressed between the two groups was calculated with the Wald test. The P value was corrected with the BH method by multiple hypothesis testing to obtain the q value (q). DESeq2 v.1.6.3  was used to analyze differential gene expression between the two groups. Genes with |log2[foldchange]| ≥ 1 and q < 0.05 in each pairwise comparison were identified as DEGs. The UniProt database was used to annotate and obtain detailed information on the DEGs.
TCseq analysis was performed using the R packages Vegan and Cairo to evaluate trends in gene expression at different developmental stages of grape berry using FPKM values, and genes with similar expression patterns were divided into sets ; the gene expression trends in the same set were similar under the same conditions. To investigate gene function, GO and KEGG enrichment analyses were performed using R package (clusterProfiler). GO terms and KEGG pathways with q < 0.05 were considered as significantly enriched.
To elucidate gene regulatory networks involved in grape berry development, WGCNA was performed for all genes (19,387) with FPKM ≥0.5 , with the soft threshold set to 1. WGCNA calculates the correlation between genes using a topological overlap method, which yields more biologically meaningful results. Cytoscape v.3.6.1 was used to analyze gene interaction networks in the module. Hub genes in a given module were screened with the MCODE module.
Validation of DEGs by qRT-PCR
To validate the accuracy of RNA-seq data, the expression levels of some DEGs, genes of cluster 1 identified in the TCseq analysis, and candidate hub genes were evaluated by qRT-PCR. Total RNA was isolated from ‘Kyoho’ grape berries in the treatment and the control groups using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (DP441; Tiangen, Beijing, China) according to the manufacturer’s instructions. HiScript II 1st Strand cDNA Synthesis Kit (R211–01; Vazyme, Nanjing, China) was used for reverse transcription. Using a suitable amount of reverse transcribed cDNA as template, qRT-PCR amplification was performed on a CFX96 Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using TransStart Top Green qRCP SuperMix (AQ131; Trans, Beijing, China) according to the manufacturer’s instructions. The reaction conditions were 95 °C (30 s), followed by 40 cycles at 95 °C (5 s) and 56 °C (30 s). The experiment was repeated three times and fold change in gene expression level was calculated with the 2−ΔΔCt method . The VvUbiquitin1 gene was used as an internal reference. Primers used in this study are shown in Additional file 7. qRT-PCR data are presented as mean ± SD; the significance of differences between groups was evaluated with the independent samples t test.
DLG and QL conceived and designed the experiment, DLG, QL and YHY analyzed and interpreted the data and wrote the manuscript. XRJ and ZGW participated in berry sample collection, RNA extraction and qRT-PCR analysis. All authors read and approved the final manuscript.
This work was supported by National Natural Science Foundation of China (NSFC:31672106), Zhongyuan Science Technology Innovation Leaders (194200510007) and National Key Research and Development Program of China (2018YFD1000105). The funding bodies had no role in the design of the study, collection, analysis, or interpretation of data, or in writing the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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