Integrated Transcriptome Analysis and Single-Base Resolution Methylomes of Watermelon (Citrullus lanatus) Reveal Epigenome Modifications in Response to Osmotic Stress

Abstract

DNA methylation plays an important role against adverse environment by reshaping transcriptional profile in plants. To better understand the molecular mechanisms of watermelon response to osmotic stress, the suspension cultured watermelon cells were treated with 100mM mannitol, and then a methylated cytosines map was generated by whole genome bisulfite sequencing (WGBS). Combined with transcriptome sequencing, the effects of osmotic stress on differentially methylated expressed genes (DMEGs) were assessed. It was found that genes related to plant hormone synthesis, signal transduction, osmoregulatory substance-related and reactive oxygen species scavenging-related enzyme could rapidly respond to osmotic stress. The overall methylation level of watermelon decreased after osmotic stress treatment, and demethylation occurred in CG, CHG, and CHH contexts. Moreover, differentially methylated expressed genes (DMEGs) were significantly enriched in RNA transport, starch and sucrose metabolism, plant hormone signal transduction and biosynthesis of secondary metabolites, especially in biosynthesis of osmolytes synthase genes. Interestingly, demethylation of a key enzyme gene Cla014489 in biosynthesis of inositol upregulated its expression and promoted accumulation of inositol, which could alleviate the inhibition of cell growth caused by osmotic stress. Meanwhile, a recombinant plasmid pET28a-Cla014489 was constructed and transferred into Escherichia coli BL21 for prokaryotic expression and the expression of ClMIPS protein could improve the tolerance of E. coli to osmotic stress. The effect of methylation level on the expression properties of inositol and its related genes was further confirmed by application of DNA methylation inhibitor 5-azacytidine. These results provide a preliminary insight into the altered methylation levels of watermelon cells in response to osmotic stress and suggest a new mechanism that how watermelon cells adapt to osmotic stress.

Keywords: Citrullus lanatus; DNA methylation; osmotic stress; transcriptome analysis; whole genome bisulfite sequencing.

Figure 1

Transcriptomic profiling of watermelon cells in response to osmotic stress. (A) Volcano maps of DEGs between different treatments. Red dots indicate up-regulated genes, green dots indicate down-regulated genes, and blue dots indicate out-of-threshold genes. (B) KEGG enrichment analysis of DEGs at 0h and 4h. The size of the circles represents the gene number and the color represents the q value. The same below. (C) GO enrichment analysis of DEGs at 0h and 4h. The x-axis represents three domains of GO while y-axis represents the gene numbers within each pathway and processes.

Figure 2

Overview of DEGs under osmotic stress in watermelon cells. Red arrows and numbers represent the number of up-regulated expressed genes and green arrows and numbers represent the number of down-regulated expressed genes.

Figure 3

The watermelon epigenome. (A) Relative proportions of mC in three sequence contexts of different species. (B) Circos plot of watermelon chromosomes. Track order: density plot of mC in CG, CHG and CHH contexts; density of TEs; gene density of each chromosome. Red represents hypermethylation while green represents hypomethylation.

Figure 4

Methylation and gene expression association analysis. (A) The relationship between methylation level and gene expression. (B) Venn diagram of DEGs and DMGs. (C) KEGG pathway enrichment of DEMGs.

Figure 5

Analysis of differentially methylated genes under osmotic stress. (A) Venn diagram between DMGs in different contexts. (B) KEGG enrichment pathway of DMGs in CHH contexts.

Figure 6

Regulation of osmotic stress by exogenous inositol in watermelon cells and analysis of Cla014489 prokaryotic expression. (A) Effect of exogenous inositol on the growth of watermelon cells under osmotic stress. (B) Effect of exogenous inositol on the MDA content of watermelon cells under osmotic stress. (C) The expression of ClMIPS in response to osmotic stress. (D) Inositol accumulation in watermelon cells under osmotic stress. (E) Droplet plate experiment of recombinant strain pET-28a-Cla014489 and control strain pET-28a under osmotic stress. (F) Time course of growth of the recombinant strain pET-28a-Cla014489 and the control strain (pET-28a) under 600mmol/l mannitol. Different uppercase and lowercase letters mean differences at p<0.05 and p<0.01 levels. Values are means ± SD of three biological replicates. * Indicates significantly difference (p<0.05) between the data point and 0 h data point.

Figure 7

The osmotic regulation of inositol on watermelon cells may be associated with DNA methylation. (A) The effect of different treatments on the expression of ClMIPS1. (B) The effect of different treatments on the accumulation of inositol. Different lowercase letters mean significant differences (p<0.05). Values are means ± SD of three biological replicates.

Figure 8

Model of regulation of ClMIPS in response to osmotic stress. “→” represents induction of phytohormone, while “⊥” stands for suppression. Solid lines indicate identified pathways and dashed lines indicate speculative pathways.