Transcriptomic Analysis of Short-Term Salt Stress Response in Watermelon Seedlings

Abstract

Watermelon (Citrullus lanatus L.) is a widely popular vegetable fruit crop for human consumption. Soil salinity is among the most critical problems for agricultural production, food security, and sustainability. The transcriptomic and the primary molecular mechanisms that underlie the salt-induced responses in watermelon plants remain uncertain. In this study, the photosynthetic efficiency of photosystem II, free amino acids, and transcriptome profiles of watermelon seedlings exposed to short-term salt stress (300 mM NaCl) were analyzed to identify the genes and pathways associated with response to salt stress. We observed that the maximal photochemical efficiency of photosystem II decreased in salt-stressed plants. Most free amino acids in the leaves of salt-stressed plants increased many folds, while the percent distribution of glutamate and glutamine relative to the amino acid pool decreased. Transcriptome analysis revealed 7622 differentially expressed genes (DEGs) under salt stress, of which 4055 were up-regulated. The GO analysis showed that the molecular function term "transcription factor (TF) activity" was enriched. The assembled transcriptome demonstrated up-regulation of 240 and down-regulation of 194 differentially expressed TFs, of which the members of ERF, WRKY, NAC bHLH, and MYB-related families were over-represented. The functional significance of DEGs associated with endocytosis, amino acid metabolism, nitrogen metabolism, photosynthesis, and hormonal pathways in response to salt stress are discussed. The findings from this study provide novel insights into the salt tolerance mechanism in watermelon.

Keywords: RNA-seq; amino acids; endocytosis; salt stress; watermelon.

Figure 1

Photosynthetic efficiency of photosystem II. The maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm) (A) and the efficiency of excitation capture of the open PSII center (Fv’/Fm’) (B) in cv. Crimson Sweet leaves under salt stress were measured using FluorPen (PAR-FluorPen FP 110/D). Asterisks ** and * represent significant difference at p < 0.05 and p < 0.1, respectively. Qy, equal to Fv/Fm in the dark (A) or light-adapted (B) samples, photosystem II efficiency. The error bars represent the standard deviation.

Figure 2

Fold-change increases in amino acids in watermelon leaves due to salt stress: The fold-change in plants treated with NaCl relative to the control group for amino acids showing significant changes (t-test, p < 0.05). The absolute amino acid quantities were normalized using internal standards and expressed as fold-change relative to control.

Figure 3

Summary of differentially expressed genes (DEGs) in the watermelon leaves during salt stress. Each point represents a gene; blue dots indicate no significant difference; red dots indicate up-regulated DEGs; green dots indicate down-regulated DEGs. The horizontal axis shows the fold change of genes between different samples (padj < 0.05), and the vertical coordinate indicates the statistically significant degree of changes in gene expression levels at −log10 (padj p-value).

Figure 4

Gene Ontology (GO) enrichment scatter plot. The GO enrichment analysis showing the top 20 enriched functions for up-regulated (A) and down-regulated (B) DEGs. The horizontal axis is GeneRatio (the ratio between the number of differentially expressed genes in each GO term, and all differentially expressed genes that can be found in the GO database). The vertical axis is the description of GO terms. The significance showing padj q-values are shown as a color scale, where the color and size of the dots represent the range of q-value, and the number of DEGs mapped to the indicated functions, respectively.

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment scatter plot. The KEGG enrichment analysis showing the top 20 enriched pathways for up-regulated (A) and down-regulated (B) DEGs. The horizontal axis is GeneRatio (the ratio between the number of differentially expressed genes in each pathway, and all differentially expressed genes that can be found in the KEGG database). The vertical axis is the description of the KEGG term. The significance showing padj q-values are shown as a color scale, where the color and size of the dots represent the range of q-value and the number of DEGs mapped to the individual pathways, respectively.

Figure 6

Distribution of transcription factor families. The red and green bars show the total number of down-regulated and up-regulated TFs in the respective family, respectively.

Figure 7

Quantitative real-time PCR (RT-qPCR) analysis.). Relative expression profiles of genes involved in the citrulline metabolic pathways in seedling leaf tissues of Crimson Sweet due to salt stress. The error bars are the means ± SE (n = 3), and asterisks (*) represent significant differences between treated and control tissues (p < 0.05).

Figure 8

DEGs mapped to the plant hormone signaling transduction pathways in watermelon leaves. (A) Auxin signaling pathway, (B) cytokinin pathway, (C) gibberellin (GA) pathway, (D) abscisic acid pathway, (E) ethylene signaling pathway, (F) brassinosteroid pathway, (G) jasmonic acid pathway, (H) salicylic acid pathway. The red and green boxes show the number of down- or up-regulated DEGs, respectively.