The Transcriptome and Metabolome Reveal Stress Responses in Sulfur-Fumigated Cucumber (Cucumis sativus L.)

Abstract

Sulfur (S) fumigation is a commonly used sterilization method in horticultural facilities against fungal diseases. S fumigation damaged cucumber leaves, although the response mechanism is unclear. This study analyzes the growth, transcriptome, and metabolomic profiles of young and mature leaves, ovaries, and commercial cucumber fruits to decipher the mechanism of cucumber stress response under S fumigation. S fumigation significantly changed the photosynthetic efficiency and reactive oxygen species (ROS) in leaves, but not fruit development, fruit mass, and peel color. Transcriptome analysis indicated that S fumigation strongly regulated stress defense genes. The weighted gene co-expression network analysis revealed that S fumigation regulated ASPG1, AMC1 defense genes, LECRK3, and PERK1 protein kinase. The abscisic acid (ABA)-mediated model of regulation under S fumigation was constructed. Metabolome analysis showed that S fumigation significantly upregulated or downregulated the contents of amino acids, organic acids, sugars, glycosides, and lipids (VIP > 1 and P-value < 0.05). The opposite Pearson's correlations of these differential metabolites implied that cucumber had different metabolic patterns in short-term and long-term S fumigation. Besides, the elevated levels of proline and triglyceride indicated that stress-responsive mechanisms existed in S-fumigated cucumber. Moreover, the comprehensive analysis indicated that S fumigation elevated secondary S-containing metabolites but decreased sulfate absorption and transportation in cucumber. Overall, our results provided a comprehensive assessment of S fumigation on cucumber, which laid the theoretical foundation for S fumigation in protected cultivation.

Keywords: cucumber; metabolome; stress response; sulfur fumigation; transcriptome.

FIGURE 1

Effects of sulfur (S) fumigation on (A) the appearance of leaves, (B) chlorophyll contents, (C) Fv/Fm and ΦPSII, (D) MDA, and (E) SOD and POD. * indicates significant difference at P < 0.05, according to Duncan’s test.

FIGURE 2

The changes in fruit quality in non-fumigated and S-fumigated treatments. (A) The appearance at F12. (B) The changes in length and diameter during fruit development. (C) The fruit mass at F12. (D) The peel luminosity and hue angle at F12. (E) The content of soluble solids in F12. * indicates significant difference at P < 0.05, according to Duncan’s test.

FIGURE 3

Overview of cucumber transcriptome responses to S fumigation. (A) The number of individual transcripts significantly upregulated or downregulated at L0, L12, F0, and F12. (B) Venn diagram illustrating the number of differentially expressed genes (DEGs) upregulated or downregulated by S fumigation at L0, L12, F0, and F12. (C) Gene ontology (GO) enrichment analysis of DEGs in the cucumber transcriptome at L0, L12, F0, and F12. Data were visualized using a scatter diagram with P-value levels indicated by −log10 (P-value) and an enrichment factor indicative of individual terms. Values in parentheses represent the number of DEGs in each term.

FIGURE 4

Weighted gene co-expression network analysis (WGCNA)-identified gene networks and hub genes involved in cucumber stress response after S fumigation. (A) Notably, 36 modules with co-expressed genes were hierarchically clustered. Each leaflet in the tree corresponds to an individual gene. (B) Module-trait associations based on Pearson’s correlation. The colors from green to red represent r² values from −1 to 1. (C) The gene network for the green-yellow module positively correlated with SL0 (the young leaves with S fumigation) (r² = 0.78 and P = 7.00E-06). (D) The gene network for the cyan module positively correlated with SL12 (the mature leaves with S fumigation) (r² = 0.76 and P = 1.00E-05). In each network, hub genes are highlighted with enlarged spots and marked red. WRKYs, WRKY transcription factors; NAC, NAC transcription factors; ERFs, ERF transcription factors; CBPs, CBP transcription factors; HSFs, HSF transcription factors; ERFs, ERF transcription factors; TALEs, TALE transcription factors; COs, CO transcription factors; MYBs, MYB transcription factors.

FIGURE 5

A model for the abscisic acid (ABA)-mediated stress signal transduction after S fumigation. The upregulated and downregulated genes after S fumigation are presented in blue and red boxes, respectively. The white boxes represent genes with unchanged expression. * indicates DEGs with | log2FoldChange| > 1 and P-value < 0.05. HOS, E3-ubiquitin-ligases; SnRK2s, SNF1-related kinases; PP2Cs, protein phosphatase 2Cs; PYLs, PYR-like genes; PUB, U-box type E3 ubiquitin ligases; RBOHF, NADPH oxidases; ASPG, aspartic protease; ABF4, ABA-responsive element binding factor 4; ABI5, ABA insensitive factor 5.

FIGURE 6

Pathway analysis of the DEMs detected in leaves and fruits under non-fumigation and S fumigation, respectively. The x-axis represents the number of DEMs annotated in each pathway. The blue portions represent differentially detected metabolites, while the green portions represent the other annotated metabolites in the pathway. The percentages are the ratios of blue to green. Only pathways with at least 10% significant regulation were included.

FIGURE 7

Changes in sugars, lipids, and amino acids in tricarboxylic acid cycle (TCA) metabolism. Blue and red boxes, respectively, represent metabolites with lower or higher abundance under S fumigation. The white boxes represent metabolites with unchanged abundances. The gray letters represent undetected metabolites. * indicates DEMs with P-value < 0.05 and VIP > 1. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol.

FIGURE 8

O2PLS loading plot of metabolites and transcripts involved in S-related metabolism. Transcripts (circles) and metabolites (triangles) represent individual transcript and metabolite loading values, respectively (Supplementary Tables 9, 10).

FIGURE 9

Changes in S metabolism in S fumigated cucumber. Upregulated and downregulated genes and metabolites due to S fumigation are represented by blue-to-red boxes and green-to-red circles, respectively. The white boxes or circles represent unchanged genes or metabolites. ^∗ indicates DEGs or DEMs with | log₂FoldChange| > 1 and P-value < 0.05, or VIP > 1 and P-value < 0.05. ST, sulfate transporters; ATPS, ATP sulfurylase; APR, adenosine phosphosulphate reductase; SIR, sulfate reductase; OASTL, O-acetylserine (thiol) lyase; SAT, serine acetyltransferase; CYSK, cysteine synthase; γ-EC, γ-glutamylcysteine synthetase; GSHB, glutathione synthetase; CGS, cystathionine γ-synthase; CBL, cysteine-S-conjugate β-lyase; MetE, L-glutamine-4-(methylsulfanyl)-2-oxobutanoate aminotransferase; MetK, S-adenosylmethionine synthetase. The red star represents the hub gene identified by WGCNA.

FIGURE 10

Sulfur fumigation modulated growth, transcription expression profiles, and metabolic pathways in cucumber leaves and fruits. Red and green arrows represent upregulated and downregulated physiological indicators, transcriptional regulation pathways, and metabolic pathways due to S fumigation.