Physiological response and molecular regulatory mechanism reveal a positive role of nitric oxide and hydrogen sulfide applications in salt tolerance of Cyclocarya paliurus

Abstract

As a multifunctional tree species, Cyclocarya paliurus leaves are rich in bioactive substances with precious healthy values. To meet the huge requirement of C. paliurus leaf production, sites with some environmental stresses would be potential land for developing its plantations due to the limitation of land resources in China. Nitric oxide (NO) and hydrogen sulfide (H₂S) are common gas messengers used to alleviate abiotic stress damage, whereas the mechanism of these messengers in regulating salt resistance of C. paliurus still remains unclear. We performed a comprehensive study to reveal the physiological response and molecular regulatory mechanism of C. paliurus seedlings to the application of exogenous NO and H₂S under salt stress. The results showed that the application of sodium hydrosulfide (NaHS) and sodium nitroprusside (SNP) not only maintained the photosynthetic capacity and reduced the loss of leaf biomass, but also promoted endogenous NO synthesis and reduced oxidative damage by activating antioxidant enzyme activity and increasing the content of soluble protein and flavonoids. Moreover, transcriptome and metabolome analysis indicated the expression of genes encoding phenylalanine ammonia lyase (PAL), cytochromeP450 (CYP), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) in flavonoid biosynthesis pathway was all up-regulated by the application of NO and H₂S. Meanwhile, 15 transcriptional factors (TFs) such as WRKY, ERF, bHLH and HY5 induced by NO were found to regulated the activities of several key enzymes in flavonoid biosynthesis pathway under salt stress, via the constructed co-expression network. Our findings revealed the underlying mechanism of NO and H₂S to alleviate salt stress and regulate flavonoid biosynthesis, which provides a theoretical basis for establishing C. paliurus plantations in the salt stress areas.

Keywords: Cyclocarya paliurus; antioxidant system; exogenous substance; photosynthetic parameter; salt stress; transcription factors.

Figure 1

Phenotypes of C. paliurus seedlings after 30 days of the treatments. Code annotation: CK (no salt addition and only irrigation with distilled water), SNP (no salt addition, spraying with 0.25mM sodium nitroprusside (a NO donor), and irrigation with distilled water), NaHS (no salt addition, spraying with 0.5mM sodium hydrosulfide (a H₂S donor), and irrigation with distilled water), NaCl (0.4% NaCl treatment and irrigation with 0.4% NaCl solution), SNP+NaCl (0.4% NaCl treatment, spraying with 0.25mM SNP and irrigation with 0.4% NaCl solution), and NaHS+NaCl (0.4% NaCl treatment, spraying with 0.5mM NaHS and irrigation with 0.4% NaCl solution), the same below.

Figure 2

Leaf biomass (A) and photosynthetic parameters (B–F) in C. paliurus seedlings of different treatments. Different small letters indicate a significant difference (P < 0.05) among the treatments.

Figure 3

NO content (A) and soluble protein content (B) in C. paliurus leaves of different treatments. Different small letters indicate a significant difference (P < 0.05) among treatments.

Figure 4

PCA score plot and correlation heat map in transcriptomic profile of C. paliurus leaf samples. (A) Each point in PCA score plot representing an independent biological replicate; (B) Pearson product-moment correlation coefficients of the gene expression profile. The blue rectangles represent the positive correlation between the samples, whereas the red rectangles represent the negative correlation.

Figure 5

The analysis of DEGs in transcriptomic profile of C. paliurus samples and the annotation of KEGG enrichment pathway. Venn diagram (A) and the number (B) of DEGs between different treatments; Top 15 of KEGG enrichment pathway of DEGs between treatments of CK and SNP (C), CK and NaHS (D), Salt and SNP + NaCl (E), and Salt and NaHS + NaCl (F).

Figure 6

PCA score plot and the number of differentially accumulated metabolites in metabolomic profile of C. paliurus leaf samples. (A) Each point in PCA score plot representing an independent biological replicate; (B) The red and blue columns represent the up-regulation and down-regulation of metabolite abundance, respectively.

Figure 7

Differential expressions of structural genes and metabolites in flavonoid biosynthesis pathway of C. paliurus leaf samples. (A) Schematic diagram of metabolic relationship and related genes in flavonoid pathway; (B) Blue and yellow blocks represent high and low expression of genes, respectively. Enzyme annotation: 4CL, 4-coumarate: coenzyme A ligase; ANR, Anthocyanidin reductase; ANS, Anthocyanidin synthase; CHI, Chalcone isomerase; CHS, Chalcone synthase; CYP, CytochromeP450; DFR, Dihydroflavonol 4-reductase; F3H, Flavonoid 3-hydroxylase; FLS, Flavonol synthase; PAL, Phenylalanine ammonia lyase; UGT, UDP-glycosyltransferase. (C) Heat map representing the abundance of metabolites in the flavonoid pathway. And the shade of red in the blocks represents the abundance of metabolites.

Figure 8

Results of the gene co-expression network analysis. (A) Cluster dendrogram showing identified modules and the cluster tree of eigengenes in each module; (B) The number of genes in each module; (C) The heatmap of correlation coefficient between flavonoid contents and module eigengenes "*", "**" and "***" indicate correlations of 0.5-0.6, 0.6-0.7 and 0.7-1, respectively; with the red and green blocks representing positive and negative correlations, respectively; (D) Network constructed by selected TFs (white circles) and structural genes (yellow circles) related to flavonoid biosynthesis pathway from orange module. The red line and blue line represent positive correlation and negative correlation, respectively.