Integrated transcriptomic and metabolomic analyses uncover the key pathways of Limonium bicolor in response to salt stress

Abstract

Salinity significantly inhibits plant growth and development. While the recretohalophyte Limonium bicolor can reduce its ion content by secreting salt, the metabolic pathways it employs to adapt to high salt stress remain unclear. This study aims to unravel this enigma through integrated transcriptomic and metabolomic analyses of L. bicolor under salt stress conditions. The results showed that compared to the control (S0), low salt treatment (S1) led to a significant increase in plant growth, photosynthesis efficiency and antioxidant enzyme activity but caused no significant changes in organic soluble substance and ROS contents. However, high salt treatments (S3 and S4) led to a significant decrease in plant growth, photosynthesis efficiency and antioxidant enzyme activity, accompanied by a significant increase in organic soluble substance and ROS contents. A significant increase in phenolic compounds, such as caffeoyl shikimic acid and coniferin, upon the treatments of S1, S3 and S4, and a decrease and increase in flavonoids upon the treatments of S1 and S3 were also observed, respectively. This study also demonstrated that the expression patterns of key genes responsible for the biosynthesis of these metabolites are consistent with the observed trends in their accumulation levels. These results suggest that under low salt stress conditions, the halophyte L. bicolor experiences minimal osmotic and oxidative stress. However, under high salt stress conditions, it suffers severe osmotic and oxidative stress, and the increase in organic soluble substances and flavonoids serves as a key response to these stresses and also represents a good strategy for the alleviation of them.

Keywords: Limonium bicolor; flavonoid; phenolic compound; plant growth; salt stress.

Figure 1

Effects of different concentrations of NaCl treatment on (a) growth phenotype, (b) growth parameters, (c) chlorophyll contents, (d) photosynthetic parameters of L. bicolor seedlings. The vertical bar indicates the standard deviation of five replications. For the same parameter, different letters above the bars indicate a significant difference at P < 0.05 level. The symbols S0, S1, S2, S3 and S4 represent NaCl treatment concentrations of 0, 100, 200, 300 and 400 mm, respectively. The following results are the same. Bar = 5 cm.

Figure 2

Effects of NaCl treatment on the contents of inorganic ions, organic solutes, ROS contents, antioxidant enzymes gene expression levels and antioxidant enzyme activities of L. bicolor seedlings. (a) Na⁺ content; (b) K⁺ content; (c) proline content; (d) soluble protein content; (e) soluble sugar content; (f) glucose content; (g) fructose content; (h) sucrose content; (i) O₂ ^•− content; (j) H₂O₂ content; (k) electrolyte leakage; (l) MDA content; (m–p) the enzyme activities of SOD, POD, CAT and APX; (q–t) the relative expression levels of SOD, POD, CAT and APX genes. The vertical bar indicates the standard deviation of five replicates. For the same parameter, different letters above the bars indicate a significant difference at P < 0.05 level.

Figure 3

Changes in differentially expressed gene (DEG) expression. (a) Venn diagram for the overlap between DEGs in each of the different treatments. (b) Regulation of DEGs, both up and down. Up‐regulation is represented by the green box and down‐regulation by the red box. (c) Heatmaps of DEGs compared between different groups.

Figure 4

Analysis of metabolome quality and differential metabolite expression. (a) Type and number of metabolites of L. bicolor. (b) Principal component analysis (PCA) of DAMs. (c) Pairwise comparison of DAMs in groups on the Venn diagram. The overlap indicates the proportion of metabolites shared by each comparison group, while the non‐overlap indicates the proportion of metabolites specific to the comparison group. (d–f) Comparison of heat maps of DAMs between different groups.

Figure 5

Analysis of differential metabolite expression. DAMs up‐ and down‐regulation in various treatment groups. (a–c) Volcano plots displaying the metabolites that are up‐ and down‐regulated. Red points, green points and grey points indicate the metabolites that were significantly up‐regulated, down‐regulated and non‐significance, respectively. (d–f) Top 10 up‐regulated (red) and down‐regulated (green) compounds.

Figure 6

Correlation analysis of transcriptomic and metabolomic data from L. bicolor at different NaCl concentrations. (a–c) The nine‐quadrant diagram for S0 vs S1, S0 vs S3 and S1 vs S3. (d–f) KEGG enrichment analysis of DEGs (blue column) and DAMs (red column) enriched in the same pathway.

Figure 7

Major metabolic pathways of L. bicolor plants following 14 days of treatment with varying concentrations of NaCl. (a) Contribution of differentially expressed genes (DEGs) and differentially abundant metabolites (DAMs) to phenylpropanoid biosynthesis pathway under salt stress. (b) Role of DEGs and DAMs in flavonoid biosynthesis pathway under salt stress. Circular diagram visually represents differential metabolites, showcasing their altered content levels. Enclosed within boxes are the relevant enzymes, which are impacted by the expression changes of differential genes. The diagram is divided into three equal segments (S0, S1 and S3), each representing a distinct aspect or condition. The vibrant colours of the rectangles signify the regulation status of the genes or metabolites under NaCl treatment, as described in the scale bar.

Figure 8

Model of the regulatory mechanisms of L. bicolor in response to low and high salt treatments. Blue and red represent low and high salt treatments, respectively. Different types of arrows indicate the inductive or inhibitory effects of stress, which are manifested to the right of the responding genes, metabolites and osmolytes.