Analysis of the Antioxidant Mechanism of Tamarix ramosissima Roots under NaCl Stress Based on Physiology, Transcriptomic and Metabolomic

Abstract

There is a serious problem with soil salinization that affects the growth and development of plants. Tamarix ramosissima Ledeb (T. ramosissima), as a halophyte, is widely used for afforestation in salinized soils. At present, there are few reports on the antioxidant mechanism of T. ramosissima under NaCl stress. In this study, we learned about the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content changes in T. ramosissima. We also mined the relevant metabolic pathways in the antioxidant mechanism, candidate key genes, and their related differential metabolites and verified them using quantitative real-time PCR (qRT-PCR). The results show that the SOD, POD, and CAT activities, and the H₂O₂ and MDA content reached the highest values in the roots of T. ramosissima. Simultaneously, 92 differentially expressed genes (DEGs) related to antioxidant enzyme activities changed during 48 and 168 h of NaCl stress, and these DEGs were mainly upregulated in 168 h. Based on the association analysis of transcriptomic and metabolomic data, we found Unigene0089358 and Unigene0007782 as genes related to key enzymes in the flavonoid biosynthesis pathway. They were located in the upstream positive regulation at 48 and 168 h under NaCl stress, and their respective related metabolites (phloretin and pinocembrin) were involved in resistance to NaCl stress, and they were significantly correlated with their respective metabolites. In conclusion, at 48 and 168 h under NaCl stress, the roots of T. ramosissima resist NaCl stress by enhancing enzymatic and nonenzymatic antioxidant mechanisms, scavenging ROS generated by high-salt stress, alleviating NaCl toxicity, and maintaining the growth of T. ramosissima. This study provides genetic resources and a scientific theoretical basis for further breeding of salt-tolerant Tamarix plants and the molecular mechanism of antioxidants to alleviate NaCl toxicity.

Keywords: NaCl stress; NaCl toxicity; antioxidant mechanism; metabolome; transcriptome.

Figure 1

Change in antioxidative enzyme activity of T. ramosissima roots under NaCl stress. (Changes in SOD, POD, and CAT activities in the roots of T. ramosissima at 0, 48, and 168 h under NaCl stress).

Figure 1

Change in antioxidative enzyme activity of T. ramosissima roots under NaCl stress. (Changes in SOD, POD, and CAT activities in the roots of T. ramosissima at 0, 48, and 168 h under NaCl stress).

Figure 2

Change in H₂O₂ and MDA content of T. ramosissima roots under NaCl stress. (Changes in H₂O₂ and MDA content in roots of T. ramosissima under different treatments at 0, 48, and 168 h).

Figure 3

Flavonoid biosynthesis pathway annotated with DEGs and differential metabolites. (DEGs and metabolites annotated on the flavonoid biosynthesis pathway in the roots of T. ramosissima at 48 and 168 h under NaCl stress. Note: blue box: DEGs regulate their related differential metabolites; red genes: DEGs upregulated; black genes: DEGs downregulated; red differential metabolites: differential metabolite accumulation; green differential metabolites: differential metabolite degradation.

Figure 3

Flavonoid biosynthesis pathway annotated with DEGs and differential metabolites. (DEGs and metabolites annotated on the flavonoid biosynthesis pathway in the roots of T. ramosissima at 48 and 168 h under NaCl stress. Note: blue box: DEGs regulate their related differential metabolites; red genes: DEGs upregulated; black genes: DEGs downregulated; red differential metabolites: differential metabolite accumulation; green differential metabolites: differential metabolite degradation.

Figure 4

Heatmap of the correlation between DEGs and differential metabolites in the flavonoid biosynthesis pathway. (Heatmap of correlations between DEGs regulating differential metabolites in the flavonoid biosynthesis pathway and their differential metabolites. Note: p ≥ 0.05 is not marked; 0.01 < p < 0.05 is marked as *; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***).

Figure 5

qRT-PCR validation of candidate key genes. (Nine candidate key genes were obtained based on antioxidant enzyme activity and gene expression in the flavonoid biosynthesis pathway. Note: p ≥ 0.05 is not marked; 0.01 < p < 0.05 is marked as *; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***; red color: numerical value is shown on the left side of the Y axis; blue color: numerical value is shown on the right side of the Y axis).

Figure 5

qRT-PCR validation of candidate key genes. (Nine candidate key genes were obtained based on antioxidant enzyme activity and gene expression in the flavonoid biosynthesis pathway. Note: p ≥ 0.05 is not marked; 0.01 < p < 0.05 is marked as *; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***; red color: numerical value is shown on the left side of the Y axis; blue color: numerical value is shown on the right side of the Y axis).