Comparative Physiological and Transcriptome Profiles Uncover Salt Tolerance Mechanisms in Alfalfa

Abstract

Salinity is a major limiting factor that affects crop production. Understanding of the mechanisms of plant salt tolerance is critical for improving crop yield on saline land. Alfalfa (Medicago sativa L.) is the most important forage crop, while its salt tolerance mechanisms are largely unknown. The physiological and transcriptomic responses in two contrasting salt tolerant cultivars to salinity stress were investigated in the present study. "Magnum Salt" showed higher salt tolerance than "Adrenalin," with higher relative germination rate, survival rate, biomass and K⁺/Na⁺ ratio after salt treatment. Activities of antioxidant enzymes SOD, CAT and GR, and proline concentrations were upregulated to higher levels in roots and shoots in Magnum Salt than in Adrenalin after salinity stress, except for no difference in GR activity in shoots, and lower levels of O₂ ^⋅- and H₂O₂ were accumulated in leaves. It was interesting to find that salinity caused a decrease in total unsaturated fatty acid in Adrenalin other than Magnum Salt, C18:2 was increased significantly after salinity in Magnum Salt, while it was unaltered in Adrenalin. High quality RNA sequencing (RNA-seq) data was obtained from samples of Magnum Salt and Adrenalin at different time points (0, 2, and 26 h). Generally, "phagosome," "TCA cycle" and "oxidative phosphorylation" pathways were inhibited by salinity stress. Upregulated DEGs in Magnum Salt were specifically enriched in "fatty acid metabolism," "MAPK signaling" and "hormone signal transduction" pathways. The DEGs involved in ionic homeostasis, reactive oxygen species (ROS) scavenging and fatty acid metabolism could partially explain the difference in salt tolerance between two cultivars. It is suggested that salt tolerance in alfalfa is associated with regulation of ionic homeostasis, antioxidative enzymes and fatty acid metabolism at both transcriptional and physiological level.

Keywords: alfalfa; antioxidant defense system; ionic homeostasis; salt tolerance; transcriptome; unsaturated fatty acids.

FIGURE 1

Evaluation of salt tolerance of 8 alfalfa cultivars by analyzing relative germination rate and survival rate under salt condition. Alfalfa seeds were treated with 0 or 150 mM NaCl solution, and relative germination rate (A) were calculated within 7 days. Four-week old seedlings treated with Hoagland nutrient solution with 500 mM NaCl (pH 5.8) weekly. Three weeks later, photographs (B) were taken and survival rates (C) were analyzed. Values are mean ± SE (n = 3 replicates). Different letters (P < 0.05, one-way ANOVA) compare different cultivars.

FIGURE 2

Effect of salt treatment on the biomass of Magnum Salt and Adrenalin. Three-week old seedlings were treated with 1/2 Hoagland solution containing 0 or 150 mM NaCl for 1 week. Photographs (A) were taken and biomass of shoot (B) and root (C) were analyzed. Values are mean ± SE (n = 10 replicates). Different letters indicate the significant differences (P < 0.05, LSD) between two cultivars or treatments.

FIGURE 3

Effect of salt treatment on the Na⁺, K⁺ and Ca²⁺ accumulation in Magnum Salt and Adrenalin. Three-week old seedlings were treated with 1/2 Hoagland solution containing 0 or 150 mM NaCl for 1 week. Na⁺, K⁺, Ca²⁺ content and Na⁺/K⁺ ratio in roots (A,C,E,G) and shoots (B,D,F,H) were analyzed. Values are mean ± SE (n = 3 replicates). Different letters indicate the significant differences (P < 0.05, LSD) between two cultivars or treatments.

FIGURE 4

Effect of salt treatment on the antioxidant enzyme activities and proline content in Magnum Salt and Adrenalin. Four-week old seedlings grown in soil with a mixture of pearlite, vermiculite and perlite (1:1:2) were treated with 0 or 500 mM NaCl for 6 days. SOD, CAT, GR, and APX activities and proline contents were analyzed in both root (A,C,E,G,I) and shoot (B,D,F,H,J). Values are mean ± SE (n = 3 replicates). Different letters indicate the significant differences (P < 0.05, LSD) between two cultivars or treatments.

FIGURE 5

Effect of salt treatment on the ROS content in Magnum Salt and Adrenalin. Four-week old seedlings grown in soil with a mixture of pearlite, vermiculite and perlite (1:1:2) were treated with 0 or 500 mM NaCl for 6 days. Leaves were sampled for NBT and DAB staining.

FIGURE 6

Effect of salt treatment to the Fatty acids content in Magnum Salt and Adrenalin. Four-week old seedlings treated with 0 or 500 mM NaCl. At 0, 3 and 6 days, roots were sampled for fatty acids content analysis (A). The upper and lower edges of the box in the boxplot represent the maximum and minimum values, respectively, and the horizontal line in the middle represents the median values. Relative abundance of saturated fatty acids (SFA), unsaturated fatty acid (UFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acid (PUFA); (B) and double bond index (DBI; number of double bonds per mole) (C) were calculated. Values are mean ± SE (n = 3 replicates). Different letters indicate the significant differences (P < 0.05, LSD) between two cultivars or treatments.

FIGURE 7

Differentially expressed genes (DEGs) in Magnum Salt (T) and Adrenalin (S) under salt stress. (A) Number of upregulated and downregulated DEGs under salt stress. (B) Venn diagram analysis of the DEGs in different comparison groups of S0-vs-S2, S0-vs-S26, T0-vs-T2 and T0-vs-T26.

FIGURE 8

Heatmap of the relative expression of the DEGs involved in Na⁺, K⁺ and Ca²⁺ transport (A), ROS-scavenging (B) and fatty acid metabolization (C) in Magnum Salt and Adrenalin under salt stress. FPKM was used for representing the expression abundance of genes. The clustered heatmap was portrayed after normalized with zero-to-one scale method using the TBtools software.

FIGURE 9

RNA-seq data validation. Random selected genes with different expression patterns were analyzed by qRT-PCR (A–K), correlation between qRT-PCR and RNA-seq data are calculated (L). Values are mean ± SE (n = 3 replicates).

FIGURE 10

A proposed model showing salt tolerance mechanisms in alfalfa. Salt tolerance in alfalfa is associated with regulation of ionic homeostasis, antioxidative enzymes and fatty acid metabolism. Red and blue genes are proposed positive and negtive regulatory genes in salt tolerance.