Overexpression of MsRCI2D and MsRCI2E Enhances Salt Tolerance in Alfalfa (Medicago sativa L.) by Stabilizing Antioxidant Activity and Regulating Ion Homeostasis

Abstract

Rare cold-inducible 2 (RCI2) genes from alfalfa (Medicago sativa L.) are part of a multigene family whose members respond to a variety of abiotic stresses by regulating ion homeostasis and stabilizing membranes. In this study, salt, alkali, and ABA treatments were used to induce MsRCI2D and MsRCI2E expression in alfalfa, but the response time and the expression intensity of the MsRCI2D,-E genes were different under specific treatments. The expression intensity of the MsRCI2D gene was the highest in salt- and alkali-stressed leaves, while the MsRCI2E gene more rapidly responded to salt and ABA treatment. In addition to differences in gene expression, MsRCI2D and MsRCI2E differ in their subcellular localization. Akin to MtRCI2D from Medicago truncatula, MsRCI2D is also localized in the cell membrane, while MsRCI2E is different from MtRCI2E, localized in the cell membrane and the inner membrane. This difference might be related to an extra 20 amino acids in the C-terminal tail of MsRCI2E. We investigated the function of MsRCI2D and MsRCI2E proteins in alfalfa by generating transgenic alfalfa chimeras. Compared with the MsRCI2E-overexpressing chimera, under high-salinity stress (200 mmol·L^-1 NaCl), the MsRCI2D-overexpressing chimera exhibited a better phenotype, manifested as a higher chlorophyll content and a lower MDA content. After salt treatment, the enzyme activities of SOD, POD, CAT, and GR in MsRCI2D- and -E-overexpressing roots were significantly higher than those in the control. In addition, after salt stress, the Na⁺ content in MsRCI2D- and -E-transformed roots was lower than that in the control; K⁺ was higher than that in the control; and the Na⁺/K⁺ ratio was lower than that in the control. Correspondingly, H⁺-ATPase, SOS1, and NHX1 genes were significantly up-regulated, and the HKT gene was significantly down-regulated after 6 h of salt treatment. MsRCI2D was also found to regulate the expression of the MsRCI2B and MsRCI2E genes, and the MsRCI2E gene could alter the expression of the MsRCI2A, MsRCI2B, and MsRCI2D genes. MsRCI2D- and -E-overexpressing alfalfa was found to have higher salt tolerance, manifested as improved activity of antioxidant enzymes, reduced content of reactive oxygen species, and sustained Na⁺ and K⁺ ion balance by regulating the expression of the H⁺-ATPase, SOS1, NHX1, HKT, and MsRCI2 genes.

Keywords: H+-ATPase; HKT; Medicago saliva L.; MsRCI2s; SOS1; salt tolerance.

Figure 1

Relative expression of MsRCI2D (A,B) and MsRCI2E (C,D) in leaves and roots under abiotic stress: 200 mmol·L⁻¹ NaCl, 200 mmol·L⁻¹ NaHCO3 (pH 8.5), and 100 μmol·L⁻¹ ABA treatment. The figure on the left shows the gene expression changes in the leaves; the figure on the right shows those in the roots. The values are the means ± SDs of three replicates; * indicates a significant difference (p < 0.05); ** indicates an extremely significant difference (p < 0.01).

Figure 2

Subcellular localization of MsRCI2D/-E–GFP in Arabidopsis protoplasts. Confocal laser scanning microscope images of Arabidopsis protoplasts expressing GFP fused to MsRCI2D,-E proteins. The red fluorescence signal represents FM4-64-stained chloroplast in a cell. The white scale bars represent 8 or 10 μm.

Figure 3

Transgenic alfalfa chimera overexpressing MsRCI2D–GFP or MsRCI2E–GFP in hairy roots due to Agrobacterium rhizogenes. (A) Vector structure; (B) green-fluorescent-signal detection in MsRCI2D/-E transgenic hairy roots. Confocal laser scanning microscope images of MsRCI2D- and -E–GFP-overexpressing transgenic alfalfa chimera. White scale bars represent 25 μm.

Figure 4

Phenotype (A), chlorophyll content (B), relative conductivity (C), malondialdehyde content (D), and soluble sugars (E) analysis of alfalfa transformed with the MsRCI2D and MsRCI2E genes on the 5th and 10th days of salt treatment. WT stands for wild type. RCI2D and RCI2E were transformed with the MsRCI2D and MsRCI2C genes in hairy roots, respectively. The values are the means ± SDs of three replicates; * indicates a significant difference (p < 0.05); ** indicates an extremely significant difference (p < 0.01).

Figure 5

Changes in the antioxidant capacity of MsRCI2D/-E transgenic alfalfa hairy roots before and after exposure to salt stress. Comparison of H₂O₂ and ORF contents (A,B), SOD activity (C), POD activity (D), CAT activity (E), and GR (F) in WT plants and transgenic alfalfa hairy roots before and after 5 and 10 days of salt treatment. The values are the means ± SDs of three replicates; * indicates a significant difference (p < 0.05); ** indicates an extremely significant difference (p < 0.01).

Figure 6

Changes in Na⁺ and K⁺ contents (A,B) and Na⁺/K⁺ ratio (C) in the hairy roots of transgenic alfalfa after salt treatment for 10 days. The values are the means ± SDs of three replicates; * indicates a significant difference (p < 0.05); ** indicates an extremely significant difference (p < 0.01).

Figure 7

Relative expression of related genes under salt stress in WT and transgenic alfalfa hairy roots. The differences in the expression of genes closely related to antioxidants are shown in (A–D) ((A) Cu/Zn-SOD; (B) CAT; (C) GR; (D) GS). The expression of H⁺-ATPase (E), SOS1 (F), NHX1 (G), and HKT1 (H) was also detected after and before salt stress at 6 h and 12 h. The values are the means ± SDs of three replicates (** p < 0.01; * p < 0.05).

Figure 8

Relative expression of MsRCI2A-F genes under salt stress in WT and transgenic hairy roots. (A–D) Differences in the expression of MsRCI2A-C and MsRCI2F (D) in MsRCI2D- and -E-overexpressing hairy roots; differences in the expression of MsRCI2D (E) and MsRCI2E (F) were also detected. The values are the means ± SDs of three replicates (** p < 0.01; * p < 0.05).

Figure 9

Functional model of the MsRCI2D/-E response to salinity stress. The main functions of MsRCI2D/-E are as follows: (1) ROS scavenging and stabilization of the antioxidant system in response to salinity stress and (2) differences in MsRCI2 gene expression and regulatory relationships with H⁺-ATPase, SOS1, NHX1, HKT1, or MsRCI2s.