Overexpression of MsRCI2A, MsRCI2B, and MsRCI2C in Alfalfa (Medicago sativ a L.) Provides Different Extents of Enhanced Alkali and Salt Tolerance Due to Functional Specialization of MsRCI2s

Abstract

Rare cold-inducible 2/plasma membrane protein 3 (RCI2/PMP3) genes are ubiquitous in plants and belong to a multigene family whose members respond to a variety of abiotic stresses by regulating ion homeostasis and stabilizing membranes, thus preventing damage. In this study, the expression of MsRCI2A, MsRCI2B, and MsRCI2C under high-salinity, alkali and ABA treatments was analyzed. The results showed that the expression of MsRCI2A, MsRCI2B, and MsRCI2C in alfalfa (Medicago sativa L.) was induced by salt, alkali and ABA treatments, but there were differences between MsRCI2 gene expression under different treatments. We investigated the functional differences in the MsRCI2A, MsRCI2B, and MsRCI2C proteins in alfalfa (Medicago sativa L.) by generating transgenic alfalfa plants that ectopically expressed these MsRCI2s under the control of the CaMV35S promoter. The MsRCI2A/B/C-overexpressing plants exhibited different degrees of improved phenotypes under high-salinity stress (200 mmol.L^-1 NaCl) and weak alkali stress (100 mmol.L^-1 NaHCO₃, pH 8.5). Salinity stress had a more significant impact on alfalfa than alkali stress. Overexpression of MsRCI2s in alfalfa caused the same physiological response to salt stress. However, in response to alkali stress, the three proteins encoded by MsRCI2s exhibited functional differences, which were determined not only by their different expression regulation but also by the differences in their regulatory relationship with MsRCI2s or H⁺-ATPase.

Keywords: Medicago saliva L.; RCI2 genes; alkali tolerance; gene overexpression; salt tolerance.

FIGURE 1

Relative expression of the MsRCI2 genes under abiotic treatment: (A) 100 mmol.L^–1 NaHCO₃ (pH 8.5) treatment, (B) 200 mmol.L^–1 NaHCO₃ (pH 8.5) treatment, (C) 200 mmol.L^–1 NaCl treatment, and (D) 100 μmol.L^–1 ABA induction. The figure on the left shows the expression changes in genes in the leaves; the figure on the right shows those in the roots. The values are the means ± SDs of three replicates (**P < 0.01; *P < 0.05).

FIGURE 2

Phenotype (A), Chl content (B), and relative conductivity (C) analysis of MsRCI2 transgenic alfalfa plants under alkali stress. WT stands for wild-type; A12 and A22, B13 and B19, and C2 and C10 were transformed with the MsRCI2A, MsRCI2B, MsRCI2C genes, respectively. The values are the means ± SDs of three replicates (Duncan test: P < 0.05).

FIGURE 3

MDA content, SOD activity, POD activity, and CAT activity in WT and transgenic alfalfa plants before and after alkali and salt treatment. Comparisons of the MDA content (A,E), SOD activity (B,F), POD activity (C,G), and CAT activity (D,H) of the WT plants and transgenic alfalfa plants before and after 6 and 12 d of alkali and salt treatments. The values are the means ± SDs of three replicates (Duncan test: P < 0.05).

FIGURE 4

Contents of soluble sugars and Pro in WT and transgenic alfalfa plants before and after alkali and salt treatments. Comparisons of soluble sugar contents (A,C) and Pro contents (B,D) of WT and transgenic alfalfa plants before and after 6- and 12-days alkali and salt treatments. The values are the means ± SDs of three replicates (Duncan test: P < 0.05).

FIGURE 5

Relative expression of stress-responsive H⁺-ATPase (A), MsRCI2A (B), MsRCI2B (C), and MsRCI2C (D) genes in WT and transgenic alfalfa plants under alkali stress. The values are the means ± SDs of three replicates (**P < 0.01; *P < 0.05).

FIGURE 6

Functional model of the MsRCI2 response to alkali and salinity stress. The main functions of MsRCI2s are as follows: (1) ROS scavenging and osmotic adjustment in response to salinity stress; (2) differences in MsRCI2 gene expression and regulatory relationships with H⁺-ATPase or MsRCI2s; and (3) overexpression of MsRCI2A/B/C genes enhanced alkali and salt tolerance.