Genome-Wide Analysis of the MsRCI2 Gene Family in Medicago sativa and Functional Characterization of MsRCI2B in Salt Tolerance

Abstract

The Rare Cold-Inducible 2 (RCI2) gene encodes a conserved hydrophobic peptide that plays a crucial role in ion homeostasis, membrane stability, and responses to abiotic stress. In this study, six members of the MsRCI2 gene family were identified in Medicago sativa L., all of which contain highly conserved PMP₃ domains. Comparative collinearity analysis revealed syntenic relationships between M. sativa and M. truncatula, with each gene displaying distinct expression profiles under various stress conditions. Among them, MsRCI2B was significantly upregulated in response to salt stress. Alfalfa plants overexpressing MsRCI2B exhibited enhanced salt tolerance, as evidenced by increased antioxidant enzyme activities and reduced accumulation of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂^-) compared to wild-type plants. Furthermore, the transgenic lines maintained better Na⁺/K⁺ homeostasis under salt stress, reflected by a lower Na⁺/K⁺ ratio and significantly elevated expression of key ion transport genes, including MsSOS1, MsAKT1, and MsNHX1. To elucidate the molecular mechanisms underlying MsRCI2B function, a yeast two-hybrid (Y2H) screen identified 151 potential interacting proteins. Gene Ontology (GO) enrichment analysis revealed that these interactors are mainly involved in antioxidant defense and ion transport. Further validation confirmed direct interactions between MsRCI2B and both calmodulin (CaM) and vacuola H⁺-ATPase (V-H⁺-ATPase), suggesting that MsRCI2B contributes to ion homeostasis through interactions with CaM and V-H⁺-ATPase, thereby promoting Na⁺/K⁺ balance and enhancing salt tolerance. These findings provide new insights into the role of MsRCI2B in salt stress responses and underscore its potential as a genetic target for enhancing salinity tolerance in forage crops.

Keywords: Medicago sativa L.; MsRCI2B; ROS scavenging; ion homeostasis; protein interaction.

Figure 1

Evolution relationship of MsRCI2 genes. (A) Phylogenetic tree of the RCI2 genes family. Red: Medicago truncatula, Dark bule: Medicago sativa, Green: Arabidopsis thaliana, Yellow: Zea mays, Pale Purple: Amborella trichopoda. Roman numerals (Ia, Ib, II) denote major clades. Bootstrap values are indicated by dot sizes. (B) Gene and protein structure analysis of MsRCI2 genes. UTR: Untranslated Regions.

Figure 2

Chromosomal Localization and Collinearity Analysis of RCI2 genes. (A) Collinearity analysis of RCI2 genes within Medicago sativa showing gene density and pairwise collinearity (gray curve whole genome, red curve: RCI2 genes). (B) Collinearity analysis between Medicago sativa and other species, highlighting RCI2 gene relationships. a: the collinear relationships between Medicago sativa and A. thaliana, b: the collinear relationships between Medicago sativa and G. max, c: the collinear relationships between Medicago sativa and G. max.

Figure 3

Analysis of cis-regulatory elements and expression patterns of MsRCI2 genes. (A) Heatmap of cis-regulatory element analysis. The color represents the number of cis-elements possessed. (B) Expression heatmap of MsRCI2 genes under salt stress (200 mM NaCl) at 0, 1, 3, and 6 h. Significance analysis was performed for different time points within the group. Different letters indicate significant differences (p < 0.05, one-way ANOVA with Tukey’s test) among time points within the same treatment. (C) Expression heatmap of MsRCI2B under various treatments (200 μM ABA, 100 μM SA, 200 mM NaCl, 200 mM NaHCO₃, and 100 μM H₂O₂) at 0, 1, 3, and 6 h. Significance analysis was performed for different time points within the group. (D) Subcellular localization of MsRCI2B-mCherry fusion protein. GFP-AtPIP2;1 was used as a membrane marker, and the mCherry empty vector served as the control.

Figure 4

Overexpression of MsRCI2B enhances salt tolerance in alfalfa via osmotic adjustment and antioxidant defense. Wilted areas are marked with red circles. (A) The phenotype of wild−type and OE#MsRCI2B lines under salt stress at 0 and 10 days. (B,C) Variations in H₂O₂, O₂⁻ in MsRCI2B−overexpressing alfalfa leaves subjected salt stress. (D–F) Activity levels of SOD, POD, and CAT enzymes. (G) Relative conductivity of WT and OE#MsRCI2B lines. (H,I) MDA content and proline content of WT and OE#MsRCI2B lines. Statistical significance was assessed using Tukey’s test to compare MsRCI2B−overexpressing lines with wild-type (WT) controls at time points (0, 5, and 10 h post-treatment). Significance thresholds are denoted as: *** p < 0.001, **** p < 0.0001. Error bars indicate standard deviation (SD) derived from three independent biological replicates, with each experiment including three technical replicates to ensure measurement consistency.

Figure 5

Changes in sodium and potassium content and differential expression of ion transport related genes in OE#MsRCI2B plants under salt stress. (A) Na⁺ content in the leaves of OE#MsRCI2B plants. (B) K⁺ content in the leaves of OE#MsRCI2B plants. (C) Na⁺/K⁺ ratios in the leaves of OE#MsRCI2B plants. (D–F) Expression levels of MsSOS1, MsAKT1, and MsNHX1 gene in the leaves of transgenic plants as measured by qPCR. Statistical significance was assessed using Tukey’s test to compare MsRCI2B-overexpressing lines with wild-type (WT) controls at time points (0, 5, and 10 h post treatment). Significance thresholds are denoted as * p < 0.05, ** p < 0.01, **** p < 0.0001. Error bars indicate standard deviation (SD) derived from three independent biological replicates, with each experiment including three technical replicates to ensure measurement consistency.

Figure 6

GO analysis of screened genes. (A) GO−CC (Cell Component) analysis of screened genes. (B) GO−BP (Biological Process) analysis of screened genes. (C) GO−MF (Molecular Function) analysis of screened gene.

Figure 7

Transcriptome analysis of candidate proteins in alfalfa under salt stress at 0, 3, 6, 12, 48 h. The heatmap was generated using TBtools and the data were normalized. Lines: Cluster genes with similar expression levels. (A) Heatmap of candidate proteins associated with photosynthetic reactions. (B) Heatmap of candidate interacting proteins associated with ion transport. (C) Heatmap of candidate interacting proteins associated with transmembrane transport. (D) Heatmap of candidate interacting proteins associated with enzyme.

Figure 8

Validation of MsRCI2B interacting protein. (A) Validation of MsRCI2B-interacting protein by yeast two-hybrid. (B) The interaction between MsRCI2B and CaM was verified by the Dual-LUC assay. Images are color coded according to the colored bars (4951 to 15,513) shown on the right side. (C) The interaction between MsRCI2B and V-H⁺-ATPase was verified by Dual-LUC assay. Images are color coded according to the colored bars (1155 to 11,429) shown on the right side. (D) Analysis of calmodulin in response to salt stress in MsRCI2B-overexpressing alfalfa in comparison with the wild-type plants. (E) Analysis of vacuolar V-H⁺-ATPase in response to salt stress in MsRCI2B-overexpressing alfalfa in comparison with the wild-type plants (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars indicate standard deviation (SD) derived from three independent biological replicates, with each experiment including three technical replicates to ensure measurement consistency.

Figure 9

Model of MsRCI2B gene response to salinity stress. This model illustrates the interactions of MsRCI2B with CaM, V-H⁺-ATPase, and redox enzyme to enhance salt tolerance by regulating ion homeostasis and ROS scavenging. Red arrows indicate upregulation.