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

Rare cold-inducible 2 (RCI2) genes from alfalfa ( 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 and expression in alfalfa, but the response time and the expression intensity of the genes were different under specific treatments. The expression intensity of the gene was the highest in salt- and alkali-stressed leaves, while the 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 , 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 -overexpressing chimera, under high-salinity stress (200 mmol·L NaCl), the 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 and -overexpressing roots were significantly higher than those in the control. In addition, after salt stress, the Na content in and 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, , , and genes were significantly up-regulated, and the gene was significantly down-regulated after 6 h of salt treatment. was also found to regulate the expression of the and genes, and the gene could alter the expression of the , , and genes. and - 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 , , , , and genes.