Calmodulin-like proteins (CMLs) are calcium (Ca²⁺) sensors involved in plant growth and development as well as adaptation to environmental stresses; however, their roles in plant responses to cold are not well understood. To reveal the role of MsCML10 from alfalfa (Medicago sativa) in regulating cold tolerance, we examined transgenic alfalfa and Medicago truncatula overexpressing MsCML10, MsCML10-RNAi alfalfa, and a M. truncatula cml10-1 mutant and identified MsCML10-interacting proteins. MsCML10 and MtCML10 transcripts were induced by cold treatment. Upregulation or downregulation of MsCML10 resulted in increased or decreased cold tolerance, respectively, while cml10-1 showed decreased cold tolerance that was complemented by expressing MsCML10, suggesting that MsCML10 regulates cold tolerance. MsCML10 interacted with glutathione S-transferase (MsGSTU8) and fructose 1,6-biphosphate aldolase (MsFBA6), and the interaction depended on the presence of Ca²⁺. The altered activities of Glutathione S-transferase and FBA and levels of ROS and sugars were associated with MsCML10 transcript levels. We propose that MsCML10 decodes the cold-induced Ca²⁺ signal and regulates cold tolerance through activating MsGSTU8 and MsFBA6, leading to improved maintenance of ROS homeostasis and increased accumulation of sugars for osmoregulation, respectively.

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid (JA), which is involved in plant growth and development as well as adaptation to environmental stresses. We identified the cold- and pathogen-responsive AOC2 gene from Medicago sativa subsp. falcata (MfAOC2) and its homolog MtAOC2 from Medicago truncatula. Heterologous expression of MfAOC2 in M. truncatula enhanced cold tolerance and resistance to the fungal pathogen Rhizoctonia solani, with greater accumulation of JA and higher transcript levels of JA downstream genes than in wild-type plants. By contrast, mutation of MtAOC2 reduced cold tolerance and pathogen resistance, with less accumulation of JA and lower transcript levels of JA downstream genes in the aoc2 mutant than in wild-type plants. The aoc2 phenotype and low levels of cold-responsive C-repeat-binding factor (CBF) transcripts could be rescued by expressing MfAOC2 in aoc2 plants or exogenous application of methyl jasmonate. Compared with wild-type plants, higher levels of CBF transcripts were observed in lines expressing MfAOC2 but lower levels of CBF transcripts were observed in the aoc2 mutant under cold conditions; superoxide dismutase, catalase, and ascorbate-peroxidase activities as well as proline concentrations were higher in MfAOC2-expressing lines but lower in the aoc2 mutant. These results suggest that expression of MfAOC2 or MtAOC2 promotes biosynthesis of JA, which positively regulates expression of CBF genes and antioxidant defense under cold conditions and expression of JA downstream genes after pathogen infection, leading to greater cold tolerance and pathogen resistance.

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.

Zinc finger proteins (ZFPs) are transcription factors involved in multiple cellular functions. We identified a C2H2 type ZFP (MtZPT2-2) in Medicago truncatula and demonstrated that it localizes to the nucleus and inhibits transcription of two genes encoding high-affinity potassium transporters (MtHKT1; 1 and MtHKT1; 2). MtZPT2-2 transcripts were detected in the stem, leaf, flower, seeds and roots, with the highest level in the xylem and phloem of roots and stems. MtZPT2-2 transcription in leaves was reduced after salt stress. Compared to the wild type (WT), transgenic lines overexpressing MtZPT2-2 had decreased salt tolerance, while MtZPT2-2-knockout mutants showed increased salt tolerance. MtHKT1; 1 and MtHKT1; 2 transcripts and Na⁺ accumulation in shoots and roots, as well as in the xylem of all genotypes of plants, were increased after salt treatment, with higher levels of MtHKT1; 1 and MtHKT1; 2 transcripts and Na⁺ accumulation in MtZPT2-2-knockout mutants and lower levels in MtZPT2-2-overexpressing lines compared with the WT. K⁺ levels showed no significant difference among plant genotypes under salt stress. Moreover, MtZPT2-2 was demonstrated to bind with the promoter of MtHKT1; 1 and MtHKT1; 2 to inhibit their expression. Antioxidant enzyme activities and the gene transcript levels were accordingly upregulated in response to salt, with higher levels in MtZPT2-2-knockout mutants and lower levels in MtZPT2-2-overexpressing lines compared with WT. The results suggest that MtZPT2-2 regulates salt tolerance negatively through down-regulating MtHKT1; 1 and MtHKT1; 2 expression directly to reduce Na⁺ unloading from the xylem and regulates antioxidant defense indirectly.

The wall-associated kinases (WAKs) can perceive and transmit extracellular signals as one kind of unique receptor-like kinases (RLKs) involved in the regulation of cell expansion, pathogen resistance and abiotic stress tolerance. To understand their potential roles and screen some key candidates in Medicago truncatula (M. truncatula), genome-wide identification and characterization of MtWAKs were conducted in this study. A total of 54 MtWAK genes were identified and classified into four groups based on their protein domains. They were distributed on all chromosomes, while most of them were clustered on chromosome 1 and 3. The synteny analysis showed that 11 orthologous pairs were identified between M. truncatula and Arabidopsis thaliana (A. thaliana) and 31 pairs between M. truncatula and Glycine max (G. max). The phylogenetic analysis showed that WAK-RLKs were classified into five clades, and they exhibited a species-specific expansion. Most MtWAK-RLKs had similar exon–intron organization and motif distribution. Multiple cis-acting elements responsive to phytohormones, stresses, growth and development were observed in the promoter regions of MtWAK-RLKs. In addition, the expression patterns of MtWAK-RLKs varied with different plant tissues, developmental stages and biotic and abiotic stresses. Interestingly, plasm membrane localized MtWAK24 significantly inhibited Phytophthora infection in tobacco. The study provides valuable information for characterizing the molecular functions of MtWAKs in regulation of plant growth, development and stress tolerance in legume plants.