Genome-wide analysis of receptor-like 2 kinase 1-like genes reveals involvement of a RLK1L member in tolerance to salt stress in Medicago sativa

The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family constitutes a specialized group within plant receptor-like protein kinases that has been detected in numerous plant varieties. However, RLK1L members have not been characterized in alfalfa. Through systematic analysis, this study examined the features and identification of RLK1L genes within alfalfa based on known gene sequences from the model plants within the Arabidopsis genus, specifically Arabidopsis thaliana, and those in the Medicago genus, such as Medicago truncatula. Phylogenetic analysis of AtCrRLK1Ls, MtCrRLK1Ls, and MsCrRLK1Ls led to the classification of 19 MsCrRLK1L genes in alfalfa into eight distinct clusters. Bioinformatic analyses revealed that the molecular masses of MsCrRLK1L proteins spanned from 29.17 to 167.36 kDa. Subcellular targeting predictions indicated predominant cell membrane localization for these polypeptides Conserved domain profiling demonstrated the ubiquitous presence of a malectin-like domain across all MsCrRLK1L members. Chromosome localization and collinearity analysis showed that the 19 MsCrRLK1L genes were unevenly distributed across five alfalfa chromosomes, with four gene pairs exhibiting intra-species collinearity. Cis-Acting element analysis identified stress- and hormone-responsive motifs, suggesting that MsCrRLK1Ls are involved in abiotic stress signaling. Gene expression profiling under salt stress (200 mM NaCl) demonstrated tissue-specific induction patterns, with six MsCrRLK1L genes significantly upregulated in aerial and root tissues. Furthermore, MsCrRLK1L2 overexpression in Arabidopsis improved the germination rate and growth status under salt stress. This study comprehensively profiles the MsCrRLK1L family in alfalfa, confirming its role in salt tolerance and suggesting genetic targets to boost crop resilience.