MtSIN1a Enhances Salinity Tolerance in Medicago truncatula and Alfalfa

<b>Background/Objectives</b>: Alfalfa is a widely cultivated high-quality forage crop, and salinity tolerance is one of the most important breeding goals. <i>Glycine max SALT INDUCED NAC 1</i> (<i>GmSIN1</i>) was found to enhance salinity tolerance in soybean plants. The phylogenetic analysis showed there were two homologs of <i>GmSIN1</i> in <i>Medicago truncatula</i>, <i>MtSIN1a</i> and <i>MtSIN1b</i>. This raised questions regarding the roles of <i>MtSIN1s</i> in alfalfa under salinity stress. <b>Methods</b>: From a <i>Tnt1</i> mutant collection, we identified the mutants of <i>MtSIN1a</i>. We recorded the survival rate and plant height of <i>mtsin1a-1</i> and <i>mtsin1a-2</i> after 100 mM NaCl treatment. Subsequently, we generated <i>35S:MtSIN1a-GFP</i> transgenic alfalfa lines via genetic transformation. Two lines with relatively high <i>MtSIN1a</i> expression, <i>35S:MtSIN1a-GFP</i>#3 and <i>35S:MtSIN1a-GFP</i>#4, were selected for gradient NaCl treatments. In addition, DAB and NBT staining were performed, and the H₂O₂ content and catalase (CAT) activity were determined. Then, we used RNA-seq analysis and RT-qPCR to study the mechanism of its tolerance. <b>Results</b>: This study found that after salt treatment, the survival rate and plant height of <i>mtsin1a-1</i> and <i>mtsin1a-2</i> were significantly lower than those of the WT. The mutants of <i>MtSIN1a</i> were sensitive to salinity stress. The transgenic alfalfa plants exhibited higher plant height, weaker DAB staining, stronger NBT staining, less H₂O₂ content, and enhanced CAT activity. The transgenic alfalfa constructed by transforming <i>MtSIN1a</i> showed enhanced salinity tolerance with elevated ROS scavenging. We identified <i>MsSOD1</i> showing elevated expression levels in transcriptomic analysis. <b>Conclusions</b>: <i>MtSIN1a</i> is a positive regulator for enhancing salinity tolerance in alfalfa with activated ROS scavenging.