# MtSIN1a Enhances Salinity Tolerance in Medicago truncatula and Alfalfa

## TL;DR

This study shows that the MtSIN1a gene helps alfalfa and Medicago truncatula tolerate salt stress by improving plant growth and reducing harmful reactive oxygen species.

## Contribution

The study identifies MtSIN1a as a novel gene that enhances salinity tolerance in alfalfa through ROS scavenging mechanisms.

## Key findings

- MtSIN1a mutants showed reduced survival and growth under salt stress compared to wild-type plants.
- Transgenic alfalfa with MtSIN1a overexpression had improved salt tolerance, reduced H2O2 content, and increased CAT activity.
- RNA-seq analysis revealed elevated expression of MsSOD1 in transgenic plants, suggesting a role in ROS scavenging.

## Abstract

Background/Objectives: Alfalfa is a widely cultivated high-quality forage crop, and salinity tolerance is one of the most important breeding goals. Glycine max SALT INDUCED NAC 1 (GmSIN1) was found to enhance salinity tolerance in soybean plants. The phylogenetic analysis showed there were two homologs of GmSIN1 in Medicago truncatula, MtSIN1a and MtSIN1b. This raised questions regarding the roles of MtSIN1s in alfalfa under salinity stress. Methods: From a Tnt1 mutant collection, we identified the mutants of MtSIN1a. We recorded the survival rate and plant height of mtsin1a-1 and mtsin1a-2 after 100 mM NaCl treatment. Subsequently, we generated 35S:MtSIN1a-GFP transgenic alfalfa lines via genetic transformation. Two lines with relatively high MtSIN1a expression, 35S:MtSIN1a-GFP#3 and 35S:MtSIN1a-GFP#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. Results: This study found that after salt treatment, the survival rate and plant height of mtsin1a-1 and mtsin1a-2 were significantly lower than those of the WT. The mutants of MtSIN1a 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 MtSIN1a showed enhanced salinity tolerance with elevated ROS scavenging. We identified MsSOD1 showing elevated expression levels in transcriptomic analysis. Conclusions: MtSIN1a is a positive regulator for enhancing salinity tolerance in alfalfa with activated ROS scavenging.