miRNA–mRNA Analysis Reveals That Alfalfa Responds to Saline‐Alkali Stress via Phenylpropanoid Biosynthesis and Starch and Sucrose Metabolism Pathways

Abstract

Alfalfa, a globally important leguminous forage crop, is valued for high yield, superior nutritional quality, and robust stress resistance. It is widely used in animal husbandry and ecological protection. However, soil salinization has become a major environmental factor limiting its yield and quality. In this study, two alfalfa cultivars, Zhongmu No. 1 (ZM) and SK3010 (SK), were subjected to saline‐alkali stress. The treatment resulted in stunted growth, leaf chlorosis, and cell membrane damage. Compared with SK, ZM exhibited less damage and stronger saline‐alkali tolerance. Transcriptomic analysis revealed that the proportion of up‐regulated differentially expressed mRNAs (DEMs) in ZM was significantly higher than in SK. The antioxidant systems and plant hormone signal transduction pathways are central to saline‐alkali stress adaptation. Integrated transcriptome and small RNA sequencing identified 59 saline‐alkali stress‐responsive differentially expressed microRNAs (DEMIs) that target 119 DEMs. Functional analysis demonstrated that the phenylpropanoid biosynthesis and the starch and sucrose metabolism pathways are critical for stress response. These results indicated that alfalfa mitigates saline‐alkali stress through the regulation of the antioxidant system, hormone biosynthesis and signaling, phenylpropanoid biosynthesis, and the hydrolysis of starch to soluble sugars. In particular, ZM exhibited greater tolerance by accumulating fewer reactive oxygen species while elevating antioxidant enzyme activity and antioxidant content and enhancing lignin and sucrose biosynthesis. Furthermore, we constructed a miRNA‐mRNA regulatory network responsive to saline‐alkali stress, which included miR164 and miR172 family members targeting CSE (MsG0880047430.01) and TPS (MsG0880045029.01), respectively. This work offers valuable molecular resources for multi‐gene improvement of saline‐alkali tolerance in alfalfa.