MsPYL6 and MsPYL9 improves drought tolerance by regulating stomata in alfalfa (Medicago sativa)

Severe drought stress can significantly reduce alfalfa production, and the phytohormone abscisic acid plays a central role in responses to abiotic stress. As abscisic acid receptors, the PYRABACTIN RESISTANCE 1/PYR1-LIKE/ABA-binding REGULATORY COMPONENT OF ABA RECEPTOR (PYR1/PYL/RCAR) family constitutes a critical component of the ABA signaling pathway, mediating adaptive responses and protecting plants under drought conditions. In this study, nine MsPYL genes were identified in alfalfa, and their expression levels were found to be significantly upregulated in response to ABA and drought stress. The overexpression of MsPYL genes in both Arabidopsis thaliana and alfalfa significantly improved drought tolerance. Among the MsPYL family genes, functional analysis of MsPYL6 and MsPYL9 revealed that both genes enhanced water-use efficiency by reducing leaf stomatal density, promoting stomatal closure, and decreasing transpiration rates when overexpressed. In contrast, the RNAi plants exhibited the opposite phenotypes, with increased stomatal density and higher transpiration rates. Furthermore, overexpression plants exhibited reduced malondialdehyde content and lower reactive oxygen species levels, indicating enhanced cellular stability under stress. Additionally, transcriptome analysis showed that drought-responsive genes related to antioxidant defense, stomatal regulation, and photosynthesis were more abundant in drought-treated OE plants and less abundant in RNAi plants. Interaction analysis revealed that all MsPYL proteins could interact with at least one MsPP2CA, indicating a conserved interaction pattern in ABA signaling. These findings confirm that MsPYLs play a crucial role in the ABA signaling pathway by modulating the expression of downstream drought-tolerant genes, thereby enabling alfalfa to effectively respond to drought stress conditions.