Alfalfa Transcription Factor <scp>MsNAC2a</scp> Orchestrates the Homeostasis of Salt Stress Responses via Reactive Oxygen Species Accumulation and Hydrogen Sulphide Depletion

Soil salinisation poses a significant threat to alfalfa (Medicago sativa L.) growth and development, limiting its productivity and hindering its widespread cultivation. Hydrogen sulphide (H₂S) serves as an important gaseous signalling molecule in plants, mediating a myriad of physiological processes like salt tolerance. However, the molecular mechanisms underlying H₂S accumulation and its regulation under salinity stress in alfalfa are still not fully elucidated. In this study, we demonstrated that MsNAC2a, a NAC transcription factor, is a negative modulator of salt stress resistance in alfalfa. Constitutive overexpression of MsNAC2a downregulated the expression of H₂S biosynthesis-related genes, such as _L-CYSTEINE DESULFHYDRASE1 (MsLCD1), and upregulated the O-ACETYLSERINE(THIOL)LYASE ISOFORM A1 (MsOASA1) gene, a key gene involved in H₂S metabolism, while also suppressing the expression of reactive oxygen species (ROS) scavenging genes like MsCOX11, leading to a reduction in hydrogen sulphide levels and an increase in ROS accumulation, ultimately impairing the plant's salt tolerance. Furthermore, the AP2/EREBP-type transcription factor MsEREBP1 physically interacts with MsNAC2a both in vivo and in vitro, influencing its transcriptional activity and modulating salt stress responses in alfalfa. Conversely, silencing MsNAC2a enhanced salt stress resistance without affecting plant growth or yield. Collectively, our study highlights that MsNAC2a precisely regulates the homeostasis of salt stress responses and provides new insights into the mechanisms by which the cooperative interaction between MsNAC2a and MsEREBP1 fine-tunes the homeostasis of endogenous H₂S levels, thereby influencing alfalfa's salt tolerance and offering valuable strategies for improving crop resilience under saline stress.