MsNAC72 and MsbZIP29 fine-tune salt tolerance in alfalfa via oppositely regulating MsSOS2 expression

Alfalfa (Medicago sativa L.), a high-quality perennial legume forage, is essential to global agriculture and ecosystems. Expanding alfalfa production on saline-alkaline soils is critical to alleviate shortages of high-quality forage in China, but progress is limited by a shortage of salt-tolerant varieties and incomplete understanding of the underlying molecular mechanisms. As one of the most well-characterized salt tolerance pathways in plants, salt-overly-sensitive (SOS) pathway plays a pivotal role in Na+ exclusion and cellular ion balance regulation. Here, we investigated the role and regulation of MsSOS2 in alfalfa salt stress responses. Overexpression of MsSOS2 enhanced salt tolerance in transgenic alfalfa plants, evidenced by a reduced Na+/K+ ratio. Then, two salt-induced transcription factors Basic leucine zipper 29 (MsbZIP29) and NAC domain-containing protein 72 (MsNAC72) were identified as upstream regulators of MsSOS2 through yeast one-hybrid screening. Further protein-DNA interaction assays demonstrated that MsbZIP29 directly activates MsSOS2 expression, whereas MsNAC72 directly represses it. Functional analyses revealed that MsbZIP29 promotes salt tolerance, while MsNAC72 functions as a transcriptional brake, together fine-tuning ionic homeostasis under salt stress. Additionally, MsbZIP29 interacted with MsbZIP1 to amplify the transcriptional activation of MsSOS2. Consistently, overexpression of MsbZIP1 improved salt tolerance while its knockdown increased salt stress sensitivity. Collectively, our results reveal that MsNAC72 and MsbZIP29 fine-tune salt stress tolerance in alfalfa through opposing roles in regulating MsSOS2 expression. This study provides novel insights into the transcriptional network underlying salt tolerance in alfalfa, offering potential molecular targets for improving alfalfa resilience in saline environments.