The HD-Zip transcription factor MsHDZ37 confers stress tolerance in alfalfa by directly activating MsBGLU13 and MsSGR1

HD-Zip (HDZ) proteins play essential roles in regulating plant growth, development and environmental responses. However, their functions in forage crops under abiotic stress remain poorly understood. In this study, A total of 42 HDZ genes were successfully identified from the genome of alfalfa ( Medicago sativa ) in the present study, and these genes displayed differentiated expression profiles across diverse tissues and under various abiotic stresses. Among them, MsHDZ20 / 21 / 37 were dramatically induced by both mannitol and NaCl treatments and co-expressed with genes involved in response to external stresses and defense response. Transient overexpression of MsHDZ20 / 21 / 37 in tobacco strengthened tolerance to PEG and NaCl stresses, accompanied by reduced reactive oxygen species accumulation and increased antioxidant enzyme activities. To further investigate their function, we generated transgenic alfalfa lines with stable overexpression or RNAi-mediated silencing of MsHDZ37 . Upon exposure to drought and salt stress conditions, MsHDZ37 -overexpressing plants exhibited significantly enhanced tolerance, lower malondialdehyde content and electrolyte leakage, and along with elevated antioxidant enzyme activities relative to wild-type plants. Conversely, MsHDZ37 -RNAi lines showed increased stress sensitivity and opposite physiological alterations. Mechanistically, yeast one-hybrid and dual-luciferase assays exhibited that MsHDZ37 directly binds to and activates the promoters of MsBGLU13 and MsSGR1 . Consistently, both target genes were upregulated in MsHDZ37 -overexpressing lines and downregulated in RNAi lines. Collectively, these findings establish MsHDZ37 positively modulates abiotic stress adaption in alfalfa and provide valuable insights for genetic improvement of forage crops.