Genome-Wide Identification of Alfalfa YABBY Genes Reveals Opposing Roles of MsYABBY8 in Salt and Low-Temperature Responses

Alfalfa (Medicago sativa L.) is a major perennial forage legume, but its cultivation is increasingly limited by soil salinization and low temperature. YABBY proteins are plant-specific transcription factors best known for lateral organ development, although their role in abiotic stress adaptation remains unclear. In this study, eight MsYABBY members, encoded by seven loci, were identified in the alfalfa genome; seven were anchored to four chromosomes (Chr2, Chr4, Chr5 and Chr8) and one to an unanchored scaffold. Phylogenetic analysis with Arabidopsis and rice YABBY proteins classified the MsYABBY members into the five canonical YABBY subfamilies (FIL/YAB3, YAB2, YAB5, INO and CRC). Transcriptome and qRT-PCR profiling further revealed stress-dependent expression, among which MsYABBY8 was most strongly induced under salt and cold stress. Subcellular localization confirmed that the MsYABBY8 protein was localized to the nucleus. To further validate its function, MsYABBY8 was heterologously overexpressed in Arabidopsis, resulting in contrasting phenotypes under the two stresses. Under salt stress, the transgenic lines were more sensitive than the wild type, with reduced chlorophyll, survival, and relative water content, but increased thiobarbituric acid-reactive substances (TBARS), electrolyte leakage, and the Na+/K+ ratio. In contrast, the same lines performed better under cold stress, with higher chlorophyll content, photosynthetic rate, and antioxidant enzyme activities, and elevated AtCBF1 and AtCOR15A expression. Overall, these results obtained in a heterologous system suggest that MsYABBY8 may act as a negative regulator of salt tolerance but a positive regulator of cold tolerance, which remains to be confirmed in alfalfa.