The down-regulation of MsWOX13-2 promotes enhanced waterlogging resilience in alfalfa

Soil waterlogging events are predicted to escalate globally as a result of climate change, threatening the sustainability of alfalfa (Medicago sativa L.) and livestock production in the future. WUSCHEL-related homeobox (WOX) transcription factors are known to play a role in numerous developmental processes and abiotic stress responses; however, their function in waterlogging resilience has not been investigated as of yet. In the present study, we functionally characterized the alfalfa MsWOX13-2 gene, which we found to be differentially expressed in response to waterlogging. Although the RNAi-mediated silencing of MsWOX13-2 in alfalfa did not affect growth or morphology under normally watered conditions, MsWOX13-2 RNAi plants exhibited higher chlorophyll retention and maximum quantum efficiency of photosystem II, as well as greater survivability, compared to empty vector genotypes under waterlogging. Subsequent analyses indicated that MsWOX13-2 RNAi leaves accumulated less H₂O₂ and displayed a greater increase in superoxide dismutase activity under waterlogging, resulting in reduced oxidative damage, which may have contributed to the enhanced waterlogging tolerance in these genotypes. RNA-Seq analysis confirmed alterations in the transcript levels of genes related to antioxidants, as well as those involved in photosynthesis, anaerobic fermentation, phytohormone-related pathways, and transcriptional regulation in the leaves of WOX13-2 RNAi genotypes compared to wild type following waterlogging stress. Bi-allelic mutation of MsWOX13-2 in alfalfa using CRISPR/Cas9 confirmed its function in waterlogging response. Overall, our findings suggest that MsWOX13-2 acts as a negative regulator of waterlogging response in alfalfa, providing a novel candidate for downstream breeding endeavors in this important species.