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

SUMMARY 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 H 2 O 2 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.