<scp>MsWRKY49</scp> Modulates Pollen Tube Elongation in Response to Boron Deficiency by Regulating Reactive Oxygen Species Homeostasis in Alfalfa

Boron, an essential micronutrient, plays a crucial role in plant reproductive processes. Boron deficiency is widespread in many regions and significantly reduces alfalfa (Medicago sativa) seed yield, yet the molecular mechanisms underlying its impact on reproductive organ development remain elusive. This study found that boron deficiency inhibited alfalfa pollen tube elongation and was accompanied by elevated reactive oxygen species (ROS) levels in the pollen tube. The application of exogenous hydrogen peroxide (H₂O₂) and ascorbic acid (AsA) indicated that maintaining ROS homeostasis was crucial for boron-mediated pollen tube elongation. Based on phylogenetic tree analysis and expression pattern analysis, MsWRKY49 was identified as a central regulator responding to low boron. Transgenic analysis showed that mswrky49 mutants alleviated the pollen tube phenotype and reduced ROS accumulation under low boron conditions, while overexpression of MsWRKY49 in alfalfa pollen tubes led to boron sensitivity declining and ROS accumulation. Furthermore, RNA-Seq analysis revealed that several antioxidant-related genes were significantly downregulated in the MsWRKY49-OE lines. Yeast one-hybrid and luciferase reporter assays demonstrated that MsWRKY49 suppressed the expression of MsAPX1, MsGSTU19, MsNADH and MsCu/Zn-SOD, thereby potentially leading to ROS overaccumulation and inhibiting pollen tube elongation. Our results reveal that MsWRKY49 inhibits pollen tube elongation under low-boron conditions by promoting ROS accumulation, providing a new perspective for understanding the molecular mechanism by which boron deficiency causes the decline of seed yield in alfalfa.