MsWRKY11, activated by MsWRKY22, functions in drought tolerance and modulates lignin biosynthesis in alfalfa (Medicago sativa L.)

Drought stress limits the productivity of alfalfa which is one of the most important forage species around the world. WRKY transcription factors are critical TFs involved in drought stress response. In this study, an osmotic stress-responsive WRKY TF gene, named MsWRKY11, was cloned from alfalfa (Medicago sativa L.), which exhibits high expression level in stem and petiole. Its biological function and gene regulation mechanism were studied via overexpressed MsWRKY11 (MsWRKY11-OE) or dominantly repressed MsWRKY11 (MsWRKY11-SRDX) in alfalfa seedlings or hairy roots of alfalfa. The results showed that overexpression of MsWRKY11 in alfalfa decreased stomatal density in leaves and enhanced water use efficiency and drought tolerance. Lignin contents increased in the stem of MsWRKY11-OE plants under normal and drought conditions, but decreased in hairy roots of MsWRKY11-SRDX lines. MsWRKY22 was demonstrated to directly bind to W-box elements in MsWRKY11 promoter through in vitro and in vivo assays and positively activate MsWRKY11 expression. These results provide a new insight into the role of MsWRKY11 in regulating drought tolerance through lignin biosynthesis and stomatal formation in alfalfa.