The R2R3-MYB transcription factor ClMS188 targets ClMS1L1 to orchestrate tapetum degradation and pollen formation in watermelon

Male-sterile lines are crucial genetic materials for investigating the developmental and abortion mechanisms of anthers, and they serve as key parental lines in hybrid seed production. However, research to date has primarily focused on model plants such as Arabidopsis, rice and maize, whereas the regulatory mechanisms underlying this process remain largely unexplored in Cucurbitaceae. Here, we identified an R2R3-MYB transcription factor, ClMS188, as a key regulator of tapetum and pollen development. The clms188 mutants exhibited complete male sterility due to delayed tapetum degeneration and abnormal tetrad formation. In vitro and in vivo experiments demonstrated that ClMS188 directly binds to the TAACCA motif in the ClMS1L1 promoter and activates its transcription. Similarly, loss-of-function mutants of ClMS1L1 led to defective microspore development and delayed tapetum degeneration. Comparative transcriptomic analyses revealed that ClMS188 orchestrates anther development via distinct regulatory branches: a ClMS1L1-independent pathway controlling tetrad wall degradation, sporopollenin biosynthesis, and anther wall lignification, and a ClMS1L1-dependent pathway governing lipid metabolism for pollen coat maturation. Together, these findings establish the ClMS188-ClMS1L1 regulatory module that coordinates tapetum degeneration and pollen formation, providing insights into the tapetum and pollen development and a valuable genetic resource for hybrid seed production in watermelon.