OsALKBH9-mediated m⁶A demethylation regulates tapetal PCD and pollen exine accumulation in rice

The N-6-methyladenosine (m(6)A) mRNA modification is crucial for plant development and stress responses. In rice, the male sterility resulting from the deficiency of OsFIP37, a core component of m(6)A methyltransferase complex, emphasizes the significant role of m(6)A in male fertility. m(6)A is reversible and can be removed by m(6)A demethylases. However, whether mRNA m(6)A demethylase regulates male fertility in rice has remained unknown. Here, we identify the mRNA m(6)A demethylase OsALKBH9 and demonstrate its involvement in male fertility regulation. Knockout of OsALKBH9 causes male sterility, dependent on its m(6)A demethylation activity. Cytological analysis reveals defective tapetal programmed cell death (PCD) and excessive accumulation of microspores exine in Osalkbh9-1. Transcriptome analysis of anthers shows up-regulation of genes involved in tapetum development, sporopollenin synthesis, and transport pathways in Osalkbh9-1. Additionally, we demonstrate that OsALKBH9 demethylates the m(6)A modification in TDR and GAMYB transcripts, which affects the stability of these mRNAs and ultimately leads to excessive accumulation of pollen exine. Our findings highlight the precise control of mRNA m(6)A modification and reveal the pivotal roles played by OsALKBH9-mediated m(6)A demethylation in tapetal PCD and pollen exine accumulation in rice.