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

The N⁶-methyladenosine (m⁶A) 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 m⁶A methyltransferase complex, emphasizes the significant role of m⁶A in male fertility. m⁶A is reversible and can be removed by m⁶A demethylases. However, whether mRNA m⁶A demethylase regulates male fertility in rice has remained unknown. Here, we identify the mRNA m⁶A demethylase OsALKBH9 and demonstrate its involvement in male fertility regulation. Knockout of OsALKBH9 causes male sterility, dependent on its m⁶A 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 m⁶A 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 m⁶A modification and reveal the pivotal roles played by OsALKBH9-mediated m⁶A demethylation in tapetal PCD and pollen exine accumulation in rice.