Abstract

A novel allelic variant of the heat shock protein 101, designated neo-tetraploid rice fertility gene 1 ( NTRF1 ), has been identified and is implicated in regulating fertility in neo-tetraploid rice (NTR); however, its regulatory mechanism remains unclear. In this study, we identified the ntrf1 mutant and demonstrated that its significantly reduced seed-setting rate was due to pollen developmental defects. Mechanistically, NTRF1 deficiency disrupts reactive oxygen species (ROS) homeostasis in anthers, thereby delaying the progression of programmed cell death (PCD) in tapetal cells. RNA-seq analysis of mutant anthers revealed dysregulated expression of abscisic acid (ABA) signaling components ( OsPP2C49 , OsbZIP23 ) and ROS-related genes ( OsRBOH1 , OsRBOH8 ), along with a significant downregulation of key tapetal developmental regulators ( OsGAmyb , CYP703A3 ). Integrated multi-omics analysis showed that the reduced pollen viability in the ntrf1 mutant is associated with the pyruvate metabolic pathway. Protein interaction assays confirmed that NTRF1 directly binds SAPK2, a core kinase in ABA signaling transduction. This interaction explained how exogenous ABA application partially restored the reduced seed-setting rate in ntrf1 mutants. Collectively, our findings elucidated an NTRF1 -centered regulatory network that coordinates ABA signaling with ROS homeostasis to ensure timely tapetal PCD and subsequent pollen maturation. This study provides valuable molecular targets for advancing the genetic improvement of polyploid rice.