The cysteine protease PAI1 acts as a quantitative metabolic coordinator of amino acid flux for pollen maturation and anther dehiscence in rice.

The coordination of pollen maturation and anther dehiscence is essential for cereal fertility, yet the metabolic mechanisms linking these processes remain poorly understood. Here, we demonstrate that the rice cysteine protease PAI1 (Pollen Anther Integrator 1) functions as a quantitative metabolic coordinator during rice male reproduction. Unlike canonical cysteine protease mutants that exhibit complete male sterility due to blockage of programmed cell death (PCD), pai1 mutants display partial male sterility characterized by 2 distinct metabolic defects: approximately 50% pollen abortion and defective anther dehiscence, resulting in an approximately 75% reduction in seed set. PAI1 localizes to the endoplasmic reticulum (ER) and plasma membrane (PM), where its proteolytic activity generates free amino acid pools required for maintaining amino acid homeostasis. Integrated metabolomic and transcriptomic analyses revealed that PAI1 loss-of-function is associated with selective depletion of protein-derived and aromatic amino acids during the critical developmental transition (stages 10 to 11), which correlates with reduced phenylpropanoid flux for endothecium lignification and fatty acid metabolism for pollen wall formation, without blocking PCD execution. The transcriptional regulators TDR, bHLH142, and AIP1 directly activate PAI1 expression by binding to overlapping cis-regulatory elements. Relative expression analysis further showed that OsCP1 and other network components are upregulated in pai1 mutants, reflecting an incomplete compensatory response, while the residual approximately 25% fertility likely arises from partial functional redundancy within the broader PAI1-like gene family. Together, these findings support a working model in which PAI1-mediated proteolysis quantitatively coordinates amino acid flux toward specific anabolic pathways, revealing a distinct class of cysteine proteases that may regulate developmental metabolic transitions beyond their established role in PCD execution.