Glycolysis dominates over photorespiration in governing oxalate accumulation in rice

Oxalate is widely distributed and serves various functional roles in plants. However, its biosynthetic pathway and regulatory mechanisms remain poorly defined. In this study, we further investigated the mechanism of oxalate accumulation using rice (Oryza sativa) CRISPR-Cas9-generated nitrate reductase (NR) and nitrite reductase (NiR) mutants, combined with different treatments. Both NR and NiR mutants exhibited significantly reduced oxalate levels. The reduced oxalate in NR and NiR mutants was restored by nitrite and nitrate, respectively, whereas ammonium had no effect, indicating that both nitrate and nitrite reduction, rather than the subsequent ammonium assimilation, are involved in modulating oxalate accumulation in rice. Furthermore, various organic acids, including glycolate, glyoxylate, glycerate, and oxaloacetate, markedly stimulated oxalate content. Transcriptomic and metabolomic analyses revealed that oxalate levels were closely and positively associated with the expression of glycolytic and tricarboxylic acid cycle genes and the content of their intermediate metabolites. Treatment with glycolytic intermediates (phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PGA)) and inhibitors (IOA and 3-bromopyruvate) further confirmed that glycolysis drives oxalate synthesis in rice. Nicotinamide, a NAD precursor, also restored the oxalate content, suggesting that NAD/NADH may play a mediatory role in oxalate regulation through both nitrate and nitrite reduction. Collectively, our results demonstrate that oxalate accumulation is intimately associated with glycolysis in rice, rather than with photorespiration, which instead regulates oxalate accumulation indirectly via the glycerate-mediated impact on glycolysis.