Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H₂O₂) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn²⁺)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H₂O₂ homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H₂O₂-scavenging capacity and salt tolerance, but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.

Summary: Populations of Polypogon fugax have developed resistance to many acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST‐based metabolic resistance to the widely used ACCase‐inhibiting herbicide quizalofop‐p‐ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop‐p‐ethyl and haloxyfop‐R‐methyl. This reflects similar cross‐resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop‐p‐ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop‐p‐ethyl. LC–MS analysis of in vitro herbicide metabolism by Escherichia coli‐expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH‐quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD‐Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD‐Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.