<i>EcCYP89B16</i> confers metabolic herbicide resistance and is associated with Calvin cycle adjustments in <i>Echinochloa crus-galli</i>

Echinochloa crus-galli is a problematic weed that affects rice yield. Understanding the mechanisms underlying herbicide resistance in E. crus-galli is essential for sustainable rice cultivation. This study demonstrated that the R-HY population of E. crus-galli developed a moderate (3.4-fold) resistance to cyhalofop-butyl following prolonged selection pressure. This population also displayed cross-resistance to pinoxaden and multiple resistance to penoxsulam and tripyrasulfone. No mutations were identified in the acetyl-CoA carboxylase gene in plants that survived cyhalofop-butyl treatment. However, the cytochrome P450 inhibitor malathion effectively reversed the resistance of the R-HY population to cyhalofop-butyl. Transcriptome and RT-qPCR analyses revealed that one cytochrome P450 family gene, EcCYP89B16, is a key contributor to resistance. Rice seedlings overexpressing EcCYP89B16 exhibited resistance to cyhalofop-butyl, pinoxaden, and penoxsulam. Differential expression of EcCYP89B16 between resistant and susceptible populations was associated with sequence variation in the adjacent long terminal repeat region. Integrating transcriptomic and metabolomic analyses revealed 903 co-expressed differentially expressed genes and 186 differentially accumulated metabolites between the GFP-OE and EcCYP89B16-OE rice. Integrated analysis revealed that EcCYP89B16 may confer herbicide resistance in E. crus-galli by regulating the "Purine metabolism", "Glycerophospholipid metabolism", and "alpha-Linolenic acid metabolism" pathways, while also being associated with transcriptional and metabolic shifts linked to the Calvin cycle, suggesting a potential role in coordinating stress responses. Our findings reveal that overexpression of EcCYP89B16 is a key factor in the metabolic resistance of E. crus-galli to herbicides, providing a theoretical framework and herbicide resistance gene resources for understanding weed resistance evolution.