TSR and peroxidase genes confer resistance to fenoxaprop‐P‐ethyl and mesosulfuron‐methyl in Alopecurus aequalis

Abstract BACKGROUND Alopecurus aequalis poses severe threat to global wheat production due to evolving resistance to acetyl‐CoA carboxylase (ACCase)‐ and acetolactate synthase (ALS)‐inhibiting herbicides. In this study, the resistance mechanisms of a field‐evolved resistant population (R) were systematically investigated using dose–response bioassays, target‐site gene sequencing, inhibitor assays, antioxidant enzyme activity measurements, RNA sequencing (RNA‐seq), quantitative real‐time reverse transcription polymerase chain reaction (qRT–PCR), and yeast functional validation. RESULTS Dose–response results revealed that the R population exhibited moderate resistance to fenoxaprop‐P‐ethyl (RI = 9.58) and low‐level resistance to mesosulfuron‐methyl (RI = 3.07). Cross‐resistance testing indicated that the R population was resistant to other ACCase‐inhibiting herbicides (haloxyfop‐P‐methyl, clodinafop‐propargyl, clethodim, and pinoxaden) and the ALS‐inhibiting herbicide rimsulfuron. Target‐site sequence analysis identified two mutations in the R population: Ile‐1781‐Leu ( ACCase ) and Pro‐197‐Ser ( ALS1 ). Pretreatment with the cytochrome P450 and GST inhibitor did not reverse resistance to fenoxaprop‐P‐ethyl or mesosulfuron‐methyl. Compared to the susceptible (S) population, the R population had significantly lower H 2 O 2 content and higher activities of peroxidase (POD) and catalase (CAT), indicating an enhanced reactive oxygen species (ROS) scavenging capacity. RNA‐seq and qRT–PCR analyses identified three POD‐annotated contigs ( PODSPC4 , POD12‐1 , POD12‐2 ) that were upregulated in the R population. Yeast heterologous expression validated that AaPOD12‐1 and AaPOD12‐2 significantly increased yeast resistance to fenoxaprop‐P‐ethyl and mesosulfuron‐methyl. CONCLUSION These results demonstrate that resistance in the R population is co‐mediated by target‐site mutations and non‐target‐site resistance involving enhanced ROS scavenging, with AaPOD12‐1 and AaPOD12‐2 representing the first functionally characterized antioxidant enzyme genes associated with herbicide resistance in A. aequalis . © 2026 Society of Chemical Industry.