<i>psbA</i> gene overexpression induces isoproturon resistance in Italian ryegrass ( <i>Lolium perenne</i> ssp. <i>multiflorum</i> )

Abstract Isoproturon is widely used to control Italian ryegrass [ Lolium perenne (L.) ssp. multiflorum (L.) Husnot] in wheat fields across China. Here, we identified a highly resistant population (HR) from 87 populations collected from wheat fields, which showed 4.6-fold resistance to isoproturon compared to susceptible control (HS). DNA sequencing of the full-length psbA gene revealed no sequence differences between HR and HS plants. However, psbA expression in the HR population was significantly higher than in the HS population, both before and after isoproturon application. Transgenic assays confirmed that psbA gene overexpression in rice plants confers resistance to PSII inhibitors, including isoproturon. Under isoproturon application, the HR population also demonstrated elevated antioxidant enzyme activities and maintained higher chlorophyll and carotenoid levels. Furthermore, the HR population remained susceptible to pyroxsulam and pinoxaden, suggesting that these herbicides are practical alternatives for control. These findings indicate that psbA gene overexpression contributes to isoproturon resistance in L. perenne ssp. multiflorum , likely through the overproduction of the D1 protein to mitigate herbicide-induced PSII dysfunction. Our study provides the first confirmation and mechanistic explanation of isoproturon resistance in L. perenne ssp. multiflorum , revealing psbA gene overexpression as the key driver.