Haloacid dehalogenase–mediated dehalogenation and hydrolysis of oxadiazon and oxyfluorfen mitigate environmental risks in rice production

Protoporphyrinogen oxidase herbicides, such as oxadiazon (ODZ) and oxyfluorfen (OFF), were widely used to control field weeds for crop production. However, their excessive use can lead to its accumulation in soils and crops, affecting crop yields and human health. Therefore, developing a method to degrade ODZ and OFF in rice is crucial for ensuring food safety and agricultural productivity. A phase I reaction enzyme, haloacid dehalogenase-2 (HAD-2), has not been identified facilitates this breakdown. To assess the detoxifying and metabolic functions of HAD-2 on ODZ and OFF, we generated CRISPR-Cas9-based knockout mutant lines and genetically edited rice cultivars that overexpress OsHAD-2 (OE). The metabolites and conjugates of these compounds were identified via high-performance liquid chromatography (HPLC) coupled to a linear ion trap (LTQ) tandem mass spectrometry (HPLC-LTQ-MS/MS). Our results indicated that the OsHAD-2 was localized on the plasma membrane and significantly induced under ODZ/OFF stress. The overexpression of OsHAD-2 conferred resistance to ODZ and OFF toxicity in rice compared with the wild-type (WT), as demonstrated by increased biomass and elongation of rice plants, reduced cellular damage, higher chlorophyll accumulation, and enhanced antioxidant and detoxification enzyme activities. The OE-16 rice seedlings removed 14 % and 32 % more ODZ/OFF from their growth medium, respectively, than the WT and accumulated considerably lower quantity of parent compounds. They also exhibited a greater abundance of degradative metabolites through dehalogenation and hydrolysis. The concentration of dechlorination-hydrolysis-oxadiazon ( m / z 327) and hydrolysis-nitroreduction-oxyfluorfen (m/z 314) in the OE-16 rice grains were 2.13- and 2.70-fold higher than those in WT. In contrast, OsHAD-2 knockout lines exhibited increased accumulation of toxic ODZ and OFF in rice, increased toxic symptoms under ODZ/OFF stress, and reduced growth performance. These rice seedlings also showed reduced levels of ODZ and OFF hydrolysis and dehalogenation metabolites, indicating a functional loss of OsHAD-2 . These findings highlight the role of OsHAD-2 expression in detoxifying protoporphyrinogen oxidase herbicides in rice, primarily through dehalogenation and hydrolysis mechanisms. • Haloacid dehalogenase gene ( OsHAD-2 ) participated in ODZ/OFF metabolism in rice. • Lower accumulation of ODZ/OFF in rice of OsHAD-2 over-expression than WT. • Higher ODZ/OFF removal of OsHAD-2 over-expression rice form growth medium than WT. • Higher content of ODZ/OFF-hydrolysis metabolites in OsHAD-2 over-expression rice than WT. • Hydrolysis and dehalogenation is a detoxification pathway for crops to metabolism pesticides.