A Novel High-Throughput Colorimetric Assay to Assess Glutathione <i>S</i>-Transferase-Catalyzed Herbicide Metabolism

The glutathione <i>S</i>-transferase (GST) family is a multifunctional enzyme superfamily, and its members perform important functions in plant development, environmental adaptation, and herbicide metabolism. However, the high cost and low efficiency of currently available methods limit the elucidation of the GST function. Here, we developed a novel high-throughput colorimetric assay based on the glutathione (GSH)-5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) colorimetric reaction for detecting GST-mediated herbicide metabolism in plants. Both the chlorine substitution of five chloroacetamides and the cleavage of two aryloxyphenoxypropionate (AOPP)-type herbicides (fenoxaprop-<i>P</i>-ethyl and metamifop) were detected with GSTU1, GSTU4, GSTU33, and GSTU50 from rice. Furthermore, the transformation of triketone-like quintrione into quinclorac was first discovered, and this process was confirmed in rice and barnyard grass seedlings. Moreover, the key active amino acids in GSTU4, namely Ser16, Ile40, Ile57, and Trp116, were identified via molecular docking and point mutation. Because this novel method is time-efficient and easy to conduct, it will greatly advance our understanding of GST-catalyzed herbicide metabolism and GST-mediated environmental adaptation in plants.