Glutathione transferase photoaffinity labeling displays GST induction by safeners and pathogen infection.
Farre MF., Brown D., König M., Killinger BJ., Kaschani F., Kaiser M., Wright AT., Burton J., van der Hoorn RAL.
Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. Here, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small molecule probe contains glutathione, a photoreactive group, and a minitag for coupling to reporter tags via click chemistry. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts of different plant species. Purification and MS analysis of labeled proteins from Arabidopsis identified ten enriched GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST induction in wheat seedlings upon treatment with safeners, and in Arabidopsis leaves upon infection with avirulent bacteria. Treatment of Arabidopsis with salicylic acid (SA) analog benzothiadiazole (BTH) induces GST labeling independent of NPR1, the master regulator of salicylic acid (SA). Six Phi- and Tau-class GSTs that are induced upon BTH treatment were identified and their labeling was confirmed upon transient overexpression. These data demonstrate that GST photoaffinity labeling is a useful approach to study GST induction in crude extracts of different plants species upon different types of stress.