Biochemical and molecular basis for metabolism-based atrazine resistance in Amaranthus tuberculatus

Atrazine, a photosystem II inhibitor, is a commonly used herbicide for selective broadleaf weed control in maize (Zea mays). Rapid detoxification of s-triazine herbicides is the result of increased glutathione S-transferase (GST) activity in atrazine-tolerant crops such as maize and grain sorghum (Sorghum bicolor), or in atrazine-tolerant weeds such as Panicum spp. In previous research, two atrazine-resistant populations of tall waterhemp (Amaranthus tuberculatus) from central Illinois (designated ACR and MCR) did not possess a point mutation in psbA (encoding the D1 protein, the target site for atrazine), but rapid production of atrazine-glutathione conjugates indicated that elevated rates of metabolism may be a major resistance mechanism. My objective was to utilize traditional protein purification techniques combined with proteomic methods to further investigate the hypothesis that enhanced metabolic detoxification of atrazine, catalyzed by distinct GST isozymes, may confer resistance in ACR and MCR. ACR and MCR showed higher GST specific activities towards atrazine in partially-purified ammonium sulfate and glutathione affinity-purified fractions when compared with an atrazine-sensitive waterhemp population (WCS). One-dimensional gel electrophoresis displayed a large band at approximately 26 kDa, which is the typical subunit mass of GST subunits. Identification of several GSTs by LC-MS/MS of glutathione affinity-purified fractions from each population led to the identification of a phi class GST (ArGSTF1) and two tau class GSTs, based on peptide sequence similarity with GSTs from Arabidopsis. Elevated constitutive expression levels of ArGSTF1 in ACR and MCR correlated with atrazine resistance, while expression of each tau class GST was highest in MCR but low in ACR and WCS