ABC transporters linked to multiple herbicide resistance in blackgrass ().

Enhanced detoxification is a prominent mechanism protecting plants from toxic xenobiotics and endows resistance to diverse herbicide chemistries in grass weeds such as blackgrass ). The roles of enzyme families which impart enhanced metabolic resistance (EMR) to herbicides through hydroxylation (phase 1 metabolism) and/or conjugation with glutathione or sugars (phase 2) have been well established. However, the functional importance of herbicide metabolite compartmentalisation into the vacuole as promoted by active transport (phase 3), has received little attention as an EMR mechanism. ATP-binding cassette (ABC) transporters are known to be important in drug detoxification in fungi and mammals. In this study, we identified three distinct C-class ABCCs transporters namely ABCC1, ABCC2 and ABCC3 in populations of blackgrass exhibiting EMR and resistance to multiple herbicides. Uptake studies with monochlorobimane in root cells, showed that the EMR blackgrass had an enhanced capacity to compartmentalize fluorescent glutathione-bimane conjugated metabolites in an energy-dependent manner. Subcellular localisation analysis using transient expression of GFP-tagged ABCC2 assays in demonstrated that the transporter was a membrane bound protein associated with the tonoplast. At the transcript level, as compared with herbicide sensitive plants, and were positively correlated with EMR in herbicide resistant blackgrass being co-expressed with , a glutathione transferase (GST) involved in herbicide detoxification linked to resistance. As the glutathione conjugates generated by GSTs are classic ligands for ABC proteins, this co-expression suggested and the two ABCC transporters delivered the coupled rapid phase 2/3 detoxification observed in EMR. A role for the transporters in resistance was further confirmed in transgenic yeast by demonstrating that the expression of either ABCC1 or ABCC2, promoted enhanced tolerance to the sulfonylurea herbicide, mesosulfuron-methyl. Our results link the expression of ABCC transporters to enhanced metabolic resistance in blackgrass through their ability to transport herbicides, and their metabolites, into the vacuole.