The Role of Glutathione Transferases in Multiple Herbicide Resistance in Grass Weeds

Modern agriculture relies on chemical herbicides to control weedy species that compete with crops. In the UK, an estimated 80 % of cropland is infested with the weed species black-grass (Alopecurus myosuroides) that has evolved resistance to multiple herbicides with different modes-of-action. Studies in resistant black-grass identified a phi (F) class glutathione transferase, AmGSTF1, which was constitutively expressed. Heterologous expression of AmGSTF1 in a transgenic host plant granted a multiple herbicide resistant (MHR) phenotype and it was found that the enzyme induced the activities of endogenous detoxification enzymes as well as catalytically detoxifying damaging hydroperoxides in vitro, which can form as a downstream consequence of herbicide treatment. In the current work, AmGSTF1 mutants have been derived and exploited to better understand the function of AmGSTF1 in eliciting MHR. Using a catalytically-retarded mutant, it is shown that the enzyme elicited MHR without requiring catalysis. Instead, the mutant induced the activities of endogenous detoxification enzymes. Another mutant, lacking a cysteinyl residue (Cys120), has demonstrated that Cys120 plays a key role in the interaction of AmGSTF1 with xenobiotics. In particular, Cys120 can be alkylated and inhibited by 4-chloro-7-nitro-benzoxadiazole (NBD-Cl), a compound that can reverse MHR when sprayed on black-grass plants. Enzyme inhibition and alkylation studies found that AmGSTF1 could be alkylated by other chemicals but that this did not induce notable inhibition of the protein. The cysteinyl mutant also induced MHR in a transgenic host plant by inducing the activities of endogenous detoxification enzymes. The properties of AmGSTF1 orthologues from annual rye-grass (Lolium rigidum) and maize (Zea mays) were also explored and found to display very similar functional properties as AmGSTF1. Transcriptome profiling demonstrated that AmGSTF1 did not induce changes in host plant biochemistry by perturbing gene expression. These studies have therefore demonstrated a central regulatory role for GSTF1 enzymes in co-ordinating MHR associated with manipulating host detoxification pathways and challenges the scientific dogma that glutathione transferases require catalytic activity to elicit herbicide resistance.