SummaryBlack-grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide-susceptible blackgrass, populations showing herbicide cross-resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide-resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicidesusceptible populations but not in herbicide-resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in blackgrass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide-susceptible black-grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.

Multiple-herbicide resistance (MHR) in black-grass ( Alopecurus myosuroides ) and annual rye-grass ( Lolium rigidum ) is a global problem leading to a loss of chemical weed control in cereal crops. Although poorly understood, in common with multiple-drug resistance (MDR) in tumors, MHR is associated with an enhanced ability to detoxify xenobiotics. In humans, MDR is linked to the overexpression of a pi class glutathione transferase (GSTP1), which has both detoxification and signaling functions in promoting drug resistance. In both annual rye-grass and black-grass, MHR was also associated with the increased expression of an evolutionarily distinct plant phi (F) GSTF1 that had a restricted ability to detoxify herbicides. When the black-grass A. myosuroides ( Am ) Am GSTF1 was expressed in Arabidopsis thaliana, the transgenic plants acquired resistance to multiple herbicides and showed similar changes in their secondary, xenobiotic, and antioxidant metabolism to those determined in MHR weeds. Transcriptome array experiments showed that these changes in biochemistry were not due to changes in gene expression. Rather, Am GSTF1 exerted a direct regulatory control on metabolism that led to an accumulation of protective flavonoids. Further evidence for a key role for this protein in MHR was obtained by showing that the GSTP1- and MDR-inhibiting pharmacophore 4-chloro-7-nitro-benzoxadiazole was also active toward Am GSTF1 and helped restore herbicide control in MHR black-grass. These studies demonstrate a central role for specific GSTFs in MHR in weeds that has parallels with similar roles for unrelated GSTs in MDR in humans and shows their potential as targets for chemical intervention in resistant weed management.

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

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Discovered 40 years ago, plant glutathione transferases (GSTs) now have a well-established role in determining herbicide metabolism and selectivity in crops and weeds. Within the GST superfamily, the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification and, more rarely, bioactivating isomerizations. In total, the crystal structures of 10 plant GSTs have been solved and a highly conserved N-terminal glutathione binding domain and structurally diverse C-terminal hydrophobic domain identified, along with key coordinating residues. Unlike drug-detoxifying mammalian GSTs, plant enzymes utlilize a catalytic serine in place of a tyrosine residue. Both GSTFs and GSTUs undergo changes in structure during catalysis indicative of an induced fit mechanism on substrate binding, with an understanding of plant GST structure/function allowing these proteins to be engineered for novel functions in detoxification and ligand recognition. Several major crops produce alternative thiols, with GSTUs shown to use homoglutathione in preference to glutathione, in herbicide detoxification reactions in soybeans. Similarly, hydroxymethylglutathione is used, in addition to glutathione in detoxifying the herbicide fenoxaprop in wheat. Following GST action, plants are able to rapidly process glutathione conjugates by at least two distinct pathways, with the available evidence suggesting these function in an organ- and species-specific manner. Roles for GSTs in endogenous metabolism are less well defined, with the enzymes linked to a diverse range of functions, including signaling, counteracting oxidative stress, and detoxifying and transporting secondary metabolites.

Glutathione S-transferases (GSTs) from the phi (GSTF) and tau (GSTU) classes are unique to plants and play important roles in stress tolerance and secondary metabolism as well as catalyzing the detoxification of herbicides in crops and weeds. We have cloned and functionally characterized a group of GSTUs from wheat treated with fenchlorazole-ethyl, a herbicide safener. One of these enzymes, TaGSTU4-4, was highly active in conjugating the chemically distinct wheat herbicides fenoxaprop and dimethenamid. The structure of TaGSTU4-4 has been determined at 2.2 A resolution in complex with S-hexylglutathione. This enzyme is the first tau class GST structure to be determined and most closely resembles the omega class GSTs, but without the unique N-terminal extension or active site cysteine. The X-ray structure identifies key amino acid residues in the hydrophobic binding site and provides insights into the substrate specificity of these enzymes.

SummarySafeners enhance the selectivity of graminicidal herbicides such as fenoxaprop ethyl in cereals, by increasing their rates of detoxification in the crop. While studying the selectivity of fenoxaprop ethyl in wheat, we determined that the safeners mefenpyr diethyl and fenchlorazole ethyl also enhanced herbicide tolerance in the competing weed black-grass (Alopecurus myosuroides). Fenoxaprop ethyl was detoxified by conjugation with glutathione in both wheat and black-grass, with the resulting metabolites processed to the respective cysteine derivatives, which were then N-glycosylated. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabolism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydroxymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respectively. These safener-induced changes closely mirrored those determined in two independent black-grass populations that had acquired multiple herbicide resistance (MHR) in the field. In addition to enhanced glutathione metabolism, both safener treatment and MHR resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Our results demonstrate that safening in a grass weed is associated with an inducible activation in antioxidant and secondary metabolism which mirrors the biochemical phenotype exhibited in plants that are resistant to multiple classes of herbicides.

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