Genome-wide study of glutathione transferases and their regulation in flufenacet susceptible and resistant black-grass (Alopecurus myosuroides Huds.).

BACKGROUND: Glutathione transferases (GSTs) are enzymes with a wide range of functions, including herbicide detoxification. Up-regulation of GSTs and their detoxification activity enables the grass weed black-grass (Alopecurus myosuroides Huds.) to metabolize the very-long-chain fatty acid synthesis inhibitor flufenacet and other herbicides leading to multiple herbicide resistance. However, the genomic organization and regulation of GSTs genes is still poorly understood. RESULTS: In this genome-wide study the location and expression of 115 GSTs were investigated using a recently published black-grass genome. Particularly, the most abundant GSTs of class tau and phi were typically clustered and often followed similar expression patterns but possessed divergent upstream regulatory regions. Similarities were found in the promoters of the most up-regulated GSTs, which are located next to each other in a cluster. The binding motif of the E2F/DP transcription factor complex in the promoter of an up-regulated GST was identical in susceptible and resistant plants, however, adjacent sequences differed. This led to a stronger binding of proteins to the motif of the susceptible plant, indicating repressor activity. CONCLUSIONS: This study constitutes the first analysis dealing with the genomic investigation of GST genes found in black-grass and their transcriptional regulation. It highlights the complexity of the evolution of GSTs in black-grass, their duplication and divergence over time. The large number of GSTs allows weeds to detoxify a broad spectrum of herbicides. Ultimately, more research is needed to fully elucidate the regulatory mechanisms of GST expression. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

BACKGROUND: Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. RESULTS: The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. CONCLUSIONS: As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enhanced metabolism causes reduced flufenacet sensitivity in black-grass (Alopecurus myosuroides Huds.) field populations.

BACKGROUND: Black-grass (Alopecurus myosuroides Huds.) is a frequent grass weed that commonly occurs in winter wheat in temperate Europe. Evolving resistance to post-emergence herbicides, e.g. acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors requires more complex weed management strategies and ensuring good efficacy of pre-emergence treatments becomes increasingly important. Flufenacet, in particular, has become a key herbicide for the control of multiple-resistant A. myosuroides. However, in some of those populations, reduced flufenacet efficacy was already observed. RESULTS: In a screening of black-grass populations from several European countries, most populations were controlled with the registered field rate of flufenacet. However, differences in the level of flufenacet sensitivity were observed and correlated with glutathione S-transferase-mediated enhanced flufenacet metabolism. The efficacy of the pre-emergence herbicides pendimethalin, prosulfocarb, S-metolachlor and pethoxamid, was also significantly decreased in populations with reduced flufenacet sensitivity. The use of flufenacet in mixtures with diflufenican, particularly in combination with flurtamone or metribuzin, however, significantly improved efficacy in less susceptible black-grass populations. CONCLUSIONS: In several populations of different European origins, reduced efficacy of flufenacet was observed due to enhanced metabolism. Although differences between populations were relatively small, best weed management practices (e.g. application of full dose rates and herbicide mixtures and wide crop rotations) should be applied to reduce selection pressure and prevent flufenacet resistance from further evolving. This is particularly important as flufenacet is one of the few still-effective herbicides suitable for the control of multiple-resistant A. myosuroides genotypes in Europe, whereas alternative pre-emergence herbicides were less effective against multiple-resistant A. myosuroides populations. © 2019 Society of Chemical Industry.