Investigation of glutathione transferases at the gene regulation level in flufenacet resistant black-grass populations (Alopecurus myosuroides Huds.)

Abstract

The herbicide flufenacet, applied in pre-emergence, acts as an inhibitor of the synthesis of very long-chain fatty acids (HRAC group 15) and is an important component in the control of grasses in winter cereals, especially when resistance to other herbicide groups already exists. However, decreases in sensitivity due to increased flufenacet metabolism by glutathione transferase activity have already been described in some black-grass (Alopecurus myosuroides Huds.) populations. So far, little is known about the mechanisms of gene regulation in metabolically resistant weeds. Therefore, we aligned RNA-seq data from two sensitive black-grass populations and two black-grass populations with reduced sensitivity to flufenacet against the recently sequenced black-grass genome. In a differential gene expression analysis, an upregulation of genes involved in metabolic detoxification pathways, such as cytochrome P450 monooxygenases (CYPs), glycosyltransferases (GTs), glutathione transferases (GSTs) and ATP-binding cassette (ABC) transporters was observed. It was found that 7% of the GST genes in the two populations with reduced flufenacet sensitivity were significantly upregulated even without any herbicide application (constitutively) when compared to the two sensitive populations. Three of these genes are located next to each other on the third chromosome and represent a cluster. For each of the upregulated GST genes 3,5 kb of the upstream promoter region were investigated in silico for potential transcription factor binding sites (TFBSs). Some of them share common upstream motifs, such as an ocs element, previously described as enhancer element for GSTs. In addition, other motifs could act as potential binding sites for transcription factors (TFs) that were found upregulated. These belonged to various classes including WRKYs, basic helix-loop-helices (bHLH), basic leucine zippers (bZIP) or MADS-boxes. A better understanding of the regulation of resistanceassociated genes can contribute to improve diagnosis of herbicide resistance and help in predicting the evolution of cross-resistance, as well as contribute to the search for new active ingredients.

Keywords

• metabolic resistance
• glutathione transferase
• transcription factor binding site
• flufenacet
• blackgrass