Co-induction of glutathione- S-transferases and multidrug resistance associated protein by xenobiotics in wheat † Frederica L Theodoulou, 1 * Ian M Clark, 1 Xiao-Li He, 1 Kenneth E Pallett, 2 David J Cole 3 and David L Hallahan 1‡ 1 Crop Performance and Improvement Division, IACR-Rothamsted, Harpenden AL5 2JQ, UK 2 Aventis CropScience GmbH, Industriepark Hoechst, Frankfurt am Main, Building H872, D-65926, Germany 3 Aventis CropScience, Fyfield Road, Ongar, Essex CM5 0HW, UK Abstract: Herbicide safeners are known to protect monocotyledonous crops from herbicide injury by accelerating the metabolism of herbicides. We have investigated the effects of the safener cloquintocet- mexyl, which protects small-grain cereals against the graminicidal herbicide, clodinafop-propargyl. Subtractive suppression hybridisation was used to identify wheat genes which are up-regulated by treatment not only with cloquintocet-mexyl but also with phenobarbital, which is known to stimulate xenobiotic metabolism in animals and plants. DNA sequences of five glutathione transferases (GSTs) belonging to three different classes and a multidrug resistance associated protein (MRP) homologue were identified in the screen. The chemical inducibility of these clones was confirmed by Northern analysis. The MRP protein was shown to be induced by treatments with cloquintocet-mexyl and phenobarbital and to be localised to the tonoplast. Since clodinafop-propargyl is not known to be metabolised by glutathionylation, the significance of GST induction is interpreted in terms of a generalised response to chemical stress, particularly the generation of active oxygen species. This work establishes herbicide safeners as useful tools for the identification of genes encoding herbicide-metabolising enzymes. # 2003 Society of Chemical Industry Keywords: cloquintocet-mexyl; differential expression; GST; MRP; herbicide safener; phenobarbital; wheat Abbreviations ABC ATP Binding Cassette AOS active oxygen species BAC bacterial artificial chromosome BSA bovine serum albumin cyt P450 cytochrome(s) P450 GPox glutathione peroxidase GST glutathione transferase GS-X glutathione conjugate MRP multidrug resistance associated protein ORF open reading frame PCR polymerase chain reaction PBS phosphate buffered saline PMSF phenylmethylsulfonyl fluoride SSH subtractive suppression hybridisation TBS Tris buffered saline V-PPase vacuolar pyrophosphatase 1 INTRODUCTION Herbicide safeners form a group of structurally diverse compounds which are used to protect monocotyledo- nous crop plants from injury by certain herbicides. 1,2 Although a number of roles have been proposed, selective enhancement of herbicide metabolism is now recognised as the most common mode of safener action. However, relatively little is understood con- cerning the underlying molecular mechanisms by which herbicide metabolism is modulated. 3 Metabolism of herbicides and other foreign chemi- cals (xenobiotics) occurs in four phases in plants. 4 In phase I, an active functional group is introduced or revealed, for example by the action of esterases, amidases or cytochromes P450 (cyt P450). In phase II, this active group is conjugated to a water-soluble moiety, such as glutathione, glucose or malonate via (Received 25 February 2002; revised version received 1 April 2002; accepted 21 June 2002) * Correspondence to: Frederica L Theodoulou, Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK E-mail: freddie.theodoulou@bbsrc.ac.uk † One of a collection of papers contributed by staff of the Institute of Arable Crops Research (IACR). The papers were collected and collated by Dr Richard Bromilow ‡ Current address: Central Research and Development, The DuPont Co, Biochemical Sciences and Engineering, Wilmington, DE 19880- 0328, USA Contract/grant sponsor: Biotechnology and Biological Sciences Research Council (BBSRC) ROPA Contract/grant sponsor: Aventis CropScience SA Contract/grant sponsor: BBSRC # 2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00 202 Pest Management Science Pest Manag Sci 59:202–214 (online: 2003) DOI: 10.1002/ps.576