Differential induction of glutathione transferases and glucosyltransferases in wheat, maize and Arabidopsis thaliana by herbicide safeners.

By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: (1) The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. (2) The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. (3) The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

High-throughput mass-spectrometry monitoring for multisubstrate enzymes: determining the kinetic parameters and catalytic activities of glycosyltransferases.

A novel high-throughput screening (HTS) method with electrospray time-of-flight (ESI-TOF) mass spectrometry allows i) rapid and broad screening of multisubstrate enzyme catalytic activity towards a range of donor and acceptor substrates; ii) determination of full multisubstrate kinetic parameters and the binding order of substrates. Two representative glycosyltransferases (GTs, one common, one recently isolated, one O-glycosyltransferase (O-GT), one N-glycosyltransferase (N-GT)) have been used to validate this system: the widely used bovine beta-1,4-galactosyltransferase (EC 2.4.1.22), and the recently isolated Arabidopsis thaliana GT UGT72B1 (EC 2.4.1.-). The GAR (green/amber/red) broad-substrate-specificity screen, which is based on the mass ion abundance of product, provides a fast, high-throughput method for finding potential donors and acceptors from substrate libraries. This was evaluated by using six natural and non-natural donors (alpha-UDP-D-Glucose (UDPGlc), alpha-UDP-N-Acetyl-D-glucosamine (UDPGlcNAc), alpha-UDP-D-5-thioglucose (UDP5SGlc), alpha-GDP-L-fucose (GDPFuc), alpha-GDP-D-mannose (GDPMan), alpha,beta-UDP-D-mannose (UDPMan)) and 32 broad-ranging acceptors (sugars, plant hormones, antibiotics, flavonoids, coumarins, phenylpropanoids and benzoic acids). By using the fast-equilibrium assumption, KM, kcat and KIA were determined for representative substrates, and these values were used to determine substrate binding orders. These screening methods applied to the two very different enzymes revealed some unusual substrate specificities, thus highlighting the utility of broad-ranging substrate screening. For UGT72B1, it was shown that the donor specificity is determined largely by the nucleotide moiety. The method is therefore capable of identifying GT enzymes with usefully broad carbohydrate-transfer ability.

Partial purification and characterisation of a 2,4,5-trichlorophenol detoxifying O-glucosyltransferase from wheat.

An enzyme preparation with UDP-glucose-dependent O-glucosyltransferase (OGT; EC 2.4.1.-) activity toward 2,4,5-trichlorophenol has been purified 215-fold from wheat shoots. The OGT co-purified with the major extractable glucosylating activity toward the flavonol quercetin and was characterised as a monomeric 53 kDa protein. Among the xenobiotic phenols tested, the purified enzyme preparation showed at least a 10-fold preference for 2,4,5-trichlorophenol. When assayed with flavonoids, the OGT was active toward flavonols and coumestrol, showing a clear preference for 3-hydroxy flavone when incubated with a range of monohydroxylated flavonoids. It was concluded that the major 2,4,5-trichlorophenol-detoxifying OGT in wheat shoots is most probably a flavonol-3-O-glucosytransferase.

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean.

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.

Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4-dichloroaniline.

The pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-beta-d-glucopyranosyl-DCA exported into the medium. The N-glucosyltransferase (N-GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5-trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N-GT and O-GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4-hydroxyphenylpyruvic acid. Both O-GT and N-GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.

Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and

O-Glucosyltransferase activities toward phenolic natural products and xenobiotics in wheat and herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides).

Herbicide safeners manipulate herbicide selectivity by enhancing the activities of detoxifying enzymes, such as glutathione transferases (GSTs) and cytochrome P450 mono-oxygenases (CYPs) in cereal crops. As part of a study examining the importance of O-glucosyltransferases (OGTs) in pesticide metabolism in hexaploid bread wheat (Triticum aestivum L.), seedlings were grown in the presence of dichlormid, a safener used in maize and cloquintocet mexyl, a wheat safener. The efficacy of the treatments was confirmed by monitoring changes in the abundance of phi and tau class GSTs. OGT activities in the root and shoot tissue were assayed using phenolics of natural and xenobiotic origin to determine if they were enhanced by safeners. Cloquintocet mexyl selectively increased OGT activities toward xenobiotics (4-nitrophenol and 2,4,5-trichlorophenol) and flavonoids, (quercetin, luteolin, genistein and coumestrol) in both the roots and shoots. However, OGT activity towards simple phenols and phenylpropanoids was not enhanced by cloquintocet mexyl. Dichlormid was a much weaker enhancer of OGT activity, with the same subset of OGT activities increased as determined with cloquintocet mexyl, but with the effect being largely restricted to the roots. OGT activities were also determined in black-grass (Alopecurus myosuroides L.), an agronomically important weed in wheat. Two populations of black-grass differing in their sensitivity to herbicides were analysed. The population Peldon, which is resistant to multiple classes of herbicides due in part to the elevated expression of CYPs and GSTs active in herbicide detoxification, contained higher OGT activities than herbicide sensitive black-grass. Unlike wheat, treatment with cloquintocet mexyl or dichlormid, had no effect on OGT activities in either black-grass population.