Melissa Brazier-Hicks

The C-Glycosylation of Flavonoids in Cereals

Flavonoids normally accumulate in plants as O-glycosylated derivatives, but several species, including major cereal crops, predominantly synthesize flavone-C-glycosides, which are stable to hydrolysis and are biologically active both in planta and as dietary components. An enzyme (OsCGT) catalyzing the UDP-glucose-dependent C-glucosylation of 2-hydroxyflavanone precursors of flavonoids has been identified and cloned from rice (Oryza sativa ssp. indica), with a similar protein characterized in wheat (Triticum aestivum L.). OsCGT is a 49-kDa family 1 glycosyltransferase related to known O-glucosyltransferases. The recombinant enzyme C-glucosylated 2-hydroxyflavanones but had negligible O-glucosyltransferase activity with flavonoid acceptors. Enzyme chemistry studies suggested that OsCGT preferentially C-glucosylated the dibenzoylmethane tautomers formed in equilibrium with 2-hydroxyflavanones. The resulting 2-hydroxyflavanone-C-glucosides were unstable and spontaneously dehydrated in vitro to yield a mixture of 6C- and 8C-glucosyl derivatives of the respective flavones. In contrast, in planta, only the respective 6C-glucosides accumulated. Consistent with this selectivity in glycosylation product, a dehydratase activity that preferentially converted 2-hydroxyflavanone-C-glucosides to the corresponding flavone-6C-glucosides was identified in both rice and wheat. Our results demonstrate that cereal crops synthesize C-glucosylated flavones through the concerted action of a CGT and dehydratase acting on activated 2-hydroxyflavanones, as an alternative means of generating flavonoid metabolites.

Characterization and Engineering of the Bifunctional N- and O-Glucosyltransferase Involved in Xenobiotic Metabolism in Plants.

The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable N-glucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 Å, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.

Chemical Manipulation of Antioxidant Defences in Plants

ABSTRACT Synthetic xenobiotics such as herbicides elicit the generation of reactive oxygen intermediates (ROIs) in plants, which induce antioxidant defences, notably the production of glutathione (GSH) and glutathione transferases (GSTs). GSTs also play an important role in catalysing the conjugation of xenobiotics with GSH, which leads to their detoxification. We have been interested in the multiple roles of GSTs in counteracting xenobiotic-induced stress in plants and the induction of these enzymes by herbicide safeners, which act without the need for large-scale ROI accumulation. Safeners are compounds that enhance herbicide tolerance in cereals and are known to induce the expression of different classes of xenobiotic-detoxifying enzymes, which we have collectively termed the xenome . It has been proposed that safeners act by enhancing the detoxification of xenobiotics. However, we propose that safeners primarily protect plants by inducing a specific subset of antioxidant responses that relate to the endogenous functions of the xenome. Using GSTs as an example of multifunctional protective proteins, we examine this paradigm for safener action and what it tells us about antioxidant signalling pathways in plants.

Catabolism of Glutathione Conjugates in Arabidopsis thaliana ROLE IN METABOLIC REACTIVATION OF THE HERBICIDE SAFENER FENCLORIM

The safener fenclorim (4,6-dichloro-2-phenylpyrimidine) increases tolerance to chloroacetanilide herbicides in rice by enhancing the expression of detoxifying glutathione S-transferases (GSTs). Fenclorim also enhances GSTs in Arabidopsis thaliana, and while investigating the functional significance of this induction in suspension cultures, we determined that these enzymes glutathionylated the safener. The resulting S-(fenclorim)-glutathione conjugate was sequentially processed to S-(fenclorim)-γ-glutamyl-cysteine and S-(fenclorim)-cysteine (FC), the latter accumulating in both the cells and the medium. FC was then either catabolized to 4-chloro-6-(methylthio)-phenylpyrimidine (CMTP) or N-acylated with malonic acid. These cysteine derivatives had distinct fates, with the enzymes responsible for their formation being induced by fenclorim and FC. Fenclorim-N-malonylcysteine was formed from FC by the action of a malonyl-CoA-dependent N-malonyltransferase. A small proportion of the fenclorim-N-malonylcysteine then underwent decarboxylation to yield a putative S-fenclorim-N-acetylcysteine intermediate, which underwent a second round of GST-mediated S-glutathionylation and subsequent proteolytic processing. The formation of CMTP was catalyzed by the concerted action of a cysteine conjugate β-lyase and an S-methyltransferase, with the two activities being coordinately regulated. Although the fenclorim conjugates tested showed little GST-inducing activity in Arabidopsis, the formation of CMTP resulted in metabolic reactivation, with the product showing good enhancing activity. In addition, CMTP induced GSTs and herbicide-safening activity in rice. The bioactivated CMTP was in turn glutathione-conjugated and processed to a malonyl cysteine derivative. These results reveal the surprisingly complex set of competing catabolic reactions acting on xenobiotics entering the S-glutathionylation pathway in plants, which can result in both detoxification and bioactivation.

Xenobiotic Responsiveness of Arabidopsis thaliana to a Chemical Series Derived from a Herbicide Safener

Plants respond to synthetic chemicals by eliciting a xenobiotic response (XR) that enhances the expression of detoxifying enzymes such as glutathione transferases (GSTs). In agrochemistry, the ability of safeners to induce an XR is used to increase herbicide detoxification in cereal crops. Based on the responsiveness of the model plant Arabidopsis thaliana to the rice safener fenclorim (4,6-dichloro-2-phenylpyrimidine), a series of related derivatives was prepared and tested for the ability to induce GSTs in cell suspension cultures. The XR in Arabidopsis could be divided into rapid and slow types depending on subtle variations in the reactivity (electrophilicity) and chemical structure of the derivatives. In a comparative microarray study, Arabidopsis cultures were treated with closely related compounds that elicited rapid (fenclorim) and slow (4-chloro-6-methyl-2-phenylpyrimidine) XRs. Both chemicals induced major changes in gene expression, including a coordinated suppression in cell wall biosynthesis and an up-regulation in detoxification pathways, whereas only fenclorim selectively induced sulfur and phenolic metabolism. These transcriptome studies suggested several linkages between the XR and oxidative and oxylipin signaling. Confirming links with abiotic stress signaling, suppression of glutathione content enhanced GST induction by fenclorim, whereas fatty acid desaturase mutants, which were unable to synthesize oxylipins, showed an attenuated XR. Examining the significance of these studies to agrochemistry, only those fenclorim derivatives that elicited a rapid XR proved effective in increasing herbicide tolerance (safening) in rice.

New Perspectives on the Metabolism and Detoxification of Synthetic Compounds in Plants

In attempting to understand the mechanisms by which plants process synthetic compounds we have developed the concept of the ‘Xenome’, which we define as ‘the biosystem responsible for the detection, transport and detoxification of xenobiotics.’ In particular the last 10 years have given us unprecedented insights into the proteins responsible for the metabolism and transport of xenobiotics within plant cells and how these systems are regulated. In this review we identify recent advances in our understanding of the xenome and its role in the detoxification and processing of pollutants and pesticides. In particular, we focus on the role of the phase 1 (oxidoreductase/ hydrolytic), phase 2 (bioconjugation), phase 3 (transport) and phase 4 (metabolic recycling) stages of xenobiotic metabolism and the biosensing systems which control their expression. Ultimately, by understanding the capability of the plant xenome to detoxify xenobiotics, we may be able to predict the likely fate and environmental risk of new synthetic compounds entering the environment and food chain.

Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology

C-Glycosylated flavonoids are biologically active plant natural products linked to dietary health benefits. We have used polyprotein expression technology to reconstruct part of the respective biosynthetic pathway in tobacco and yeast, such that dihydrochalcone and flavanone precursors are directly converted to C-glycosides. The polyprotein system developed facilitated the simple and efficient co-expression of pathway enzymes requiring different sub-cellular localization in both plants and yeast. The pathway to flavone-C-glucosides comprised a flavanone 2-hydroxylase (F2H), co-expressed with a C-glucosyltransferase (CGT). While pathway engineering in tobacco resulted in only minor C-glycoside formation, when fed with the flavanone naringenin, yeast transformed with the F2H-CGT polyprotein construct produced high concentrations of 2-hydroxynaringenin-C-glucoside in the medium. These fermentation products could then be readily chemically converted to the respective flavone-C-glucosides. The efficiency of the biosynthesis was optimal when both the F2H and CGT were obtained from the same species (rice). These results confirm the coupled roles of the F2H and CGT in producing C-glucosides in vivo, with the use of the polyprotein expression system in yeast offering a useful system to optimize the synthesis of these natural products in quantities suitable for dietary studies.

Elucidation of the biosynthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. that inhibits Striga spp. development.

Isoschaftoside, an allelopathic di-C-glycosylflavone from Desmodium spp. root exudates, is biosynthesised through sequential glucosylation and arabinosylation of 2-hydroxynaringenin with UDP-glucose and UDP-arabinose. Complete conversion to the flavone requires chemical dehydration implying a dehydratase enzyme has a role in vivo to complete the biosynthesis. The C-glucosyltransferase has been partially characterised and its activity demonstrated in highly purified fractions.

A kinetic model for the metabolism of the herbicide safener fenclorim in Arabidopsis thaliana.

Glutathione transferases (GSTs) catalyse the detoxification of a range of xenobiotics, including crop protection agents in plants. Recent studies in cultures of the model plant Arabidopsis thaliana have shown that the herbicide safener fenclorim (4,6-dichloro-2-phenylpyrimidine) is conjugated by GSTs acting in the cytosol which are induced in response to this chemical treatment. The primary glutathione conjugates are then hydrolyzed to S-(4-chloro-2-phenylpyrimidin-6-yl)-cysteine, which after accumulating transiently in the cells and medium is then metabolized by a series of competing lyases and transferases, including GSTs, to a series of polar derivatives. This system therefore represents an example of an inducible metabolic pathway, where GSTs are involved in multiple steps and where detailed information on the content of intermediates is available. Using this data, a kinetic model describing the biotransformations of differing concentrations of fenclorim in Arabidopsis has been established, which was able to quantitatively analyse fluxes and changes in metabolite levels over time as a function of the induction of GSTs by the safener. The model confirmed a regulatory role for GSTs and the hydrolytic enzymes acting on the resulting glutathione conjugates. In addition, model analysis indicated that fenclorim metabolism is capable of generating oscillations if kinetic parameters are allowed to vary. The model offers new insights into the metabolic regulation of inducible xenobiotic metabolism in plants which is important in both determining herbicide selectivity in cereal crops and the remediation of organic pollutants by plants.

Testing a chemical series inspired by plant stress oxylipin signalling agents for herbicide safening activity

Abstract BACKGROUND Herbicide safening in cereals is linked to a rapid xenobiotic response (XR), involving the induction of glutathione transferases (GSTs). The XR is also invoked by oxidized fatty acids (oxylipins) released during plant stress, suggesting a link between these signalling agents and safening. To examine this relationship, a series of compounds modelled on the oxylipins 12‐oxophytodienoic acid and phytoprostane 1, varying in lipophilicity and electrophilicity, were synthesized. Compounds were then tested for their ability to invoke the XR in Arabidopsis and protect rice seedlings exposed to the herbicide pretilachlor, as compared with the safener fenclorim. RESULTS Of the 21 compounds tested, three invoked the rapid GST induction associated with fenclorim. All compounds possessed two electrophilic carbon centres and a lipophilic group characteristic of both oxylipins and fenclorim. Minor effects observed in protecting rice seedlings from herbicide damage positively correlated with the XR, but did not provide functional safening. CONCLUSION The design of safeners based on the characteristics of oxylipins proved successful in deriving compounds that invoke a rapid XR in Arabidopsis but not in providing classical safening in a cereal. The results further support a link between safener and oxylipin signalling, but also highlight species‐dependent differences in the responses to these compounds.