Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Molecular diagnostics for real-time determination of herbicide resistance in wild grasses.

The growth of resistance to multiple herbicides in grass weeds is a major threat to global cereal production and in the UK, is epitomized by the loss of control of blackgrass (Alopecurus myosuroides), causing losses in winter wheat production equating to 5% of national consumption. With an urgent need to develop new black-grass management tools, we have developed a lateral flow assay (LFA) that can predict resistance to multiple herbicides within 10 min.

Characterization of Cytochrome P450s with Key Roles in Determining Herbicide Selectivity in Maize.

Safeners such as metcamifen and benoxacor are widely used in maize to enhance the selectivity of herbicides through the induction of key detoxifying enzymes, notably cytochrome P450 monooxygenases (CYPs). Using a combination of transcriptomics, proteomics, and functional assays, the safener-inducible CYPs responsible for herbicide metabolism in this globally important crop have been identified. A total of 18 CYPs belonging to clans 71, 72, 74, and 86 were safener-induced, with the respective enzymes expressed in yeast and screened for activity toward thiadiazine (bentazon), sulfonylurea (nicosulfuron), and triketone (mesotrione and tembotrione) chemistries. Herbicide metabolism was largely restricted to family CYP81A members from clan 71, notably CYP81A9, CYP81A16, and CYP81A2. Quantitative transcriptomics and proteomics showed that CYP81A9/CYP81A16 were dominant enzymes in safener-treated field maize, whereas only CYP81A9 was determined in sweet corn. The relationship between CYP81A sequence and activities were investigated by splicing CYP81A2 and CP81A9 together as a series of recombinant chimeras. CYP81A9 showed wide ranging activities toward the three herbicide chemistries, while CYP81A2 uniquely hydroxylated bentazon in multiple positions. The plasticity in substrate specificity of CYP81A9 toward multiple herbicides resided in the second quartile of its N terminal half. Further phylogenetic analysis of CYP81A9 showed that the maize enzyme was related to other CYP81As linked to agrochemical metabolism in cereals and wild grasses, suggesting this clan 71 CYP has a unique function in determining herbicide selectivity in arable crops.

Flavonoid-based inhibitors of the Phi-class glutathione transferase from black-grass to combat multiple herbicide resistance.

The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.

Non-target Site Herbicide Resistance Is Conferred by Two Distinct Mechanisms in Black-Grass (Alopecurus myosuroides).

Non-target site resistance (NTSR) to herbicides in black-grass (Alopecurus myosuroides) results in enhanced tolerance to multiple chemistries and is widespread in Northern Europe. To help define the underpinning mechanisms of resistance, global transcriptome and biochemical analysis have been used to phenotype three NTSR black-grass populations. These comprised NTSR1 black-grass from the classic Peldon field population, which shows broad-ranging resistance to post-emergence herbicides; NTSR2 derived from herbicide-sensitive (HS) plants repeatedly selected for tolerance to pendimethalin; and NTSR3 selected from HS plants for resistance to fenoxaprop-P-ethyl. NTSR in weeds is commonly associated with enhanced herbicide metabolism catalyzed by glutathione transferases (GSTs) and cytochromes P450 (CYPs). As such, the NTSR populations were assessed for their ability to detoxify chlorotoluron, which is detoxified by CYPs and fenoxaprop-P-ethyl, which is acted on by GSTs. As compared with HS plants, enhanced metabolism toward both herbicides was determined in the NTSR1 and NTSR2 populations. In contrast, the NTSR3 plants showed no increased detoxification capacity, demonstrating that resistance in this population was not due to enhanced metabolism. All resistant populations showed increased levels of AmGSTF1, a protein functionally linked to NTSR and enhanced herbicide metabolism. Enhanced AmGSTF1 was associated with increased levels of the associated transcripts in the NTSR1 and NTSR2 plants, but not in NTSR3, suggestive of both pre- and post-transcriptional regulation. The related HS, NTSR2, and NTSR3 plants were subject to global transcriptome sequencing and weighted gene co-expression network analysis to identify modules of genes with coupled regulatory functions. In the NTSR2 plants, modules linked to detoxification were identified, with many similarities to the transcriptome of NTSR1 black-grass. Critical detoxification genes included members of the CYP81A family and tau and phi class GSTs. The NTSR2 transcriptome also showed network similarities to other (a)biotic stresses of plants and multidrug resistance in humans. In contrast, completely different gene networks were activated in the NTSR3 plants, showing similarity to the responses to cold, osmotic shock and fungal infection determined in cereals. Our results demonstrate that NTSR in black-grass can arise from at least two distinct mechanisms, each involving complex changes in gene regulatory networks.

Detection and characterization of resistance to acetolactate synthase inhibiting herbicides in Anisantha and Bromus species in the United Kingdom.

BACKGROUND: Anisantha and Bromus spp. are widespread and difficult to control, potentially due to the evolution of herbicide resistance. In this study, UK populations of four brome species have been tested for the early development of resistance to acetolactate synthase (ALS)-inhibiting herbicides commonly used in their control. RESULTS: Glasshouse assays confirmed reduced sensitivity to ALS-inhibiting herbicides in single populations of A. diandra, B. commutatus and B. secalinus, and in three populations of A. sterilis. By contrast, all 60 brome populations tested were sensitive to the ACCase-inhibiting herbicide propaquizafop and glyphosate. Dose-response with two ALS herbicides showed broad-ranging resistance in the A. diandra, A. sterilis and B. commutatus populations. In the B. commutatus population, this was associated with a point mutation in the ALS enzyme conferring target site resistance (TSR). Additionally, resistant populations of A. sterilis and B. commutatus populations contained enhanced amounts of an orthologue of the glutathione transferase phi (F) class 1 (GSTF1) protein, a functional biomarker of nontarget site resistance (NTSR) in Alopecurus myosuroides. There was further evidence of NTSR as these plants also demonstrated an enhanced capacity to detoxify herbicides. CONCLUSION: This study confirms the evolution of resistance to ALS inhibiting herbicides in brome species in the UK by mechanisms consistent with the evolution of both TSR and NTSR. These findings point to the need for increased vigilance in detecting and mitigating against the evolution of herbicide resistance in brome species in Northern Europe. © 2020 Society of Chemical Industry.

Chemically induced herbicide tolerance in rice by the safener metcamifen is associated with a phased stress response.

The closely related sulphonamide safeners, metcamifen and cyprosulfamide, were tested for their ability to protect rice from clodinafop-propargyl, a herbicide normally used in wheat. While demonstrating that both compounds were equally bioavailable in planta, only metcamifen prevented clodinafop from damaging seedlings, and this was associated with the enhanced detoxification of the herbicide. Transcriptome studies in rice cultures demonstrated that whereas cyprosulfamide had a negligible effect on gene expression over a 4 h exposure, metcamifen perturbed the abundance of 590 transcripts. Changes in gene expression with metcamifen could be divided into three phases, corresponding to inductions occurring over 30 min, 1.5 h and 4 h. The first phase of gene induction was dominated by transcription factors and proteins of unknown function, the second by genes involved in herbicide detoxification, while the third was linked to cellular homeostasis. Analysis of the inducible genes suggested that safening elicited similar gene families to those associated with specific biotic and abiotic stresses, notably those elicited by abscisic acid, salicylic acid, and methyl jasmonate. Subsequent experiments with safener biomarker genes induced in phase 1 and 2 in rice cell cultures provided further evidence of similarities in signalling processes elicited by metcamifen and salicylic acid.

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

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. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Substrate specificity and safener inducibility of the plant UDP-glucose-dependent family 1 glycosyltransferase super-family.

Plants contain large numbers of family 1 UDP-glucose-dependent glycosyltransferases (UGTs), including members that conjugate xenobiotics. Arabidopsis contains 107 UGT genes with 99 family members successfully expressed as glutathione transferase (GST)-fusion proteins in E. coli. A high-throughput catalytic screen was developed based on quantification of the fusion by measuring GST activity. UGT activity using UDP-glucose as donor was then determined using 11 synthetic acceptors bearing hydroxyl, amino and thiol groups that had been shown to undergo conjugation in plant extracts. In total, 44 UGTs, largely members of the D and E groups, were active towards xenobiotics, glucosylating phenol and thiol acceptors. In contrast, N-glucosyltransferase (NGT) activity was almost exclusively restricted to a single enzyme, UGT72B1. Using DNA microarrays, the induction of UGT transcripts following treatment with the herbicide safener fenclorim was compared in Arabidopsis and rice. D and L group members were the most safener-inducible UGTs in both species. The respective Arabidopsis enzymes showed low conjugating activity towards xenobiotics. Using Genevestigator, a small group of safened D and L UGTs were consistently induced in response to biotic and abiotic stress suggestive of protective activities beyond xenobiotic detoxification in both species. The induction of other detoxifying gene families following treatment with fenclorim, namely cytochromes P450 and glutathione transferases, further confirmed the selective enhancement of related subfamily members in the two species giving new insight into the safening response in cereals, where herbicide tolerance is enhanced compared with dicots, which are unresponsive to these treatments.

Protective responses induced by herbicide safeners in wheat.

Safeners are agrochemicals which enhance tolerance to herbicides in cereals including wheat (Triticum aestivum L.) by elevating the expression of xenobiotic detoxifying enzymes, such as glutathione transferases (GSTs). When wheat plants were spray-treated with three safener chemistries, namely cloquintocet mexyl, mefenpyr diethyl and fenchlorazole ethyl, an apparently identical subset of GSTs derived from the tau, phi and lambda classes accumulated in the foliage. Treatment with the closely related mefenpyr diethyl and fenchlorazole ethyl enhanced seedling shoot growth, but this effect was not determined with the chemically unrelated cloquintocet mexyl. Focussing on cloquintocet mexyl, treatments were found to only give a transient induction of GSTs, with the period of elevation being dose dependent. Examining the role of safener metabolism in controlling these responses, it was determined that cloquintocet mexyl was rapidly hydrolysed to the respective carboxylic acid. Studies with cloquintocet showed that the acid was equally effective at inducing GSTs as the ester and appeared to be the active safener. Studies on the tissue induction of GSTs showed that whilst phi and tau class enzymes were induced in all tissues, the induction of the lambda enzymes was restricted to the meristems. To test the potential protective effects of cloquintocet mexyl in wheat on chemicals other than herbicides, seeds were pre-soaked in safeners prior to sowing on soil containing oil and a range of heavy metals. Whilst untreated seeds were unable to germinate on the contaminated soil, safener treatments resulted in seedlings briefly growing before succumbing to the pollutants. Our results show that safeners exert a range of protective and growth promoting activities in wheat that extend beyond enhancing tolerance to herbicides.

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.

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.

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.

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.

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)-gamma-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 beta-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.

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 A, 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.

Selection of plants for roles in phytoremediation: the importance of glucosylation.

Over-expression and transposon mutagenesis in root cultures of Arabidopsis thaliana demonstrated the importance of the family 1 glycosyltransferase UGT72B1 in catalysing the N-glucosylation of the persistent pollutant 3,4-dichloroaniline (DCA). In phytotoxicity studies with DCA in seedlings, over-expression of UGT72B1 enhanced sensitivity, whereas the knockouts were more resistant than the controls. In contrast, manipulating the expression of UGT72B1 had no effect on the O-glucosylation, or toxicity, of chlorophenols. When N-glucosylation was disrupted in plants, radioactivity derived from [14C]-DCA became covalently bound into high molecular weight insoluble material, principally associated with the lignin fraction. This suggested that insolubilization into stable cell wall components represented a more effective mechanism of DCA detoxification than the formation of N-glycosidic conjugates. A screen of plants used in remediation, identified low levels of N-glucosyltransferase activity in switchgrass and high activities in reed canary grass. When incubated with [14C]-DCA, reed canary grass plants accumulated soluble N-glycosides of DCA, whereas switchgrass formed insoluble residues. Consistent with the results obtained in studies with Arabidopsis, phytotoxicity trials with DCA demonstrated that switchgrass was more tolerant than reed canary grass. Our studies provide a new biochemical basis for selecting plants for useful remediating traits towards specific classes of pollutants.

Selective disruption of wheat secondary metabolism by herbicide safeners.

In wheat (Triticum aestivum L.), treatment with herbicide safeners enhances the expression of enzymes involved in pesticide detoxification and reduces crop sensitivity to herbicides. Since these same enzymes are involved in plant secondary metabolism, it was of interest to determine whether or not the safener cloquintocet mexyl perturbed phenolic metabolism in wheat seedlings. LC/ESI/MS analysis identified 14 phenolic substrates in the shoots of young wheat plants. Fragmentation imposed by collision induced dissociation identified specific C-glycosidic conjugates of 4',5,7-trihydroxflavone (apigenin), 3',4',5,7-tetrahydroxyflavone (luteolin) and 3'-O-methylluteolin. Treatment of 7-day-old wheat shoots with cloquintocet mexyl resulted in an accelerated depletion of the conjugates of all three flavones, most notably with the glycosides of luteolin. In contrast, safener treatment caused the selective accumulation of 4',5,7-trihydroxy-3',5'-dimethoxyflavone (tricin) and the phenylpropanoid ferulic acid. Changes in phenolic content were associated with an increase in O-methyltransferase and C-glucosyltransferase activity toward flavonoid substrates as well as the classic enhancement of detoxifying glutathione transferases. Our results suggest that in addition to altering the capacity of wheat to metabolise herbicides and other xenobiotics, safeners can also cause a selective shift in the metabolism of endogenous phenolics.

Functional importance of the family 1 glucosyltransferase UGT72B1 in the metabolism of xenobiotics in Arabidopsis thaliana.

The Arabidopsis type 1 UDP-glucose-dependent glucosyltransferase UGT72B1 is highly active in conjugating the persistent pollutants 3,4-dichloroaniline (DCA) and 2,4,5-trichlorophenol (TCP). To determine its importance in detoxifying xenobiotics in planta, mutant plants where the respective gene has been disrupted by T-DNA insertion have been characterized. Extracts from the knockout ugt72B1 plants showed radically reduced conjugating activity towards DCA and TCP and the absence of immunodetectable UGT72B1 protein. In contrast, activities towards phenolic natural products were unaffected. When aseptic root cultures were fed [14C]-DCA, compared with wild types, the ugt72B1 plants showed a reduced rate of uptake of the xenobiotic and very little metabolism to soluble DCA-glucose or associated polar conjugates. Instead, the knockouts accumulated non-extractable radioactive residues, most probably associated with lignification. When the feeding studies were carried out with [14C]-TCP, rates and routes of metabolism were identical in the wild type and knockouts, with TCP-glucoside a major product in both cases. Similar differential effects on the metabolism of DCA and TCP were obtained in whole plant studies with wild type and ugt72B1 mutants, demonstrating that while UGT72B1 had a central role in metabolizing chloroanilines in Arabidopsis, additional UGTs could compensate for the conjugation of TCP in the knockout. TCP was equally toxic to wild type and ugt72B1 plants, while surprisingly, the knockouts were less sensitive to DCA. From this it was concluded that the glucosylation of DCA may not be as effective in xenobiotic detoxification as bound-residue formation.