The selective chemical control of wild grasses in wheat is primarily determined by the relative rates of herbicide metabolism, with the superfamily of cytochromes P450 (CYPs) playing a major role in catalyzing phase 1 detoxification reactions. This selectivity is enhanced by herbicide safeners, which induce CYP expression in cereals, or challenged by the evolution of nontarget site resistance (NTSR) in weeds such as blackgrass. Using transcriptomics, proteomics, and functional expression in recombinant yeast, CYPs linked to safener treatment and NTSR have been characterized in wheat and blackgrass. Safener treatment resulted in the induction of 13 families of CYPs in wheat and 5 in blackgrass, with CYP71, CYP72, CYP76, and CYP81 members active toward selective herbicides in the crop. Based on their expression and functional activities, three inducible TaCYP81s were shown to have major roles in safening in wheat. In contrast, a single AmCYP81 that was enhanced by NTSR, but not by safening, was found to dominate herbicide detoxification in blackgrass.

Safeners are agrochemicals co-applied with herbicides that facilitate selective control of weeds by protecting monocot crops from chemical injury through enhancing the expression of detoxifying enzymes such as glutathione transferases (GSTs). Even though the application of safeners causes the induction of genes encoding GSTs in model dicots such as Arabidopsis thaliana, safeners do not protect broadleaf crops from herbicide injury. In this study, we proposed that the localized induction of Arabidopsis GSTs and the fundamental differences in their detoxifying activity between dicot and monocot species, underpin the failure of safeners to protect Arabidopsis from herbicide toxicity. Using the herbicide safener, isoxadifen-ethyl, we showed that three tau (U) family GSTs namely AtGSTU7, AtGSTU19 and AtGSTU24 were induced with different magnitude by isoxadifen treatment in root and rosette tissues. The higher magnitude of inducibility of these AtGSTUs in the root tissues coincided with the enhanced metabolism of flufenacet, a herbicide that is active in root tissue, protecting Arabidopsis plants from chemical injury. Assay of the recombinant enzyme activities and the significant reduction in flufenacet metabolism determined in the T-DNA insertion mutant of AtGSTU7 (gstu7) in Arabidopsis plants identified an important function for AtGSTU7 protein in flufenacet detoxification. In-silico structural modeling of AtGSTU7, suggested the unique high activity of this enzyme toward flufenacet was due to a less constrained active site compared to AtGSTU19 and AtGSTU24. We demonstrate here that it is possible to induce herbicide detoxification in dicotyledonous plants by safener treatment, albeit with this activity being restricted to very specific combinations of herbicide chemistry, and the localized induction of enzymes with specific detoxifying activities.

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.

Herbicide resistance in problem weeds is now a major threat to global food production, being particularly widespread in wild grasses affecting cereal crops. In the UK, black-grass (Alopecurus myosuroides) holds the title of number one agronomic problem in winter wheat, with the loss of production associated with herbicide resistance now estimated to cost the farming sector at least £0.5 billion p.a. Black-grass presents us with many of the characteristic traits of a problem weed; being highly competitive, genetically diverse and obligately out-crossing, with a growth habit that matches winter wheat. With the UK’s limited arable crop rotations and the reliance on the repeated use of a very limited range of selective herbicides we have been continuously performing a classic Darwinian selection for resistance traits in weeds that possess great genetic diversity and plasticity in their growth habits. The result has been inevitable; the steady rise of herbicide resistance across the UK, which now affects over 2.1 million hectares of some of our best arable land. Once the resistance genie is out of the bottle, it has proven difficult to prevent its establishment and spread. With the selective herbicide option being no longer effective, the options are to revert to cultural control; changing rotations and cover crops, manual rogueing of weeds, deep ploughing and chemical mulching with total herbicides such as glyphosate. While new precision weeding technologies are being developed, their cost and scalability in arable farming remains unproven. As an agricultural scientist who has spent a working lifetime researching selective weed control, we seem to be giving up on a technology that has been a foundation stone of the green revolution. For me it begs the question, are we really unable to use modern chemical and biological technology to counter resistance? I would argue the answer to that question is most patently no; solutions are around the corner if we choose to develop them.

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.

Cyanogenic glucosides are nitrogen-containing specialized metabolites that provide chemical defense against herbivores and pathogens via the release of toxic hydrogen cyanide. It has been suggested that cyanogenic glucosides are also a store of nitrogen that can be remobilized for general metabolism via a previously unknown pathway. Here we reveal a recycling pathway for the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor) that avoids hydrogen cyanide formation. As demonstrated in vitro, the pathway proceeds via spontaneous formation of a dhurrin-derived glutathione conjugate, which undergoes reductive cleavage by glutathione transferases of the plant-specific lambda class (GSTLs) to produce p-hydroxyphenyl acetonitrile. This is further metabolized to p-hydroxyphenylacetic acid and free ammonia by nitrilases, and then glucosylated to form p-glucosyloxyphenylacetic acid. Two of the four GSTLs in sorghum exhibited high stereospecific catalytic activity towards the glutathione conjugate, and form a subclade in a phylogenetic tree of GSTLs in higher plants. The expression of the corresponding two GSTLs co-localized with expression of the genes encoding the p-hydroxyphenyl acetonitrile-metabolizing nitrilases at the cellular level. The elucidation of this pathway places GSTs as key players in a remarkable scheme for metabolic plasticity allowing plants to reverse the resource flow between general and specialized metabolism in actively growing tissue.

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

Herbicide resistance in wild grasses is widespread in the UK, with non-target site resistance (NTSR) to multiple chemistries being particularly problematic in weed control. As a complex trait, NTSR is driven by complex evolutionary pressures and the growing awareness of the role of the phytobiome in plant abiotic stress tolerance, led us to sequence the transcriptomes of herbicide resistant and susceptible populations of black-grass and annual rye-grass for the presence of endophytes. Black-grass (Alopecurus myosuroides; Am) populations, displaying no overt disease symptoms, contained three previously undescribed viruses belonging to the Partititiviridae (AMPV1 and AMPV2) and Rhabdoviridae (AMVV1) families. These infections were widespread in UK black-grass populations and evidence was obtained for similar viruses being present in annual rye grass (Lolium rigidum), perennial rye-grass (Lolium perenne) and meadow fescue (Festuca pratensis). In black-grass, while no direct causative link was established linking viral infection to herbicide resistance, transcriptome sequencing showed a high incidence of infection in the NTSR Peldon population. The widespread infection of these weeds by little characterised and persistent viruses and their potential evolutionary role in enhancing plant stress tolerance mechanisms including NTSR warrants further investigation.

The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.

Thirty years after the production of the first generation of genetically modified plants we are now set to move into a new era of recombinant crop technology through the application of synthetic biology to engineer new and complex input and output traits. The use of synthetic biology technologies will represent more than incremental additions of transgenes, but rather the directed design of completely new metabolic pathways, physiological traits, and developmental control strategies. The need to enhance our ability to improve crops through new engineering capability is now increasingly pressing as we turn to plants not just for food, but as a source of renewable feedstocks for industry. These accelerating and diversifying demands for new output traits coincide with a need to reduce inputs and improve agricultural sustainability. Faced with such challenges, existing technologies will need to be supplemented with new and far-more-directed approaches to turn valuable resources more efficiently into usable agricultural products. While these objectives are challenging enough, the use of synthetic biology in crop improvement will face public acceptance issues as a legacy of genetically modified technologies in many countries. Here we review some of the potential benefits of adopting synthetic biology approaches in improving plant input and output traits for their use as industrial chemical feedstocks, as linked to the rapidly developing biorefining industry. Several promising technologies and biotechnological targets are identified along with some of the key regulatory and societal challenges in the safe and acceptable introduction of such technology.

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.

BACKGROUND:Gut microbes influence animal health and thus, are potential targets for interventions that slow aging. Live E. coli provides the nematode worm Caenorhabditis elegans with vital micronutrients, such as folates that cannot be synthesized by animals. However, the microbe also limits C. elegans lifespan. Understanding these interactions may shed light on how intestinal microbes influence mammalian aging. RESULTS:Serendipitously, we isolated an E. coli mutant that slows C. elegans aging. We identified the disrupted gene to be aroD, which is required to synthesize aromatic compounds in the microbe. Adding back aromatic compounds to the media revealed that the increased C. elegans lifespan was caused by decreased availability of para-aminobenzoic acid, a precursor to folate. Consistent with this result, inhibition of folate synthesis by sulfamethoxazole, a sulfonamide, led to a dose-dependent increase in C. elegans lifespan. As expected, these treatments caused a decrease in bacterial and worm folate levels, as measured by mass spectrometry of intact folates. The folate cycle is essential for cellular biosynthesis. However, bacterial proliferation and C. elegans growth and reproduction were unaffected under the conditions that increased lifespan. CONCLUSIONS:In this animal:microbe system, folates are in excess of that required for biosynthesis. This study suggests that microbial folate synthesis is a pharmacologically accessible target to slow animal aging without detrimental effects.

Discovered 40 years ago, plant glutathione transferases (GSTs) now have a well-established role in determining herbicide metabolism and selectivity in crops and weeds. Within the GST superfamily, the numerous and plant-specific phi (F) and tau (U) classes are largely responsible for catalyzing glutathione-dependent reactions with xenobiotics, notably conjugation leading to detoxification and, more rarely, bioactivating isomerizations. In total, the crystal structures of 10 plant GSTs have been solved and a highly conserved N-terminal glutathione binding domain and structurally diverse C-terminal hydrophobic domain identified, along with key coordinating residues. Unlike drug-detoxifying mammalian GSTs, plant enzymes utlilize a catalytic serine in place of a tyrosine residue. Both GSTFs and GSTUs undergo changes in structure during catalysis indicative of an induced fit mechanism on substrate binding, with an understanding of plant GST structure/function allowing these proteins to be engineered for novel functions in detoxification and ligand recognition. Several major crops produce alternative thiols, with GSTUs shown to use homoglutathione in preference to glutathione, in herbicide detoxification reactions in soybeans. Similarly, hydroxymethylglutathione is used, in addition to glutathione in detoxifying the herbicide fenoxaprop in wheat. Following GST action, plants are able to rapidly process glutathione conjugates by at least two distinct pathways, with the available evidence suggesting these function in an organ- and species-specific manner. Roles for GSTs in endogenous metabolism are less well defined, with the enzymes linked to a diverse range of functions, including signaling, counteracting oxidative stress, and detoxifying and transporting secondary metabolites.

Pre-lamin A and progerin have been implicated in normal aging, and the pathogenesis of age-related degenerative diseases is termed 'laminopathies'. Here, we show that mature lamin A has an essential role in cellular fitness and that oxidative damage to lamin A is involved in cellular senescence. Primary human dermal fibroblasts (HDFs) aged replicatively or by pro-oxidants acquire a range of dysmorphic nuclear shapes. We observed that conserved cysteine residues in the lamin A tail domain become hyperoxidized in senescent fibroblasts, which inhibits the formation of lamin A inter- and intramolecular disulfide bonds. Both in the absence of lamin A and in the presence of a lamin A cysteine-to-alanine mutant, which eliminates these cysteine residues (522, 588, and 591), mild oxidative stress induced nuclear disorganization and led to premature senescence as a result of decreased tolerance to ROS stimulators. Human dermal fibroblasts lacking lamin A or expressing the lamin A cysteine-to-alanine mutant displayed a gene expression profile of ROS-responsive genes characteristic of chronic ROS stimulation. Our findings suggest that the conserved C-terminal cysteine residues are essential for lamin A function and that loss or oxidative damage to these cysteine residues promotes cellular senescence.

Uniquely among the plant glutathione transferases, two classes possess a catalytic cysteine capable of performing glutathione-dependent reductions. These are the dehydroascorbate reductases (DHARs) and the lambda-class glutathione transferases (GSTLs). Using immobilized GSTLs probed with crude plant extracts we have identified flavonols as high affinity ligands and subsequently demonstrated a novel glutathione-dependent role for these enzymes in recycling oxidized quercetin. By comparing the activities of DHARs and GSTLs we now propose a unified catalytic mechanism that suggests oxidized anthocyanidins and tocopherols may be alternative polyphenolic substrates of GSTLs.

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.

Sessions C308for both prokaryotic and eukaryotic enzymes is largely conserved.However there are several structural differences between prokaryotic UGM and eukaryotic AfUGM mainly because of five additional inserts in AfUGM, two of with play an important role in tetramerization.Comparing the un-complexed, native AfUGM structure and the ligandbound AfUGM structure (both oxidized and reduced FAD) allowed us to address structural changes (loop movements) that play an important role in substrate binding and redox state.Structural binding studies on AfUGM revealed a unique substrate binding mechanism significantly different from proUGMs.This has helped us in identifying important conserved residues in AfUGM substrate binding and recognition.Based on the crystal structure we have made mutants of important active site residues.

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 he able to predict the likely fate and environmental risk of new synthetic compounds entering the environment and food chain.

The plant-specific phi class of glutathione transferases (GSTFs) are often highly stress-inducible and expressed in a tissue-specific manner, suggestive of them having important protective roles. To date, these functions remain largely unknown, although activities associated with the binding and transport of reactive metabolites have been proposed. Using a sensitive and selective binding screen, we have probed the Arabidopsis thaliana GSTFs for natural product ligands from bacteria and plants. Uniquely, when overexpressed in bacteria, family members GSTF2 and GSTF3 bound a series of heterocyclic compounds, including lumichrome, harmane, norharmane and indole-3-aldehyde. When screened against total metabolite extracts from A. thaliana, GSTF2 also selectively bound the indole-derived phytoalexin camalexin, as well as the flavonol quercetin-3-O-rhamnoside. In each case, isothermal titration calorimetry revealed high-affinity binding (typically Kd<1 μM), which was enhanced in the presence of glutathione and by the other heterocyclic ligands. With GSTF2, these secondary ligand associations resulted in an allosteric enhancement in glutathione-conjugating activity. Together with the known stress responsiveness of GSTF2 and its association with membrane vesicles, these results are suggestive of roles in regulating the binding and transport of defence-related compounds in planta.

Depletion of oil reserves and the associated effects on climate change have prompted a re-examination of the use of plant biomass as a sustainable source of organic carbon for the large-scale production of chemicals and materials. While initial emphasis has been placed on biofuel production from edible plant sugars, the drive to reduce the competition between crop usage for food and non-food applications has prompted massive research efforts to access the less digestible saccharides in cell walls (lignocellulosics). This in turn has prompted an examination of the use of other plant-derived metabolites for the production of chemicals spanning the high-value speciality sectors through to platform intermediates required for bulk production. The associated science of biorefining, whereby all plant biomass can be used efficiently to derive such chemicals, is now rapidly developing around the world. However, it is clear that the heterogeneity and distribution of organic carbon between valuable products and waste streams are suboptimal. As an alternative, we now propose the use of synthetic biology approaches to ‘re-construct’ plant feedstocks for optimal processing of biomass for non-food applications. Promising themes identified include re-engineering polysaccharides, deriving artificial organelles, and the reprogramming of plant signalling and secondary metabolism.