MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE-TO-FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination-based mapping and whole-genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC-MS and polyacrylamide gel electrophoresis to measure cell-wall fucose and boron (B)-dependent dimerization of the cell-wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate-bridging of RGII, we assessed freeze-induced damage in wild-type and sfr8 plants by measuring electrolyte leakage from freeze-thawed leaf discs. We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP-d-mannose-4,6-dehydratase. sfr8 cell walls exhibited low cell-wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing-sensitive. Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.

Rational Development of Novel Activity Probes for the Analysis of Human Cytochromes P450.

The identification and quantification of functional cytochromes P450 (CYPs) in biological samples is proving important for robust analyses of drug efficacy and metabolic disposition. In this study, a novel CYP activity-based probe was rationally designed and synthesised, demonstrating selective binding of CYP isoforms. The dependence of probe binding upon the presence of NADPH permits the selective detection of functionally active CYP. This allows the detection and analysis of these enzymes using biochemical and proteomic methodologies and approaches.

5'-deoxy-5'-hydrazinylguanosine as an initiator of T7 Rna polymerase-catalyzed transcriptions for the preparation of labeling-ready RNAs.

5'-deoxy-5'-hydrazinylguanosine was incorporated into the 5'-termini of RNA transcripts using T7 RNA polymerase. Transcriptions provided 5'-hydrazinyl-RNA that was readily labeled and purified. The use of fluorophore-labeled material was validated in an endoribonuclease activity assay.

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.

Multiple roles for plant glutathione transferases in xenobiotic detoxification.

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.

Roles for glutathione transferases in plant secondary metabolism.

Plant glutathione transferases (GSTs) are classified as enzymes of secondary metabolism, but while their roles in catalysing the conjugation and detoxification of herbicides are well known, their endogenous functions are largely obscure. Thus, while the presence of GST-derived S-glutathionylated xenobiotics have been described in many plants, there is little direct evidence for the accumulation of similarly conjugated natural products, despite the presence of a complex and dichotomous metabolic pathway which processes these reaction products. The conservation in glutathione conjugating and processing pathways, the co-regulation of GSTs with inducible plant secondary metabolism and biochemical studies showing the potential of these enzymes to conjugate reactive natural products are all suggestive of important endogenous functions. As a framework for addressing these enigmatic functions we postulate that either: (a) the natural reaction products of GSTs are unstable and undergo reversible S-glutathionylation; (b) the conjugation products of GSTs are very rapidly processed to derived metabolites; (c) GSTs do not catalyse conventional conjugation reactions but instead use glutathione as a cofactor rather than co-substrate; or (d) GSTs are non-catalytic and function as transporter proteins for secondary metabolites and their unstable intermediates. In this review, we describe how enzyme biochemistry and informatics are providing clues as to GST function allowing for the critical evaluation of each of these hypotheses. We also present evidence for the involvement of GSTs in the synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates, and the conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids.

Stress-induced protein S-glutathionylation in Arabidopsis.

S-Glutathionylation (thiolation) is a ubiquitous redox-sensitive and reversible modification of protein cysteinyl residues that can directly regulate their activity. While well established in animals, little is known about the formation and function of these mixed disulfides in plants. After labeling the intracellular glutathione pool with [35S]cysteine, suspension cultures of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labeling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation as well as proteins complexed with thiolated polypeptides. The mechanism of thiolation of five proteins, dehydroascorbate reductase (AtDHAR1), zeta-class glutathione transferase (AtGSTZ1), nitrilase (AtNit1), alcohol dehydrogenase (AtADH1), and methionine synthase (AtMetS), was studied using the respective purified recombinant proteins. AtDHAR1, AtGSTZ1, and to a lesser degree AtNit1 underwent spontaneous thiolation with GSSG-biotin through modification of active-site cysteines. The thiolation of AtADH1 and AtMetS required the presence of unidentified Arabidopsis proteins, with this activity being inhibited by S-modifying agents. The potential role of thiolation in regulating metabolism in Arabidopsis is discussed and compared with other known redox regulatory systems operating in plants.

Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays).

Unlike animals which accumulate glutathione (gamma-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or gamma-glutamyl-L-cysteinyl-beta-alanine)], wheat (hydroxymethylglutathione or gamma-glutamyl-L-cysteinyl-serine) and maize (gamma-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into gamma-Glu-Cys. The soya bean enzyme primarily incorporated beta-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and alpha-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor.

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.

Manipulation of plant tolerance to herbicides through co-ordinated metabolic engineering of a detoxifying glutathione transferase and thiol cosubstrate.

The diphenyl ether herbicide fomesafen can be used selectively in soybean (Glycine max) due to its rapid detoxification by tau class glutathione transferases (GmGSTUs) which preferentially utilize the endogenous thiol homoglutathione (hGSH) as cosubstrate. Soybean cDNAs encoding GmGSTU21, which is highly active in detoxifying fomesafen, and an hGSH synthetase (GmhGS) have been cloned and functionally identified in Escherichia coli. Tobacco plants, which have limited GST activities towards fomesafen and which accumulate glutathione (GSH), rather than hGSH, have been transformed with either GmhGS alone, or a dual construct of GmhGS-GmGSTU21, both under the control of constitutive promoters. Using either construct, the transgenic tobacco accumulated hGSH, with a concomitant increase in GSH content. Segregating T1 plants were analysed for thiol content and GST activity towards fomesafen with GSH and hGSH as cosubstrates, and then scored for photobleaching injury caused by applications of fomesafen. These studies showed that hGSH accumulation alone gave no significant protection against the herbicide and that tolerance was only seen in plants which contained appreciable concentrations of hGSH and GmGSTU21 activity. Tolerance in the dual transformants was associated with the metabolism of radiolabelled fomesafen to inactive hGSH-derived conjugates, while susceptible lines were unable to detoxify the herbicide. These studies confirm the combined importance of specific GSTs and their preferred thiol cosubstrates in conferring herbicide selectivity traits in planta.