Biochemical and Structural Characterization of Chi-Class Glutathione Transferases: A Snapshot on the Glutathione Transferase Encoded by sll0067 Gene in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in detoxification processes and/or in specialized metabolism. In the cyanobacterium Synechocsytis sp. PCC 6803, SynGSTC1, a chi-class GST (GSTC), is thought to participate in the detoxification process of methylglyoxal, a toxic by-product of cellular metabolism. A comparative genomic analysis showed that GSTCs were present in all orders of cyanobacteria with the exception of the basal order Gloeobacterales. These enzymes were also detected in some marine and freshwater noncyanobacterial bacteria, probably as a result of horizontal gene transfer events. GSTCs were shorter of about 30 residues compared to most cytosolic GSTs and had a well-conserved SRAS motif in the active site (10SRAS13 in SynGSTC1). The crystal structure of SynGSTC1 in complex with glutathione adopted the canonical GST fold with a very open active site because the α4 and α5 helices were exceptionally short. A transferred multipolar electron-density analysis allowed a fine description of the solved structure. Unexpectedly, Ser10 did not have an electrostatic influence on glutathione as usually observed in serinyl-GSTs. The S10A variant was only slightly less efficient than the wild-type and molecular dynamics simulations suggested that S10 was a stabilizer of the protein backbone rather than an anchor site for glutathione.

Biochemical and Structural Insights on the Poplar Tau Glutathione Transferase GSTU19 and 20 Paralogs Binding Flavonoids.

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in xenobiotic detoxification and/or in specialized metabolism. Populus trichocarpa genome (V4.1 assembly, Phytozome 13) consists of 74 genes coding for full-length GSTs and ten likely pseudogenes. These GSTs are divided into 11 classes, in which the tau class (GSTU) is the most abundant with 54 isoforms. PtGSTU19 and 20, two paralogs sharing more than 91% sequence identity (95% of sequence similarity), would have diverged from a common ancestor of P. trichocarpa and P. yatungensis species. These enzymes display the distinctive glutathione (GSH)-conjugation and peroxidase activities against model substrates. The resolution of the crystal structures of these proteins revealed significant structural differences despite their high sequence identity. PtGSTU20 has a well-defined deep pocket in the active site whereas the bottom of this pocket is disordered in PtGSTU19. In a screen of potential ligands, we were able to identify an interaction with flavonoids. Some of them, previously identified in poplar (chrysin, galangin, and pinocembrin), inhibited GSH-conjugation activity of both enzymes with a more pronounced effect on PtGSTU20. The crystal structures of PtGSTU20 complexed with these molecules provide evidence for their potential involvement in flavonoid transport in P. trichocarpa.

Expression Patterns of Drosophila Melanogaster Glutathione Transferases.

Glutathione transferases (GSTs) are ubiquitous enzymes that catalyze the conjugation of glutathione to various molecules. Among the 42 GSTs identified in Drosophila melanogaster, Delta and Epsilon are the largest classes, with 25 members. The Delta and Epsilon classes are involved in different functions, such as insecticide resistance and ecdysone biosynthesis. The insect GST number variability is due mainly to these classes. Thus, they are generally considered supports during the evolution for the adaptability of the insect species. To explore the link between Delta and Epsilon GST and their evolution, we analyzed the sequences using bioinformatic tools. Subgroups appear within the Delta and Epsilon GSTs with different levels of diversification. The diversification also appears in the sequences showing differences in the active site. Additionally, amino acids essential for structural stability or dimerization appear conserved in all GSTs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the transcripts corresponding to these two classes are heterogeneously expressed within D. melanogaster. Some GSTs, such as GSTD1, are highly expressed in all tissues, suggesting their general function in detoxification. Conversely, some others, such as GSTD11 or GSTE4, are specifically expressed at a high level specifically in antennae, suggesting a potential role in olfaction.

Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.

Methylglyoxal Detoxification Revisited: Role of Glutathione Transferase in Model Cyanobacterium Synechocystis sp. Strain PCC 6803.

Methylglyoxal (MG) is a detrimental metabolic by-product that threatens most organisms (in humans MG causes diabetes). MG is predominantly detoxified by the glyoxalase pathway. This process begins with the conjugation of MG with glutathione (GSH), yielding a hemithioacetal product that is subsequently transformed by the glyoxalase enzymes into d-lactate and GSH. MG has been overlooked in photosynthetic organisms, although they inevitably produce it not only by the catabolism of sugars, lipids, and amino acids, as do heterotrophic organisms, but also by their active photoautotrophic metabolism. This is especially true for cyanobacteria that are regarded as having developed photosynthesis and GSH-dependent enzymes to detoxify the reactive oxygen species produced by their photosynthesis (CO2 assimilation) and respiration (glucose catabolism), which they perform in the same cell compartment. In this study, we used a combination of in vivo and in vitro approaches to characterize a logical, but as yet never described, link between MG detoxification and a (prokaryotic) representative of the evolutionarily conserved glutathione transferase (GST) detoxification enzymes. We show that the Sll0067 GST of the model cyanobacterium Synechocystis sp. strain PCC 6803 plays a prominent role in MG tolerance and detoxification, unlike the other five GSTs of this organism. Sll0067 catalyzes the conjugation of MG with GSH to initiate its elimination driven by glyoxalases. These results are novel because the conjugation of MG with GSH is always described as nonenzymatic. They will certainly stimulate the analysis of Sll0067 orthologs from other organisms with possible impacts on human health (development of biomarkers or drugs) and/or agriculture.IMPORTANCE In most organisms, methylglyoxal (MG), a toxic metabolite by-product that causes diabetes in humans, is predominantly detoxified by the glyoxalase enzymes. This process begins with the so-called "spontaneous" conjugation of MG with the cytoprotectant metabolite glutathione (GSH). In this study, we unravel a logical, but as yet unsuspected, link between MG detoxification and a (prokaryotic) representative of the ubiquitous glutathione transferase (GST) enzymes. We show that a GST of a model cyanobacterium plays a prominent role in the detoxification of MG in catalyzing its conjugation with GSH. This finding is important because this reaction, always regarded as nonenzymatic, could exist in plants and/or human and thus have an impact on agriculture and/or human health.

Is there a role for tau glutathione transferases in tetrapyrrole metabolism and retrograde signalling in plants?

In plants, tetrapyrrole biosynthesis occurs in chloroplasts, the reactions being catalysed by stromal and membrane-bound enzymes. The tetrapyrrole moiety is a backbone for chlorophylls and cofactors such as sirohaems, haems and phytochromobilins. Owing to this diversity, the potential cytotoxicity of some precursors and the associated synthesis costs, a tight control exists to adjust the demand and the fluxes for each molecule. After synthesis, haems and phytochromobilins are incorporated into proteins found in other subcellular compartments. However, there is only very limited information about the chaperones and membrane transporters involved in the trafficking of these molecules. After summarizing evidence indicating that glutathione transferases (GST) may be part of the transport and/or degradation processes of porphyrin derivatives, we provide experimental data indicating that tau glutathione transferases (GSTU) bind protoporphyrin IX and haem moieties and use structural modelling to identify possible residues responsible for their binding in the active site hydrophobic pocket. Finally, we discuss the possible roles associated with the binding, catalytic transformation (i.e. glutathione conjugation) and/or transport of tetrapyrroles by GSTUs, considering their subcellular localization and capacity to interact with ABC transporters. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds.

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

Laccaria bicolor MiSSP8 is a small-secreted protein decisive for the establishment of the ectomycorrhizal symbiosis.

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small-Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

Functional, Structural and Biochemical Features of Plant Serinyl-Glutathione Transferases.

Glutathione transferases (GSTs) belong to a ubiquitous multigenic family of enzymes involved in diverse biological processes including xenobiotic detoxification and secondary metabolism. A canonical GST is formed by two domains, the N-terminal one adopting a thioredoxin (TRX) fold and the C-terminal one an all-helical structure. The most recent genomic and phylogenetic analysis based on this domain organization allowed the classification of the GST family into 14 classes in terrestrial plants. These GSTs are further distinguished based on the presence of the ancestral cysteine (Cys-GSTs) present in TRX family proteins or on its substitution by a serine (Ser-GSTs). Cys-GSTs catalyze the reduction of dehydroascorbate and deglutathionylation reactions whereas Ser-GSTs catalyze glutathione conjugation reactions and eventually have peroxidase activity, both activities being important for stress tolerance or herbicide detoxification. Through non-catalytic, so-called ligandin properties, numerous plant GSTs also participate in the binding and transport of small heterocyclic ligands such as flavonoids including anthocyanins, and polyphenols. So far, this function has likely been underestimated compared to the other documented roles of GSTs. In this review, we compiled data concerning the known enzymatic and structural properties as well as the biochemical and physiological functions associated to plant GSTs having a conserved serine in their active site.

Advances in understanding obligate biotrophy in rust fungi.

Contents Summary 1190 I. Introduction 1190 II. Rust fungi: a diverse and serious threat to agriculture 1191 III. The different facets of rust life cycles and unresolved questions about their evolution 1191 IV. The biology of rust infection 1192 V. Rusts in the genomics era: the ever-expanding list of candidate effector genes 1195 VI. Functional characterization of rust effectors 1197 VII. Putting rusts to sleep: Pucciniales research outlooks 1201 Acknowledgements 1202 References 1202 SUMMARY: Rust fungi (Pucciniales) are the largest group of plant pathogens and represent one of the most devastating threats to agricultural crops worldwide. Despite the economic importance of these highly specialized pathogens, many aspects of their biology remain obscure, largely because rust fungi are obligate biotrophs. The rise of genomics and advances in high-throughput sequencing technology have presented new options for identifying candidate effector genes involved in pathogenicity mechanisms of rust fungi. Transcriptome analysis and integrated bioinformatics tools have led to the identification of key genetic determinants of host susceptibility to infection by rusts. Thousands of genes encoding secreted proteins highly expressed during host infection have been reported for different rust species, which represents significant potential towards understanding rust effector function. Recent high-throughput in planta expression screen approaches (effectoromics) have pushed the field ahead even further towards predicting high-priority effectors and identifying avirulence genes. These new insights into rust effector biology promise to inform future research and spur the development of effective and sustainable strategies for managing rust diseases.

Oak genome reveals facets of long lifespan.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

The Rust Fungus Melampsora larici-populina Expresses a Conserved Genetic Program and Distinct Sets of Secreted Protein Genes During Infection of Its Two Host Plants, Larch and Poplar.

Mechanisms required for broad-spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici-populina infects two taxonomically unrelated plants, larch, on which sexual reproduction is achieved, and poplar, on which clonal multiplication occurs, leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia, and aecia, and we performed comparative transcriptomics of infection on poplar and larch hosts, using available expression data. Secreted protein was the only significantly overrepresented category among differentially expressed M. larici-populina genes between the basidial, the pycnial, and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes were commonly expressed on the two hosts and a fraction exhibited host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Structural plasticity among glutathione transferase Phi members: natural combination of catalytic residues confers dual biochemical activities.

The glutathione transferase (GST) gene family is divided into 14 classes in photosynthetic organisms. Among them, the Phi class (GSTF) is composed of a large number of genes that are often induced in response to environmental constraints due to their ability to detoxify xenobiotics, to their peroxidase activity and to their involvement in the biosynthesis and/or transport of secondary metabolites. However, the exact functions of GSTFs from many plants including Populus trichocarpa are unknown. Here, following GSTF1 characterization, we have performed a comparative analysis of the seven other GSTFs found in poplar by systematically evaluating the biochemical and enzymatic properties of the corresponding recombinant proteins and of variants mutated for active site residues and by determining the three-dimensional structures of several representatives. Owing to the presence of a cysteine with a pKa value around 5 in their active site, GSTF3, F7, and F8 displayed a thiol transferase activity in addition to the usual glutathione transferase and peroxidase activities. From structural analyses, it appeared that these dual biochemical properties originate from the existence of a certain variability in the ß1-α1 loop. This allows positioning of several active site residues at proximity of the glutathione molecule, which itself remains unchanged in GSTF three-dimensional structures. These results highlight the promiscuity of some GSTFs and that changes of active site residues in some isoforms during evolution generated functional diversity by modifying their activity profile. DATABASE: Structural data are available in the PDB under the accession numbers 5EY6, 5F05, 5F06, and 5F07.

Dithiol disulphide exchange in redox regulation of chloroplast enzymes in response to evolutionary and structural constraints.

Redox regulation of chloroplast enzymes via disulphide reduction is believed to control the rates of CO2 fixation. The study of the thioredoxin reduction pathways and of various target enzymes lead to the following guidelines.

Crystal Structure of Saccharomyces cerevisiae ECM4, a Xi-Class Glutathione Transferase that Reacts with Glutathionyl-(hydro)quinones.

Glutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosynthesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging. The work here reports its crystallographic study. In addition to exhibiting the general GSTX structural features, ScECM4 shows extensions including a huge loop which contributes to the quaternary assembly. These structural extensions are probably specific to Saccharomycetaceae. Soaking of ScECM4 crystals with GS-menadione results in a structure where glutathione forms a mixed disulfide bond with the cysteine 46. Solution studies confirm that ScECM4 has reductase activity for GS-menadione in presence of glutathione. Moreover, the high resolution structures allowed us to propose new roles of conserved residues of the active site to assist the cysteine 46 during the catalytic act.

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories.

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

The Poplar Rust-Induced Secreted Protein (RISP) Inhibits the Growth of the Leaf Rust Pathogen Melampsora larici-populina and Triggers Cell Culture Alkalinisation.

Plant cells secrete a wide range of proteins in extracellular spaces in response to pathogen attack. The poplar rust-induced secreted protein (RISP) is a small cationic protein of unknown function that was identified as the most induced gene in poplar leaves during immune responses to the leaf rust pathogen Melampsora larici-populina, an obligate biotrophic parasite. Here, we combined in planta and in vitro molecular biology approaches to tackle the function of RISP. Using a RISP-mCherry fusion transiently expressed in Nicotiana benthamiana leaves, we demonstrated that RISP is secreted into the apoplast. A recombinant RISP specifically binds to M. larici-populina urediniospores and inhibits their germination. It also arrests the growth of the fungus in vitro and on poplar leaves. Interestingly, RISP also triggers poplar cell culture alkalinisation and is cleaved at the C-terminus by a plant-encoded mechanism. Altogether our results indicate that RISP is an antifungal protein that has the ability to trigger cellular responses.

Insights into ascorbate regeneration in plants: investigating the redox and structural properties of dehydroascorbate reductases from Populus trichocarpa.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs.

Effector-Mining in the Poplar Rust Fungus Melampsora larici-populina Secretome.

The poplar leaf rust fungus, Melampsora larici-populina has been established as a tree-microbe interaction model. Understanding the molecular mechanisms controlling infection by pathogens appears essential for durable management of tree plantations. In biotrophic plant-parasites, effectors are known to condition host cell colonization. Thus, investigation of candidate secreted effector proteins (CSEPs) is a major goal in the poplar-poplar rust interaction. Unlike oomycetes, fungal effectors do not share conserved motifs and candidate prediction relies on a set of a priori criteria established from reported bona fide effectors. Secretome prediction, genome-wide analysis of gene families and transcriptomics of M. larici-populina have led to catalogs of more than a thousand secreted proteins. Automatized effector-mining pipelines hold great promise for rapid and systematic identification and prioritization of CSEPs for functional characterization. In this review, we report on and discuss the current status of the poplar rust fungus secretome and prediction of candidate effectors from this species.