Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings.

I Aller, N Rouhier, A Meyer. Frontiers in Plant Science.

Abstract

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (EGSH) of more negative than -310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase. Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signalling mechanisms it is essential to be able to measure EGSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between -280 and -290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of EGSH values that can be measured in vivo with roGFP2-based probes from about -320 mV for GRX1-roGFRP2 down to about -210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an EGSH of about -260 mV in this mutant.

The genome of an arbuscular mycorrhizal fungus provides insights into the oldest plant symbiosis. E Tisserant, M Malbreil, A Kuo, A Kohler, et al. Proceedings of the National Academy of Sciences

Significance

The arbuscular mycorrhizal symbiosis between fungi of the Glomeromycota phylum and plants involves more than two-thirds of all known plant species, including important crop species. This mutualistic symbiosis, involving one of the oldest fungal lineages, is arguably the most ecologically and agriculturally important symbiosis in terrestrial ecosystems. The Glomeromycota are unique in that their spores and coenocytic hyphae contain hundreds of nuclei in a common cytoplasm, which raises important questions about the natural selection, population genetics, and gene expression of these highly unusual organisms. Study of the genome of Rhizophagus irregularis provides insight into genes involved in obligate biotrophy and mycorrhizal symbioses and the evolution of an ancient asexual organism, and thus is of fundamental importance to the field of genome evolution.

Abstract

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularisgenes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Structure and function of bacterial communities in ageing soils: Insights from the Mendocino ecological staircase
S Uroz, JJ Tech, NA Sawaya, P Frey-Klett, JHJ Leveau
Soil Biology and Biochemistry

Highlights

• Soil and microbial characteristic were determined in the Mendocino site.
• Each terrace is derived from the same mineralogical parent material but differ in age.
• Soil and microbiological approaches were performed on the first three terraces.
• Bacterial communities appeared significantly different along the chronosequence.
• Nutrient availability affects the diversity and function of the bacterial communities.

Abstract

Diversification of Fungal Specific Class A Glutathione Transferases in Saprotrophic Fungi Yann Mathieu, Pascalita Prosper, Frédérique Favier, Luc Harvengt, Claude Didierjean, Jean-Pierre Jacquot, Mélanie Morel-Rouhier, Eric Gelhaye

Abstract

Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms fromPhanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry.

Density mapping of decaying wood using X-ray computed tomography. V Hervé, F Mothe, C Freyburger, E Gelhaye and P. Frey-Klett. International Biodeterioration and Biodegradation

Abstract

Evolution of a symbiotic receptor through gene duplications in the legume–rhizobium mutualism. De Mita S., A. Streng, T. Bisseling and R. Geurts. New phytologist

Abstract:

• The symbiosis between legumes and nitrogen-fixing rhizobia co-opted pre-existing endomycorrhizal features. In particular, both symbionts release lipo-chitooligosaccharides (LCOs) that are recognized by LysM-type receptor kinases. We investigated the evolutionary history of rhizobial LCO receptor genes MtLYK3–LjNFR1 to gain insight into the evolutionary origin of the rhizobial symbiosis.
• We performed a phylogenetic analysis integrating gene copies from nonlegumes and legumes, including the non-nodulating, phylogenetically basal legume Cercis chinensis. Signatures of differentiation between copies were investigated through patterns of molecular evolution.
• We show that two rounds of duplication preceded the evolution of the rhizobial symbiosis in legumes. Molecular evolution patterns indicate that the resulting three paralogous gene copies experienced different selective constraints. In particular, one copy maintained the ancestral function, and another specialized into perception of rhizobial LCOs. It has been suggested that legume LCO receptors evolved from a putative ancestral defense-related chitin receptor through the acquisition of two kinase motifs. However, the phylogenetic analysis shows that these domains are actually ancestral, suggesting that this scenario is unlikely.
• Our study underlines the evolutionary significance of gene duplication and subsequent neofunctionalization in MtLYK3-LjNFR1genes. We hypothesize that their ancestor was more likely a mycorrhizal LCO receptor, than a defense-related receptor kinase.

Monothiol glutaredoxin-BolA interactions: redox control of Arabidopsis thaliana BolA2 and SufE1

J Couturier, HC Wu, T Dhalleine, H Pégeot, D Sudre… – Molecular Plant, 2013.

Abstract

A functional relationship between monothiol glutaredoxins and BolA has been unravelled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of GFP fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist inArabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic and BolA4 dual-targeted to mitochondria and plastids. Binary yeast-two hybrid experiments demonstrated that all BolAs and SufE1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same sub-cellular compartment have been confirmed by bimolecular fluorescence complementation. In vitroexperiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desulfurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionylated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several sub-cellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.

Two papers involving IAM members have been accepted in Frontiers in Plant Sciences :

Biochemical and catalytic properties of classical dithiol plant glutaredoxins. J Couturier, JP Jacquot, N Rouhier.  Frontiers in Plant Science

Abstract: Glutaredoxins (Grxs) are small oxidoreductases particularly specialized in the reduction of protein-glutathione adducts. Compared to other eukaryotic organisms, higher plants present an increased diversity of Grxs which can be classified into four classes. This work aimed at comparing the biochemical properties of dithiol class I Grxs, namely GrxC1, GrxC2, GrxC3 and GrxC4, in order to understand the existence of several isoforms presenting such a high sequence identity. In vitro measurements of oxidoreductase activity using the physiological glutathione reducing system and classical Grx substrates revealed that they display significantly different kinetic parameters which could be partly linked to differences in the redox potential and in the pKa value of the catalytic cysteine. In addition, gel filtration experiments coupled with site-directed mutagenesis indicated that GrxC1 and GrxC2, but not GrxC3 and GrxC4, can form covalent homodimers owing to the presence of a conserved C-terminal cysteine. Finally, the role of active site cysteine residues and of this C-terminal cysteine for the catalytic mechanism was deciphered by investigating the sensitivity of each isoform to several oxidants such as hydrogen peroxide, oxidized glutathione and nitrosoglutathione and by identifying the resulting oxidized forms by mass spectrometry.

Characterisation of a roGFP2-derived fluorescent probe for direct measurements of the glutathione redox potential in oxidising cellular compartments. I Aller, N Rouhier, A Meyer. Frontiers in Plant Science

Abstract: Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the production of reactive oxygen species that need to be detoxified to prevent severe damages of cellular structures. This detoxification is at least partially based on glutathione as the major low molecular weight thiol redox buffer in the ER. Due to the lack of efficient reducing systems, however, the luminal glutathione pool is far more oxidised than in other compartments. The oxidising redox potential in the lumen supports protein folding whereas deviations from steady-state redox conditions induce an unfolded protein response. Vice versa, malfunction in oxidative protein folding is assumed to affect the redox potential of the glutathione pool. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the oxidative protein folding became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have a rather negative midpoint potential rendering them fully oxidised in the ER. Even less negative roGFP1-iX probes still appear fully oxidised in the ER lumen at steady-state, which restricts their applicability for dynamic measurements. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -235 mV similar to the roGFP1-iX probes, but different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterised in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements in the oxidising ER lumen and in the cytosol of severe GSH-deficient mutants where the original roGFP2 probes are completely oxidised.

Transcriptome analysis of poplar rust telia reveals overwintering adaptation and tightly coordinated karyogamy and meiosis processes.
S Hacquart, C Delaruelle, P Frey, E Tisserant, A Kohler, S Duplessis
Frontiers in Plant Science

Abstract:

Most rust fungi have a complex life cycle involving up to five different spore-producing stages. The telial stage that produces melanised overwintering teliospores is one of these and plays a fundamental role for generating genetic diversity as karyogamy and meiosis occur at that stage. Despite the importance of telia for the rust life cycle, almost nothing is known about the fungal genetic programs that are activated in this overwintering structure. In the present study, the transcriptome of telia produced by the poplar rust fungus M. larici-populina has been investigated using whole genome exon oligoarrays and RT-qPCR. Comparative expression profiling at the telial and uredinial stages identifies genes specifically expressed or up-regulated in telia including osmotins/thaumatin-like proteins and aquaporins that may reflect specific adaptation to overwintering as well numerous lytic enzymes acting on plant cell wall, reflecting extensive cell wall remodelling at that stage. The temporal dynamics of karyogamy was followed using combined RT-qPCR and DAPI-staining approaches. This reveals that fusion of nuclei and induction of karyogamy-related genes occur simultaneously between the 25-39 days post inoculation time frame. Transcript profiling of conserved meiosis genes indicate a preferential induction right after karyogamy and corroborate that meiosis begins prior to overwintering and is interrupted in Meiosis I (prophase I, diplonema stage) until teliospore germination in early spring.

Keywords: Melampsora larici-populina, obligate biotrophic fungus, rust lifecycle, teliospores, Gene Expression, Microarray

Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (< i> Hordeum vulgare</i> cv. Nure) roots
M Cardi, K Chibani, D Castiglia, D Cafasso, E Pizzo, N Rouhier, JP Jacquot …
Plant Physiology and Biochemistry

Abstract

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms.

The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction.

The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH.

This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.

Keywords

• OPPP;
• G6PDH;
• Barley roots;
• Plastidial isoform