Repression of the Arabidopsis thaliana Jasmonic Acid/Ethylene-Induced Defense Pathway by TGA-Interacting Glutaredoxins Depends on Their C-Terminal ALWL Motif

Glutaredoxins are small heat-stable oxidoreductases that transfer electrons from glutathione （GSH） to oxi- dized cysteine residues, thereby contributing to protein integrity and regulation. In Arabidopsis thaliana, floral glutare- doxins ROXY1 and ROXY2 and pathogen-induced ROXY19/GRX480 interact with bZIP transcription factors of the TGACG （TGA） motif-binding family. ROXY1, ROXY2, and TGA factors PERIANTHIA, TGA9, and TGA10 play essential roles in floral development. In contrast, ectopically expressed ROXY19/GRX480 negatively regulates expression of jasmonic acid （JA）/ ethylene （ET）-induced defense genes through an unknown mechanism that requires clade II transcription factors TGA2, TGA5, and/or TGA6. Here, we report that at least 17 of the 21 land plant-specific glutaredoxins encoded in the Arabidopsis genome interact with TGA2 in a yeast-two-hybrid system. To investigate their capacity to interfere with the expression of JA/ET-induced genes, we developed a transient expression system. Activation of the ORA59 （OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF-domain protein 59） promoter by transcription factor EIN3 （ETHYLENE INSENSITVE 3） was sup- pressed by co-expressed ROXY19/GRX480. Suppression depended on the L＊＊LL motif in the C-terminus of ROXY19/ GRX480. This putative protein interaction domain was recently described as being essential for the TGA/ROXY interaction. Ten of the 17 tested ROXY proteins suppressed ORA59 promoter activity, which correlated with the presence of the C-terminal ALWL motif, which is essential for ROXY1 function in flower development. ROXY19/GRX480-mediated repres- sion depended on the GSH binding site, suggesting that redox modification of either TGA factors or as yet unknown target proteins is important for the suppression of ORA59 promoter activity.

Conserved Functions of Arabidopsis and Rice CC-Type Glutaredoxins in Flower Development and Pathogen Response

Glutaredoxins （GRXs） are ubiquitous oxidoreductases that play a crucial role in response to oxidative stress by reducing disulfides in various organisms. In planta, three different GRX classes have been identified according to their active site motifs. CPYC and CGFS classes are found in all organisms, whereas the CC-type class is specific for higher land plants. Recently, two Arabidopsis CC-type GRXs, ROXY1 and ROXY2, were shown to exert crucial functions in petal and anther initiation and differentiation. To analyze the function of CC-type GRXs in the distantly related monocots, we isolated and characterized OsROXY1 and OsROXY2-two rice homologs of ROXY1. Both genes are expressed in vegetative and reproductive stages. Although rice flower morphology is distinct from eudicots, OsROXY1/2 floral expression patterns are similar to their Arabidopsis counterparts ROXY1/2. Complementation experiments demonstrate that OsROXY1 and OsROXY2 can fully rescue the roxyl floral mutant phenotype. Overexpression of OsROXY1, OsROXY2, and ROXY1 in Arabidopsis causes similar vegetative and reproductive plant developmental defects. ROXY1 and its rice homologs thus exert a conserved function during eudicot and monocot flower development. Strikingly, overexpression of these CC-type GRXs also leads to an increased accumulation of hydrogen peroxide levels and hyper-susceptibility to infection from the necrotrophic pathogen Botrytis cinerea, revealing the importance of balanced redox processes in flower organ develop- ment and pathogen defence.

Glutathione regulates the transfer of iron-sulfur cluster from monothiol and dithiol glutaredoxins to apo ferredoxin

Holo glutaredoxin(Grx)is a homo-dimer that bridges a[2Fe-2S]cluster with two glutathione(GSH)ligands.In this study,both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur(FeS)cluster to an apo ferredoxin(Fdx)through direct interaction,regardless of FeS cluster stability in holo Grxs.The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH,which inhibits the FeS cluster transfer from holo Grxs to apo Fdx.This phenomenon suggests a novel role of GSH in FeS cluster trafficking.

Morphological alterations and redox changes associated with hepatic warm ischemia-reperfusion injury

AIM To study the effects of warm ischemia-reperfusion(I/R) injury on hepatic morphology at the ultrastructural level and to analyze the expression of the thioredoxin(TRX)and glutaredoxin(GRX) systems.METHODS Eleven patients undergoing liver resection were subjected to portal triad clamping(PTC). Liver biopsies were collected at three time points; first prior to PTC(baseline), 20 min after PTC(post-ischemia) and 20 min after reperfusion(post-reperfusion). Electron microscopy and morphometry were used to study and quantify ultrastructural changes, respectively. Additionally, gene expression analysis of TRX and GRX isoforms was performed by quantitative PCR. For further validation of redox protein status, immunogold staining was performed for the isoforms GRX1 and TRX1.RESULTS Post-ischemia, a significant loss of the liver sinusoidal endothelial cell(LSEC) lining was observed(P = 0.0003) accompanied by a decrease of hepatocyte microvilli in the space of Disse. Hepatocellular morphology was well preserved apart from the appearance of crystalline mitochondrial inclusions in 7 out of 11 patients. Postreperfusion biopsies had similar features as post-ischemia with the exception of signs of a reactivation of the LSECs. No changes in the expression of redox-regulatory genes could be observed at mR NA level of the isoforms of the TRX family but immunoelectron microscopy indicated a redistribution of TRX1 within the cell.CONCLUSION At the ultrastructural level, the major impact of hepatic warm I/R injury after PTC was borne by the LSECs with detachment and reactivation at ischemia and reperfusion, respectively. Hepatocytes morphology were well preserved. Crystalline inclusions in mitochondria were observed in the hepatocyte after ischemia.

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.

Selenium and the Thioredoxin and Glutaredoxin Systems

Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FADcontaining enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a Km-value (6μmol·L-1 ) and a high turnover number (kcat, 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the hut-nan plasrna glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furtherrnore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SEClS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of seleflium for cell proliferation

PICOT promotes T lymphocyte proliferation by down-regulating cyclin D2 expression

The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes.PICOT is required for normal and transformed cell growth and is critical for embryonic development.Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development,a polycomb group protein and a core component of the polycomb repressive complex 2,which contributes to the maintenance of transcriptional repression and chromatin remodeling.Furthermore,PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes.PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2,a cell cycle-regulating gene which encodes the cyclin D2 protein.As a result,the expression levels of CCND2 mRNA and protein levels were reduced,concomitantly with inhibition of the cell growth rate.Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers.In addition,this parameter correlated with poor patient survival,suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.

Monothiol Glutaredoxin-BolA Interactions： Redox Control of Arabidopsis thaliana BolA2 and SufE1

A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluo- rescent protein （GFP） fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis 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 SufE 1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments 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 desuifurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionyiated 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 subcellular 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.

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions,and oxidative damage has been implicated in several neurodegenerative diseases.End-binding protein 1(EB1)is a master regulator of microtubule plus-end tracking proteins(+TIPs)and is critically involved in the control of microtubule dynamics and cellular processes.However,the impact of glutathionylation on EB1 functions remains unknown.Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation.In vitro biochemical and cellular assays reveal that EB1 is glutathionylated.Diamide,a mild oxidizing reagent,reduces EB1 comet number and length in cells,indicating the impairment of microtubule dynamics.Three cysteine residues of EB1 are glutathionylated,with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics.In addition,glutaredoxin 1(Grx1)deglutathionylates EB1,and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration,suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively,these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.

Molecular characterization of glutaredoxin 2 from Ostrinia furnacalis

Glutaredoxins(GRXs)play very important roles in maintaining intracellular redox homeostasis.In the present study,the full-length cDNA sequence encoding GRX2,named OfurGRX2(GenBank accession no.GU393246),was obtained from Ostrinia furnacalis,using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends.Sequence analysis revealed that the open reading frame of OfurGRX2 consists of 351 nucleotides encoding 116 amino acid residues with a predicted molecular weight of 12.6 kDa.Homolog research revealed that OfurGRX2 shares a common active site,CPYC/CPFC,with other insect counterparts.Expression profiles revealed that OfurGRX2 is a ubiquitous gene expressed in insect heads,fat bodies,epidermises,mid guts and muscles.The OfurGRX2 transcript peaked in 36-h larvae of 4th instars,and then suddenly declined in the molting stage.Hormone treatment experiments revealed that 20-hydroxyecodyson(20e)significantly induces the expression of the OfurGRX2 transcript,whereas juvenile hormone(JH)counteracts 20e effects.Adverse stress factors(including starvation,ultraviolet light,mechanical injury,Escherichia coli exposure,and high and low temperatures)dramatically induced OfurGRXGRX2 transcript expression,which confirmed for the first time that GRX2 play important roles in insecta during exposure to adverse environments.