Whole Transcriptome Analyses of Apricots and Japanese Plum Fruits after 1-MCP (Ethylene-Inhibitor) and Ethrel (Ethylene-Precursor) Treatments Reveal New Insights into the Physiology of the Ripening Process.

The physiology of Prunus fruit ripening is a complex and not completely understood process. To improve this knowledge, postharvest behavior during the shelf-life period at the transcriptomic level has been studied using high-throughput sequencing analysis (RNA-Seq). Monitoring of fruits has been analyzed after different ethylene regulator treatments, including 1-MCP (ethylene-inhibitor) and Ethrel (ethylene-precursor) in two contrasting selected apricot (Prunus armeniaca L.) and Japanese plum (P. salicina L.) cultivars, 'Goldrich' and 'Santa Rosa'. KEEG and protein-protein interaction network analysis unveiled that the most significant metabolic pathways involved in the ripening process were photosynthesis and plant hormone signal transduction. In addition, previously discovered genes linked to fruit ripening, such as pectinesterase or auxin-responsive protein, have been confirmed as the main genes involved in this process. Genes encoding pectinesterase in the pentose and glucuronate interconversions pathway were the most overexpressed in both species, being upregulated by Ethrel. On the other hand, auxin-responsive protein IAA and aquaporin PIP were both upregulated by 1-MCP in 'Goldrich' and 'Santa Rosa', respectively. Results also showed the upregulation of chitinase and glutaredoxin 3 after Ethrel treatment in 'Goldrich' and 'Santa Rosa', respectively, while photosystem I subunit V psaG (photosynthesis) was upregulated after 1-MCP in both species. Furthermore, the overexpression of genes encoding GDP-L-galactose and ferredoxin in the ascorbate and aldarate metabolism and photosynthesis pathways caused by 1-MCP favored antioxidant activity and therefore slowed down the fruit senescence process.

Intrinsically Disordered Region Modulates Ligand Binding in Glutaredoxin 1 from Trypanosoma Brucei.

Glutaredoxins are small proteins that share a common well-conserved thioredoxin-fold and participate in a wide variety of biological processes. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (Fe-S) metabolism. In the present work, we report different structural and dynamics aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei that differentiate it from other orthologues by the presence of a parasite-specific unstructured N-terminal extension whose role has not been fully elucidated yet. Previous nuclear magnetic resonance (NMR) studies revealed significant differences with respect to the mutant lacking the disordered tail. Herein, we have performed atomistic molecular dynamics simulations that, complementary to NMR studies, confirm the intrinsically disordered nature of the N-terminal extension. Moreover, we confirm the main role of these residues in modulating the conformational dynamics of the glutathione-binding pocket. We observe that the N-terminal extension modifies the ligand cavity stiffening it by specific interactions that ultimately modulate its intrinsic flexibility, which may modify its role in the storage and/or transfer of preformed iron-sulfur clusters. These unique structural and dynamics aspects of Trypanosoma brucei 1CGrx1 differentiate it from other orthologues and could have functional relevance. In this way, our results encourage the study of other similar protein folding families with intrinsically disordered regions whose functional roles are still unrevealed and the screening of potential 1CGrx1 inhibitors as antitrypanosomal drug candidates.

Ginsenoside F2 induces cellular toxicity to glioblastoma through the impairment of mitochondrial function.

BACKGROUND: Glioblastoma (GBM) is the most aggressive tumor residing within the central nervous system, with extremely poor prognosis. Although the cytotoxic effects of ginsenoside F2 (GF2) on GBM were previously suggested, the precise anti-GBM mechanism of GF2 remains unclear. The aim of this study was to explore the anti-cancer molecular mechanism of GF2 toward human GBM. METHODS: GF2-driven cellular toxicity was confirmed in two different GBM cells, U373 and Hs683. To test mitochondrial impairment driven by GF2, we examined the mitochondrial membrane potential, OCR, and ATP production. An intracellular redox imbalance was identified by measuring the relative ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), glutaredoxin (GLRX) mRNA expression, intracellular NAD+ level, and AMPK phosphorylation status. RESULTS: GF2 increased the percentage of cleaved caspase 3-positive cells and γH2AX signal intensities, confirming that GF2 shows the cytotoxicity against GBM. GO enrichment analysis suggested that the mitochondrial function could be negatively influenced by GF2. GF2 reduced the mitochondrial membrane potential, basal mitochondrial respiratory rate, and ATP production capacity. Our results showed that GF2 downregulated the relative GSH/GSSG, intracellular NAD+ level, and GLRX expression, suggesting that GF2 may alter the intracellular redox balance that led to mitochondrial impairment. CONCLUSION: GF2 reduces mitochondrial membrane potential, inhibits cellular oxygen consumption, activates AMPK signaling, and induces cell death. Our study examined the potential vulnerability of mitochondrial activity in GBM, and this may hold therapeutic promise.

Substrate Metabolism-Driven Assembly of High-Quality CdS xSe1- x Quantum Dots in Escherichia coli: Molecular Mechanisms and Bioimaging Application.

Biosynthesis offers opportunities for cost-effective and sustainable production of semiconductor quantum dots (QDs), but is currently restricted by poor controllability on the synthesis process, resulting from limited knowledge on the assembly mechanisms and the lack of effective control strategies. In this work, we provide molecular-level insights into the formation mechanism of biogenic QDs (Bio-QDs) and its connection with the cellular substrate metabolism in Escherichia coli. Strengthening the substrate metabolism for producing more reducing power was found to stimulate the production of several reduced thiol-containing proteins (including glutaredoxin and thioredoxin) that play key roles in Bio-QDs assembly. This effectively diverted the transformation route of the selenium (Se) and cadmium (Cd) metabolic from Cd3(PO4)2 formation to CdS xSe1- x QDs assembly, yielding fine-sized (2.0 ± 0.4 nm), high-quality Bio-QDs with quantum yield (5.2%) and fluorescence lifetime (99.19 ns) far exceeding the existing counterparts. The underlying mechanisms of Bio-QDs crystallization and development were elucidated by density functional theory calculations and molecular dynamics simulation. The resulting Bio-QDs were successfully used for bioimaging of cancer cells and tumor tissue of mice without extra modification. Our work provides fundamental knowledge on the Bio-QDs assembly mechanisms and proposes an effective, facile regulation strategy, which may inspire advances in controlled synthesis and practical applications of Bio-QDs as well as other bionanomaterials.

Copper accumulation in senescent cells: Interplay between copper transporters and impaired autophagy.

Cellular senescence is characterized by irreversible growth arrest incurred through either replicative exhaustion or by pro-oncogenic cellular stressors (radioactivity, oxidative stress, oncogenic activation). The enrichment of senescent cells in tissues with age has been associated with tissue dyshomeostasis and age-related pathologies including cancers, neurodegenerative disorders (e.g. Alzheimer's, Parkinson's, etc.) and metabolic disorders (e.g. diabetes). We identified copper accumulation as being a universal feature of senescent cells [mouse embryonic fibroblasts (MEF), human prostate epithelial cells and human diploid fibroblasts] in vitro. Elevated copper in senescent MEFs was accompanied by elevated levels of high-affinity copper uptake protein 1 (Ctr1), diminished levels of copper-transporting ATPase 1 (Atp7a) (copper export) and enhanced antioxidant defence reflected by elevated levels of glutathione (GSH), superoxide dismutase 1 (SOD1) and glutaredoxin 1 (Grx1). The levels of intracellular copper were further increased in senescent MEFs cultured in copper supplemented medium and in senescent Mottled Brindled (Mobr) MEFs lacking functional Atp7a. Finally, we demonstrated that the restoration/preservation of autophagic-lysosomal degradation in senescent MEFs following rapamycin treatment correlated with attenuation of copper accumulation in these cells despite a further decrease in Atp7a levels. This study for the first time establishes a link between Atp7a and the autophagic-lysosomal pathway, and a requirement for both to effect efficient copper export. Such a connection between cellular autophagy and copper homeostasis is significant, as both have emerged as important facets of age-associated degenerative disease.

Structural insights into the binding of buckwheat glutaredoxin with GSH and regulation of its catalytic activity.

Glutaredoxins (Grxs) are ubiquitous thioltransferases and members of the thioredoxin (Trx) fold superfamily. They have multiple functions in cells including oxidative stress responses and cell signaling. A novel glutaredoxin from buckwheat (rbGrx) with higher catalytic activity was identified, cloned, and purified. The structures of glutathionylated rbGrx and an rbGrx mutant, in which cysteine 39 was mutated to alanine, were solved by x-ray diffraction at a resolution of 2.05Å and 2.29Å, respectively. In rbGrx, GSH (glutathione) is bound at the conserved GSH-binding site, and its structure shows that it has the potential to function as a scaffold protein for the assembly and delivery of GSH. The crystal structure shows that GSH does not bind to the C39A rbGrx mutant, and the C39A mutant had no catalytic activity, indicating that C39 is a key residue that is involved in both the binding of rbGrx to GSH and the regulation of its catalytic activity. The model showing the binding of GSH with rbGrx provides a basis for understanding its molecular function and its potential future applications in medicinal food science.

Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

Characterization of Three New Glutaredoxin Genes in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis: Putative Role of RiGRX4 and RiGRX5 in Iron Homeostasis.

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

Functional Analysis of GLRX5 Mutants Reveals Distinct Functionalities of GLRX5 Protein.

Glutaredoxin 5 (GLRX5) is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different amino acids of the protein will lead to distinct effects on downstream Fe/S biosynthesis.

Effect of dehydroepiandrosterone treatment on hormone levels and antioxidant parameters in aged rats.

The aim of the current study was to evaluate the effect of chronic dehydroepiandrosterone (DHEA) administration on steroid hormones and antioxidant parameters in aged rats. To this end, three groups of Sprague-Dawley rats were compared: young (3 months of age) untreated; aged (19 months old) untreated; and aged rats treated with 20 mg/kg DHEA for 8 weeks. Major organs of aged rats in the untreated group demonstrated physiological atrophy, compared to those of young rats; this effect appeared to have been partially reversed by DHEA treatment. Testosterone and estradiol contents were significantly decreased and aldosterone significantly increased in aged untreated, compared to young untreated rats. Steroid hormone levels were obviously reversed, however, in aged rats treated with DHEA. Additionally, superoxide dismutase activity in serum, brain, heart, and liver was decreased, and maleic dialdehyde content in heart was markedly increased in untreated aged, compared to young, rats. Importantly, these changes in brain and heart of aged rats were reversed by DHEA treatment. Heme oxygenase mRNA levels were increased and inducible nitric oxide synthase mRNA levels decreased in aged, compared to young, rats; DHEA treatment appeared to reverse these changes. These results indicate that chronic DHEA administration may have effects on steroid hormone levels and antioxidant parameters in aged rats and result in postponement of the aging process.

Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4.

AIMS: Dietary supplementation with ursolic acid (UA) prevents monocyte dysfunction in diabetic mice and protects mice against atherosclerosis and loss of renal function. The goal of this study was to determine the molecular mechanism by which UA prevents monocyte dysfunction induced by metabolic stress. METHODS AND RESULTS: Metabolic stress sensitizes or "primes" human THP-1 monocytes and murine peritoneal macrophages to the chemoattractant MCP-1, converting these cells into a hyper-chemotactic phenotype. UA protected THP-1 monocytes and peritoneal macrophages against metabolic priming and prevented their hyper-reactivity to MCP-1. UA blocked the metabolic stress-induced increase in global protein-S-glutathionylation, a measure of cellular thiol oxidative stress, and normalized actin-S-glutathionylation. UA also restored MAPK phosphatase-1 (MKP1) protein expression and phosphatase activity, decreased by metabolic priming, and normalized p38 MAPK activation. Neither metabolic stress nor UA supplementation altered mRNA or protein levels of glutaredoxin-1, the principal enzyme responsible for the reduction of mixed disulfides between glutathione and protein thiols in these cells. However, the induction of Nox4 by metabolic stress, required for metabolic priming, was inhibited by UA in both THP-1 monocytes and peritoneal macrophages. CONCLUSION: UA protects THP-1 monocytes against dysfunction by suppressing metabolic stress-induced Nox4 expression, thereby preventing the Nox4-dependent dysregulation of redox-sensitive processes, including actin turnover and MAPK-signaling, two key processes that control monocyte migration and adhesion. This study provides a novel mechanism for the anti-inflammatory and athero- and renoprotective properties of UA and suggests that dysfunctional blood monocytes may be primary targets of UA and related compounds.

The Synechocystis PCC6803 MerA-like enzyme operates in the reduction of both mercury and uranium under the control of the glutaredoxin 1 enzyme.

In a continuing effort to analyze the selectivity/redundancy of the three glutaredoxin (Grx) enzymes of the model cyanobacterium Synechocystis PCC6803, we have characterized an enzyme system that plays a crucial role in protection against two toxic metal pollutants, mercury and uranium. The present data show that Grx1 (Slr1562 in CyanoBase) selectively interacts with the presumptive mercuric reductase protein (Slr1849). This MerA enzyme plays a crucial role in cell defense against both mercuric and uranyl ions, in catalyzing their NADPH-driven reduction. Like MerA, Grx1 operates in cell protection against both mercury and uranium. The Grx1-MerA interaction requires cysteine 86 (C86) of Grx1 and C78 of MerA, which is critical for its reductase activity. MerA can be inhibited by glutathionylation and subsequently reactivated by Grx1, likely through deglutathionylation. The two Grx1 residues C31, which belongs to the redox active site (CX(2)C), and C86, which operates in MerA interactions, are both required for reactivation of MerA. These novel findings emphasize the role of glutaredoxins in tolerance to metal stress as well as the evolutionary conservation of the glutathionylation process, so far described mostly for eukaryotes.

Physical and functional interactions of a monothiol glutaredoxin and an iron sulfur cluster carrier protein with the sulfur-donating radical S-adenosyl-L-methionine enzyme MiaB.

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-L-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

Somatic and reproductive cell development in rice anther is regulated by a putative glutaredoxin.

The switch from mitosis to meiosis is one of the most pivotal events in eukaryotes undergoing sexual reproduction. However, the mechanisms orchestrating meiosis initiation remain elusive, particularly in plants. Flowering plants are heterosporous, with male and female spore genesis adopting different developmental courses. We show here that plant pollen mother cells contain a specific meiosis initiation machinery through characterization of a rice (Oryza sativa) gene, MICROSPORELESS1 (MIL1). The mil1 mutant does not produce microspores in anthers but has the normal female fertility. Detailed molecular and cytological investigations demonstrate that mil1 anthers are defective in the meiotic entry of sporogenous cell progenies and in the differentiation of surrounding somatic cell layers, resulting in locules filled with somatic cells instead of microspores. Furthermore, analysis of mil1 msp1 double mutants reveals that due to the absence of MIL1, the cells in their anther locule center do not activate meiotic cell cycle either, generating a similar anther phenotype to mil1. MIL1 encodes a plant-specific CC-type glutaredoxin, which could interact with TGA transcription factors. These results suggest meiotic entry in microsporocytes is directed by an anther-specific mechanism, which requires MIL1 activity, and redox regulation might play important roles in this process.

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins.

The ß(2) subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe(2)(III)-Tyr(•)) that is essential for nucleotide reduction. The ß(2) subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (ß) and Rnr4 (ß'). Although only ß is capable of iron binding and Tyr(•) formation, cells lacking ß' are either dead or exhibit extremely low Tyr(•) levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that ß' is required for iron loading and Tyr(•) formation in ß in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent ß and are hypersensitive to iron depletion and the Tyr(•)-reducing agent hydroxyurea. Transient induction of ß' in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr(•) levels in ß. Tyr(•) can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant ß' and is inhibited by adding an iron chelator prior to, but not after, ß' supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr(•) levels and ßß' activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr(•) cofactor in RNR.

Tinospora cordifolia attenuates oxidative stress and distorted carbohydrate metabolism in experimentally induced type 2 diabetes in rats.

Diabetes is a chronic metabolic disorder affecting a vast number of people worldwide. Oxidative stress is the causative agent amplifying diabetic complications in various organs by generating noxious amount of free radicals. A huge interest always exists in exploring nutraceuticals from plant materials to replace synthetic drugs in order to overcome their adverse effects and also for economic reasons. The anti-diabetic efficiency of a medicinal plant, Tinospora cordifolia (TC) was studied in experimentally induced type 2 diabetes in Sprague-Dawley rats. Diabetes was induced by a combination of high fat diet (HFD) for a period of 10 weeks followed by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg of body weight). Oral treatment of TC (100 and 200 mg/kg body weight) for 14 days regulated blood glucose, provoked insulin secretion and also suppressed oxidative stress marker, thiobarbituric acid reactive substances (TBARS), formation and restored cellular defence anti-oxidant markers including superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH), in liver. Treatment with TC (100 and 200 mg/kg) also inhibited glucose 6-phosphatase and fructose 1,6-diphosphatase (p < 0.001); and restored glycogen content in liver (p < 0.005), which was also studied by histopathological staining with periodic acid-Schiff stain. In conclusion, the traditional plant Tinospora cordifolia mediates its anti-diabetic potential through mitigating oxidative stress, promoting insulin secretion and also by inhibiting gluconeogenesis and glycogenolysis, thereby regulating blood glucose.

Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10 interact with floral glutaredoxins ROXY1 and ROXY2 and are redundantly required for anther development.

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

Penultimate selenocysteine residue replaced by cysteine in thioredoxin reductase from selenium-deficient rat liver.

Selenium is an essential micronutrient for humans and animals, and its deficiency can predispose to the development of pathological conditions. This study evaluates the effect of selenium deficiency on the thioredoxin system, consisting of NADPH, selenoprotein thioredoxin reductase (TrxR), and thioredoxin (Trx); and the glutathione system, including NADPH, glutathione reductase, glutathione, and glutaredoxin coupled with selenoprotein glutathione peroxidase (GPx). We particularly investigate whether inactive truncated TrxR is present under selenium-starvation conditions due to reading of the UGA codon as stop. Feeding rats a selenium-deficient diet resulted in a large decrease in activity of TrxR and GPx in rat liver but not in the levels of Trx1 and Grx1. However, selenium deficiency induced mitochondrial Grx2 10-fold and markedly changed the expression of some flavoproteins that are involved in the cellular folate, glucose, and lipid metabolism. Liver TrxR mRNA was nearly unchanged, but no truncated enzyme was found. Instead, a low-activity form of TrxR with a cysteine substituted for the penultimate selenocysteine in the C-terminal active site was identified in selenium-deficient rat liver. These results show a novel mechanism for decoding the UGA stop codon, inserting cysteine to make a full-length enzyme that may be required for selenium assimilation.

Identification, expression pattern, and characterization of mouse glutaredoxin 2 isoforms.

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase involved in the maintenance of mitochondrial redox homeostasis. Grx2 was first characterized as mitochondrial protein, but alternative mRNA variants lacking the transit peptide-encoding first exon were demonstrated for human and proposed for mouse. We systematically screened for alternative transcript variants of mouse Grx2. We identified a total of six exons, three constitutive (II, III, and IV), two alternative first exons (exons Ia and Ic), and one single-cassette exon (exon IIIb) located between exons III and IV. Exons Ic and IIIb are not present in the human genome; mice lack human exon Ib. The six exons give rise to five transcript variants that encode three protein isoforms: mitochondrial Grx2a, a cytosolic isoform that is homologous to the cytosolic/nuclear human Grx2c and present in specific cells of many tissues and the testis-specific isoform Grx2d that is unique to mice. Mouse Grx2c can form an iron/sulfur cluster-bridged dimer, is enzymatically active as a monomer, and can donate electrons to ribonucleotide reductase. Testicular cells lack mitochondrial Grx2a but contain cytosolic Grx2. Prominent immunostaining was detected in spermatogonia and spermatids. These results provide evidence for additional functions of Grx2 in the cytosol, in cell proliferation, and in cellular differentiation.