NADPH Thioredoxin Reductase C Controls the Redox Status of Chloroplast 2-Cys Peroxiredoxins in Arabidopsis thaliana

Chloroplast 2-Cys peroxiredoxins （2-Cys Prxs） are efficiently reduced by NADPH Thioredoxin reductase C （NTRC）. To investigate the effect of light/darkness on NTRC function, the content of abundant plastidial enzymes, Rubisco, glutamine synthetase （GS）, and 2-Cys Prxs was analyzed during two consecutive days in Arabidopsis wild-type and ntrc mutant plants. No significant difference of the content of these proteins was observed during the day or the night in wildtype and mutant plants. NTRC deficiency caused a lower content of fully reduced 2-Cys Prxs, which was undetectable in darkness, suggesting that NTRC is the most important pathway for 2-Cys Prx reduction, probably the only one during the night. Arabidopsis contains two plastidial 2-Cys Prxs, A and B, for which T-DNA insertion lines were characterized showing the same phenotype as wild-type plants. Two-dimensional gel analysis of leaf extracts from these mutants allowed the identification of basic and acidic isoforms of 2-Cys Prx A and B. In-vitro assays and mass spectrometry analysis showed that the acidic isoform of both proteins is produced by overoxidation of the peroxidatic Cys residue to sulfinic acid. 2-Cys Prx overoxidation was lower in the NTRC mutant. These results show the important function of NTRC to maintain the redox equilibrium of chloroplast 2-Cys Prxs.     

Inositol Polyphosphate Phosphatidylinositol 5-Phosphatase9 （At5PTase9） Controls Plant Salt Tolerance by Regulating Endocytosis

Phosphatidylinositol 5-phosphatases （5PTases） components of membrane trafficking system. Recently, we that hydrolyze the 5＇ position of the inositol ring are key reported that mutation in AtSPTase7 gene reduced produc- tion of reactive oxygen species （ROS） and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5＇ phos- phate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS produc- tion and Ca2~ influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the AtSptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound Ptdlns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca2＋ influx, and induction of stress-responsive genes.     

NADPH Thioredoxin Reductase C Is Involved in Redox Regulation of the Mg-Chelatase I Subunit in Arabidopsis thaliana Chloroplasts

Dear Editor,
The synthesis of tetrapyrroles, including chlorophylls, is central for chloroplast function. The metabolic pathway of tetrapyrrole biosynthesis in Arabidopsis is initiated with the formation of amino levulinic acid （ALA）, which is con- verted by a series of common reactions to protoporphy- tin IX （Proto IX） （Tanaka et al., 2011）. Then the pathway diverges into two branches： the synthesis of heme/bilin and chlorophylls. The insertion of Mg2＋ into Proto IX, cata- lyzed by Mg-chelatase, is the first committed reaction of the chlorophyll branch and is considered a key step for the regulation of the whole pathway. Mg-chelatase is a het- erotrimeric enzyme composed of subunits CHLI, CHLD, and CHLH, the reaction mechanism of which has been estab- lished. It is a two-step process consisting in the Mg-ATP- dependent activation of the enzyme, which implies the formation of a ternary complex of subunits CHLI and CHLD with ATP-Mg2＋, and Mg2＋ chelation, which is catalyzed by CHLH driven by ATP hydrolysis, CHLI providing ATPase activity to the complex （Tanaka et al., 2011）. In Arabidopsis, CHLH and CHLD are encoded by single genes, whereas two genes, CHLI-I and CHLI-2, encode the two isoforms of CHLI.     

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.     

Comparative expression analysis of genes encoding metallothioneins in response to heavy metals and abiotic stresses in rice (Oryza sativa) and Arabidopsis thaliana

To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H₂O₂ or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H₂O₂) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance.     

Heat Shock Factors HsfB 1 and HsfB2b Are Involved in the Regulation of Pdf1.2 Expression and Pathogen Resistance in Arabidopsis

In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdfl.2a/b in mutant plants. The Pdfexpression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB 1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdfgenes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.     

叠氮钠、过氧化氢对SH-SY5Y细胞内硫氧还蛋白还原酶的影响

过度氧化应激是诱发许多神经退变病的重要因素。叠氮钠(NaN3)是线粒体有氧呼吸链细胞色素c氧化酶(COX)的特异性抑制剂,过氧化氢(H2O2)释放氧自由基造成氧化损伤,两者都可以用于氧化应激情况下神经元损伤模型的建立。硫氧还蛋白还原酶(thioredoxin reductase,TR)特异性的还原氧化型的硫氧还蛋白(thioredoxin,TRx),调节细胞中氧化还原的平衡。现以不同浓度NaN3或H2O2,处理人神经母细胞瘤细胞(SH-SY5Y细胞),建立损伤模型。通过MTT法、形态学方法检测SH-SY5Y细胞损伤程度。同时,通过Western blot定量法、免疫细胞化学法,检测损伤的SH-SY5Y细胞中TR含量的改变,观察TR在胞内的分布。实验表明,NaN3、H2O2,均以浓度依赖方式损伤SH-SY5Y细胞;TR分布于SH-SY5Y细胞的胞浆,表明TR是一种分泌蛋白,损伤后分布无明显变化。但一定浓度的NaN3作用后3h,胞内TR水平显著降低,即神经系统内呼吸链受损可抑制TR的表达,为神经退变病的防治提供了新的思路。     

The chloroplast NADPH thioredoxin reductase C,NTRC, controls non‐photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis

High irradiances may lead to photooxidative stress in plants, and non‐photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light‐limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans‐thylakoid ΔpH and have 10‐fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc–psbs double‐knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc–psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield.     

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys⁵⁹/Cys⁶⁴), and a C-terminal cysteine/selenocysteine motif (Cys⁴⁹⁷/Sec⁴⁹⁸). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO^•) to the hydroxyl radical adduct (DEPMPO/HO^•). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O. Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys⁵⁹/Cys⁶⁴ dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys⁵⁹/Cys⁶⁴ dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys⁴⁹⁷/Sec⁴⁹⁸ active site. Although the bulk of the DEPMPO/HO^• signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO^• could be generated by the enzyme in an H₂O₂-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.     

Between a Rock and a Dry Place： The Water-Stressed Moss

The earliest land plants faced a suite of abiotic stresses largely unknown to their aquatic algal ancestors. The descendants of these plants evolved two general mechanisms for survival in the relatively arid aerial environment. While the vascular plants or ＇tracheophytes＇ developed tissue specializations to transport and retain water, the other main lineages of land plants, the bryophytes, retained a simple, nonvascular morphology. The bryophytes--mosses, hornworts, and liverworts--continually undergo a co-equilibration of their water content with the surrounding environment and rely to a great extent on intrinsic cellular mechanisms to mitigate damage due to water stress. This short review will focus on the cellular and molecular responses to dehydration and rehydration in mosses, and offer insights into general plant responses to water stress.     

Effect of Vitamin E on Serum Aminotransferase and Thioredoxin Levels in Patients with Viral Hepatitis C

Objectives: Oxidative stress induces cellular responses such as cell death, gene activation and cell proliferation, in the liver. Vitamin E (Vit. E) has been found to protect the liver against oxidative stress in animal experiments. Thioredoxin (TRX) is a stress inducible, multifunctional protein, secreted during oxidative stress. This study evaluated effects of Vit. E on serum TRX and aminotransferase levels in hepatitis C virus (HCV) patients, partly non-responsive to initial interferon (IFN), with higher than average level of serum alanine aminotransferase (ALT) after receiving anti-inflammatory drug treatment. Methods: Seventeen HCV patients (male=3; female=14) of age 62±7.65 years receiving anti-inflammatory drug therapy, at least 6 months prior to Vit. E administration, were given d-α´-tocopherol 500?mg/day, orally, for a period of 3 months. ALT, aspartate aminotransferase (AST), TRX and Vit. E were measured at 0, 1, 2 and 3 months and 1 month after end of treatment. As controls, the same patients biochemical data, 3 months from the start of therapy were used. Patients were divided into three categories: total patients “T”, low ALT group “L” (ALT<70?IU/l) and high ALT group “H”(ALT>70?IU/l), respectively.Results: The ALT level was lowered, significantly in group H, in the 1st, 2nd, 3rd and 1-month post therapy, compared to the initial value. But group L showed little or no change in ALT. Post Vit. E therapy, in groups T and H, the TRX level was elevated but remained below initial levels, whereas in group L, TRX level remained significantly lower than the pretreatment value. Groups T and L, showed significant reduction (p<0.05) in serum TRX levels in the 2nd and 3rd month. Group H showed a tendency towards TRX reduction, but not significantly. Serum Vit. E levels increased significantly (p<0.0001) from the 1st to 3rd month in all three T, H and L groups. Conclusion: Oxidative stress induced liver damage is reduced by Vit. E in patients with viral hepatitis C, particularly those with initial ALT levels >70?IU/l. Vit. E treatment causes reduction of oxidative stress markers as TRX and ALT in sera. Therefore, Vit. E can act as a supportive therapy to combat liver damage caused by oxidative stress, in such patients with continuously high levels of ALT even after anti-viral and anti-inflammatory drug therapy.     

The Structure of Trypanosoma cruzitrypanothione Reductase in the Oxidized and NADPH Reduced State

The three-dimensional structure of trypanothione reductase (TR) (EC 1.6.4.8) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C. fasciculata TR as a starting model. Elucidation of the T. cruzi TR structure represents the first step in the rational design of a drug against Chagas' disease. The structure of T. cruzi TR is compared with those of C. fasciculata TR as well as human and E. coli glutathione reductase (GR). In the FAD-binding domain, TR has two insertions, each about 10 residues long, which do not occur in GR. The first one is a rigid loop stabilizing the position of helix 91–117 which is responsible for the wider active site of TR as compared to GR. The second insertion does not occur where it is predicted by sequence alignment; rather the residues extend three strands of the 4-stranded β-sheet by one or two residues each. This increases the number of hydrogen bonds within the sheet structure. The structure of the NADPH.TR complex has been solved at 0.33 nm resolution. The nicotinamide ring is sandwiched between the flavin ring and the side chain of Phe-198 which undergoes the same conformational change upon coenzyme binding as Tyr-197 in GR. In addition to Arg-222 and Arg-228, which are conserved in TR and GR, Tyr-221—the last residue of the second β-sheet strand of the βαβ dinucleotide binding fold—is in hydrogen bonding distance to the 2′ phosphate group of NADPH. © 1994 John Wiley & Sons, Inc.     

The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress

The effect of heavy metal stress as a potent abiotic elicitor for triggering an accumulation of jasmonic acid (JA) was investigated. Copper and cadmium in in vivo conditions induced accumulation of jasmonates in mature leaves of Arabidopsis thaliana and in young and oldest Phaseolus coccineus plants. The dynamics of jasmonate accumulation showed a biphasic character in both plants. In the first phase, after 7 (A. thaliana) or 14 h (P. coccineus) of exposure to Cu or Cd, a rapid increase of JA level occurred, followed by a rapid decrease observed during 7 successive hours. In the next phase, a repeated but slow increase of JA content occurred. The heavy metal stress induced in particular a more stable (3R,7R) form of jasmonates. These results indicate that JA is connected with the mechanism of toxic action of both heavy metals in plants, differentially reacting to exogenous JA and possessing variable dynamics depending on the plants studied as well as their growth stage.