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.     

Regulation of coupling factor in field-grown sunflower: A Redox model relating coupling factor activity to the activities of other thioredoxin-dependent chloroplast enzymes

Simultaneous, non-invasive measurements were made of the rate of photosynthetic CO₂ fixation and the state of activation of the chloroplast CF₁CF₀-ATP synthase (CF) in field-grown sunflower (Helianthus annuus L.) during the dark-to-light transition at sunrise. CO₂ fixation showed a linear response with light intensity from zero to about 500–700 E m^-2 s^-1. However, at light intensities of only 5–22 E m^-2 s^-1, the energetic threshold for activation of the CF was found to be significantly lowered (as compared to the pre-dawn state), presumably through reduction of the regulatory sulfhdryl groups of the -subunit of the CF. When these studies were extended to chamber-grown plants, it was found that as little as 5 seconds of illumination at 4 E m^-2 s^-1 caused apparently full CF reduction. It is clear, therefore, that the catalytic activation of CF is not rate limiting to the induction of carbon assimilation under field conditions during a natural dark-to-light transition at sunrise. A model, based on the redox properties of the regulatory sulfhydryls, was developed to examine the significance of sulfhydryl midpoint potential in explaining the differences in light sensitivity and oxidation and reduction kinetics, between the CF and other thioredoxin-modulated chloroplast enzymes. Computer simulations of the light-induced regulation of three representative thioredoxin-modulated enzymes are presented.Abbreviations CF chloroplast CF₁-CF₀ ATP synthease or coupling factor - E_a active form of CF - E_a ⁰ active, oxidized form of CF - E_a ^r active, reduced form of CF - E_i inactive form of CF - E_i ⁰ inactive, oxidized form of CF - E_i ^r inactive, reduced form of CF - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin oxidoreductase - G6PDH glucose-6-phosphate dehydrogenase - MDH NADP-malate dehydrogenase - pmf protonmotive force - pmf_T threshold pmf required to activate CF - pmf_T ⁰ threshold pmf required to active the oxidized form of CF - pmf_T ^r threshold pmf required to activate the reduced form of CF - TR thioredoxin     

P515的测量原理、方法和应用

光谱技术已广泛应用于光合研究领域,如光吸收信号P515和P700氧化还原动力学以及叶绿素荧光等,可快速、准确地检测植物的光合活性。P515信号广泛存在于高等植物和藻类中,是类囊体膜上的色素分子吸收光能后,其吸收光谱发生位移造成。利用光诱导的P515快速和慢速动力学,可检测PSI和PSII反应中心的比值、ATP合酶的质子...     

Na+ overload during ischemia and reperfusion in rat hearts: Comparison of the Na+/H+ exchange blockers EIPA,cariporide and eniporide

Intracellular myocardial Na⁺ overload during ischemia is an important cause of reperfusion injury via reversed Na⁺/Ca²⁺ exchange. Prevention of this Na⁺ overload can be accomplished by blocking the different Na⁺ influx routes. In this study the effect of ischemic inhibition of the Na⁺/H⁺ exchanger (NHE) on [Na⁺]_i, pHi and post-ischemic contractile recovery was tested, using three different NHE-blockers: EIPA, cariporide and eniporide. pHi and [Na⁺]_i were measured using simultaneous ³¹P and ²³Na NMR spectroscopy, respectively, in paced (5 Hz) isolated, Langendorff perfused rat hearts while contractility was assessed by an intraventricular balloon. NHE-blockers (3 M) were administered during 5 min prior to 30 min of global ischemia followed by 30 min drug-free reperfusion. NHE blockade markedly reduced ischemic Na⁺ overload; after 30 min of ischemia [Na⁺]_i had increased to 293 ± 26, 212 ± 6, 157 ± 5 and 146 ± 6% of baseline values in untreated and EIPA (p < 0.01 vs. untreated), cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. Ischemic acidosis did not differ significantly between groups. During reperfusion, however, recovery of pHi was significantly delayed in treated hearts. The rate pressure product recovered to 12.0 ± 1.9, 12.1 ± 2.1, 19.5 ± 2.8 and 20.4 ± 2.5 × 103 mmHg/min in untreated and EIPA, cariporide (p < 0.01 vs. untreated) and eniporide (p < 0.01 vs. untreated) treated hearts, respectively. In conclusion, blocking the NHE reduced ischemic Na⁺ overload and improved post-ischemic contractile recovery. EIPA, however, was less effective and exhibited more side effects than cariporide and eniporide in the concentrations used.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.