Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings

Plants reconfigure their metabolic network under stress conditions. Changes of mitochondrial metabolism such as tricarboxylic acid (TCA) cycle and amino acid metabolism are reported in Arabidopsis roots but the exact molecular basis underlying this remains unknown. We here hypothesise the reassembly of enzyme protein complexes to be a molecular mechanism for metabolic regulation and tried in the present study to find out mitochondrial protein complexes which change their composition under oxidative stress by the combinatorial approach of proteomics and metabolomics. Arabidopsis seedlings were treated with menadione to induce oxidative stress. The inhibition of several TCA cycle enzymes and the oxidised NADPH pool indicated the onset of oxidative stress. In blue native/SDS-PAGE analysis of mitochondrial protein complexes the intensities of 18 spots increased and those of 13 spots decreased in menadione treated samples suggesting these proteins associate with, or dissociate from, protein complexes. Some spots were identified as metabolic enzymes related to central carbon metabolism such as malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, monodehydroascorbate reductase and alanine aminotransferase. The change in spot intensity was not directly correlated to the total enzyme activity and mRNA level of the corresponding enzyme but closely related to the metabolite profile, suggesting the metabolism is regulated under oxidative stress at a higher level than translation. These results are somewhat preliminary but suggest the regulation of the TCA cycle, glycolysis, ascorbate and amino acid metabolism by reassembly of plant enzyme complexes.     

A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism

In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.     

Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings

Wheat ( Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods (drought acclimation). The antioxidant defense in terms of redox metabolites and enzymes in leaf cells, chloroplasts, and mitochondria was examined in relation to ROS-induced membrane damage. Drought-acclimated seedlings modulated growth by maintaining favorable turgor potential and RWC and were able to limit H₂O₂ accumulation and membrane damage as compared with non-acclimated plants during severe water stress conditions. This was due to systematic upregulation of H₂O₂-metabolizing enzymes especially ascorbate peroxidase (APX, EC 1.11.1.11) and by maintaining ascorbate–glutathione redox pool in acclimated plants. By contrast, failure in the induction of APX and ascorbate–glutathione cycle enzymes makes the chloroplast susceptible to oxidative stress in non-acclimated plants. Non-acclimated plants protected the leaf mitochondria from oxidative stress by upregulating superoxide dismutase (SOD, EC 1.15.1.1), APX, and glutathione reductase (GR, EC 1.6.4.2) activities. Rewatering led to rapid enhancement in all the antioxidant defense components in non-acclimated plants, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or downregulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced oxidative stress tolerance by well-co-ordinated induction of antioxidant defense both at the chloroplast and at the mitochondrial level.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.     

The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development

Ascorbate peroxidase (APX) exists as several isoforms that are found in various compartments in plant cells. The cytosolic and chloroplast APXs appear to play important roles in antioxidation metabolism in plant cells, yet the function of peroxisomal APX is not well studied. In this study, the localization of a putative peroxisomal membrane-bound ascorbate peroxidase, APX3 from Arabidopsis, was confirmed by studying the green fluorescent protein (GFP)-APX3 fusion protein in transgenic plants. GFP-APX3 was found to co-localize with a reporter protein that was targeted to peroxisomes by the peroxisomal targeting signal 1. The function of APX3 in Arabidopsis was investigated by analysing an APX3 knockout mutant under normal and several stress conditions. It was found that loss of function in APX3 does not affect Arabidopsis growth and development, suggesting that APX3 may not be an important antioxidant enzyme in Arabidopsis, at least under the conditions that were tested, or the function of APX3 could be compensated by other antioxidant enzymes in plant cells.     

Operation and function of the tricarboxylic acid cycle in the illuminated leaf

Respiratory activity of plants in the light, measured as carbon dioxide release from the tricarboxylic acid (TCA) cycle or oxygen consumption by the respiratory chain, is generally reported to lie between 25 and 100% of that in the dark. While this has been interpreted as evidence for an inhibition of respiration during photosynthesis, an increasing body of evidence indicates that mitochondrial respiration plays an important role in photosynthetic tissues. Historically, the view from experiments using specific respiratory inhibitors has been that oxidative phosphorylation in the mitochondria provides the cytosol with adenosine triphosphate even in the light. However, functioning of TCA cycle reactions is also required for the export of carbon skeletons necessary for nitrate reduction in the cytosol. In addition, export of TCA cycle-derived reducing equivalents may also be necessary for photorespiration (for hydroxypyruvate reduction in the peroxisomes). The work with respiratory inhibitors has recently been complemented by a range of transgenic experiments that provide direct evidence for the importance of the TCA cycle in the illuminated leaves. These transgenesis experiments hint at an important role for ascorbate in coordinating the major pathways of energy metabolism within the leaf and are in keeping with current thinking that redox signals emanating from the mitochondria are important in setting the cellular machinery to maintain overall redox balance. In this review we intend to synthesize recent experimental data to postulate a model of the function of the TCA cycle in the illuminated leaf.     

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. ¹³C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Pyruvate is supplied by three independent processes that act synergistically to maintain metabolic flux and support plant respiration in a variety of circumstances.     

Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA‐NONOate, a pure NO slow generator, and of SIN‐1 (3‐morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non‐enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA‐NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn‐SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN‐1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn‐SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO‐resistant AP and mitochondrial APX may be important components of the H₂O₂‐signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn‐SOD, against NO and ONOO^? stress in plant mitochondria.     

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.     

Catalase Inhibition Affects Glyoxylate Cycle Enzyme Expression and Cellular Redox Control during the Functional Transition of Sunflower and Safflower Seedlings

Oilseed crops are an important natural resource because they can be used for food and renewable energy production. However, oilseed seedling establishment and vigor depend upon the capacity to overcome functional transition, a developmental stage characterized by the consumption of the remaining oil reserves, through β-oxidation and glyoxylate cycle, and the onset of autotrophic metabolism. The increased growth and the acclimation to full photosynthetic activity lead to production of reactive oxygen species and a reorganization of the cell antioxidant systems to achieve a new redox homeostasis. In the present study, catalase (CAT) was inhibited by 3-amino-1,2,4-triazole application during functional transition in sunflower and safflower seedlings to understand the effect of this antioxidant enzyme impairment on the mRNA expression of the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS), as well as the superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity and expression. CAT inhibition led to significant seedling growth reduction and increases in H₂O₂ content, SOD activity, and mRNA expression of CAT and APX in both species. However, APX activity was induced only in safflower plants. Additionally, ICL and MLS mRNA expressions were upregulated after 6 h of treatment when compared to the control values. These results indicate that under CAT impairment conditions, redox homeostasis at the functional transition phase was partially supported by the SOD and APX antioxidant systems to maintain the seedling photosynthetic establishment.     

Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.