James N. Siedow

Mitochondrial Reactive Oxygen Species. Contribution to Oxidative Stress and Interorganellar Signaling

The inner membrane of a plant mitochondrion contains the mitochondrial electron transport chain (mtETC), consisting of protein complexes that use an energy source-derived reductant to form a proton gradient across the membrane. This proton gradient drives ATP synthesis, a primary mitochondrial

The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria

In addition to possessing multiple NAD(P)H dehydrogenases, most plant mitochondria contain a cyanide- and antimycin-insensitive alternative terminal oxidase. Although the general characteristics of this terminal oxidase have been known for a considerable number of years, the mechanism by which it is regulated is unclear and until recently there has been relatively little information on its exact nature. In the past 5 years, however, the application of molecular and novel voltametric techniques has advanced our understanding of this oxidase considerably. In this article, we review briefly current understanding on the structure and function of the multiple NADH dehydrogenases and consider, in detail, the nature and regulation of the alternative oxidase. We derive a kinetic model for electron transfer through the ubiquinone pool based on a proposed model for the reduction of the oxidase by quinol and show how this can account for deviations from Q-pool behaviour. We review information on the attempts to isolate and characterise the oxidase and finally consider the molecular aspects of the expression of the alternative oxidase.

Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.

Characterization of Transformed Arabidopsis with Altered Alternative Oxidase Levels and Analysis of Effects on Reactive Oxygen Species in Tissue

The alternative oxidase (AOX) of plant mitochondria transfers electrons from the ubiquinone pool to oxygen without energy conservation. AOX can use reductant in excess of cytochrome pathway capacity, preventing reactive oxygen species (ROS) formation from an over-reduced ubiquinone pool, and thus may be involved in acclimation to oxidative stresses. The AOX connection with mitochondrial ROS has been investigated only in isolated mitochondria and suspension culture cells. To study ROS and AOX in whole plants, transformed lines of Arabidopsis (Arabidopsis thaliana) were generated: AtAOX1a overexpressors, AtAOX1a anti-sense plants, and overexpressors of a mutated, constitutively active AtAOX1a. In the presence of KCN, leaf tissue of either mutant or wild-type AOX overexpressors showed no increase in oxidative damage, whereas anti-sense lines had levels of damage greater than those observed for untransformed leaves. Similarly, ROS production increased markedly in anti-sense and untransformed, but not overexpressor, roots with KCN treatment. Thus, AOX functions in leaves and roots, as in suspension cells, to ameliorate ROS production when the cytochrome pathway is chemically inhibited. However, in contrast with suspension culture cells, no changes in leaf transcript levels of selected electron transport components or oxidative stress-related enzymes were detected under nonlimiting growth conditions, regardless of transformation type. Further, a microarray study using an anti-sense line showed AOX influences outside mitochondria, particularly in chloroplasts and on several carbon metabolism pathways. These results illustrate the value of expanding AOX transformant studies to whole tissues.

Plant Mitochondrial Electron Transfer and Molecular Biology.

Severa1 types of reduced carbon com- pounds, including fatty acids, organic acids, and amino acids, can serve as the primary reducing substrates for plant respi- ration. However, the most common substrate used by plant tissues for respiration is carbohydrate (CHpO). The complete oxidation of a carbohydrate releases a large amount of free energy, much of which is coupled to the conversion of ADP and Pi to ATI? When sucrose (Cl2H=0,1) is the substrate, aer- obic respiration can be divided into three distinct phases: glycolysis, the tricarboxylic acid (TCA) cycle, and the coupled processes of mitochondrial electron transfer and ATP synthe- sis. Except for glycolysis, all of the metabolic steps of aerobic respiration take place in the mitochondrion. Like mitochon- dria found in other eukaryotes, plant mitochondria are roughly spherical subcellular organelles that usually range from

The mitochondrial cyanide-resistant oxidase: structural conservation amid regulatory diversity

Mitochondria from all plants, many fungi and some protozoa contain a cyanide-resistant, alternative oxidase that functions in parallel with cytochrome c oxidase as the terminal oxidase on the electron transfer chain. Characterization of the structural and potential regulatory features of the alternative oxidase has advanced considerably in recent years. The active site is proposed to contain a di-iron center belonging to the ribonucleotide reductase R2 family and modeling of a four-helix bundle to accommodate this active site within the C-terminal two-thirds of the protein has been carried out. The structural features of this active site are conserved among all known alternative oxidases. The post-translational regulatory features of the alternative oxidase are more variable among organisms. The plant oxidase is dimeric and can be stimulated by either alpha-keto acids or succinate, depending upon the presence or absence, respectively, of a critical cysteine residue found in a conserved block of amino acids in the N-terminal region of the plant protein. The fungal and protozoan alternative oxidases generally exist as monomers and are not subject to organic acid stimulation but can be stimulated by purine nucleotides. The origins of these diverse regulatory features remain unknown but are correlated with sequence differences in the N-terminal third of the protein.

The Alternative Oxidase of Plant Mitochondria Is Involved in the Acclimation of Shoot Growth at Low Temperature. A Study of Arabidopsis AOX1a Transgenic Plants

The alternative oxidase (AOX) pathway of plant mitochondria uncouples respiration from mitochondrial ATP production and may ameliorate plant performance under stressful environmental conditions, such as cold temperatures, by preventing excess accumulation of reactive oxygen species. We tested this model in whole tissues by growing AtAOX1a-transformed Arabidopsis (Arabidopsis thaliana) plants at 12°C. For the first time, to our knowledge, in plants genetically engineered for AOX, we identified a vegetative shoot growth phenotype. Compared with wild type at day 21 after sowing, anti-sense and overexpressing lines showed, on average, 27% reduced leaf area and 25% smaller rosettes versus 30% increased leaf area and 33% larger rosette size, respectively. Lines overexpressing a mutated, constitutively active AOX1a showed smaller phenotypic effects. These phenotypic differences were not the result of a major alteration of the tissue redox state because the changes in levels of lipid peroxidation products, reflecting oxidative damage, and the expression of genes encoding antioxidant and electron transfer chain redox enzymes did not correspond with the shoot phenotypes. However, the observed phenotypes were correlated with the amount of total shoot anthocyanin at low temperature and with the transcription of the flavonoid pathway genes PAL1 and CHS. These results demonstrate that (1) AOX activity plays a role in shoot acclimation to low temperature in Arabidopsis, and that (2) AOX not only functions to prevent excess reactive oxygen species formation in whole tissues under stressful environmental conditions but also affects metabolism through more pervasive effects, including some that are extramitochondrial.

Regulation of the Cyanide-resistant Alternative Oxidase of Plant Mitochondria IDENTIFICATION OF THE CYSTEINE RESIDUE INVOLVED IN α-KETO ACID STIMULATION AND INTERSUBUNIT DISULFIDE BOND FORMATION

The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by α-keto acids. After determining that theArabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala. Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization. The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate. Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with α-keto acid activation occurring through a thiohemiacetal. These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by α-keto acids. In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of α-keto acid activation and of the regulatory sulfhydryl/disulfide system.

Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria

Two factors known to regulate plant mitochondrial cyanide‐resistant alternative oxidase activity, pyruvate and the redox status of the enzymes intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated alternative oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced alternative oxidase protein, a wide range of enzyme activity is possible in vivo.

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.