In vivo cytochrome and alternative pathway respiration in leaves of Arabidopsis thaliana plants with altered alternative oxidase under different light conditions.

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition.

Temperature-sensitive alternative oxidase protein content and its relationship to floral reflectance in natural Plantago lanceolata populations.

In many plant species, the alternative respiratory pathway consisting of alternative oxidase (AOX) is affected by growth temperature. The adaptive significance of this temperature-sensitivity is unresolved. Here, leaf and spike (flower cluster) AOX protein content and spike/floral reflectance of genotypes from European Plantago lanceolata populations found in regions differing in reproductive season temperatures were measured. Cloned genotypes grown at controlled warm and cool temperatures were used to assess the natural within- and between-population variation in AOX content, temperature-sensitive phenotypic plasticity in content, and the relationship between AOX and temperature-sensitive floral/spike reflectance. AOX content and plasticity were genetically variable. Leaf AOX content, although greater at cool temperature, was relatively low and not statistically different across populations. Spike AOX content was greater than in leaves. Spike AOX plasticity differed significantly among populations and climate-types and showed significant negative correlation with floral reflectance plasticity, which also varied among populations. Genotypes with more AOX at cool than at warm temperature had greater floral reflectance plasticity; genotypes with relatively more AOX at warm temperature had less floral reflectance plasticity. The data support the hypothesis that plasticity of AOX content in reproductive tissues is associated with long-term thermal acclimatization.

Regulation of plant alternative oxidase activity: a tale of two cysteines.

Two Cys residues, Cys(I) and Cys(II), are present in most plant alternative oxidases (AOXs). Cys(I) inactivates AOX by forming a disulfide bond with the corresponding Cys(I) residue on the adjacent subunit of the AOX homodimer. When reduced, Cys(I) associates with alpha-keto acids, such as pyruvate, to activate AOX, an effect mimicked by charged amino acid substitutions at the Cys(I) site. Cys(II) may also be a site of AOX activity regulation, through interaction with the small alpha-keto acid, glyoxylate. Comparison of Arabidopsis AOX1a (AtAOX1a) mutants with single or double substitutions at Cys(I) and Cys(II) confirmed that glyoxylate interacted with either Cys, while the effect of pyruvate (or succinate for AtAOX1a substituted with Ala at Cys(I)) was limited to Cys(I). A variety of Cys(II) substitutions constitutively activated AtAOX1a, indicating that neither the catalytic site nor, unlike at Cys(I), charge repulsion is involved. Independent effects at each Cys were suggested by lack of Cys(II) substitution interference with pyruvate stimulation at Cys(I), and close to additive activation at the two sites. However, results obtained using diamide treatment to covalently link the AtAOX1a subunits by the disulfide bond indicated that Cys(I) must be in the reduced state for activation at Cys(II) to occur.

Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis.

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.

Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes.

The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

Comparison of intact Arabidopsis thaliana leaf transcript profiles during treatment with inhibitors of mitochondrial electron transport and TCA cycle.

Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

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 degrees 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.

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.