Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco)

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.     

Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase

The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.     

Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 [mu]M antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme’s apparent V_max.     

Plasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: The logic of Aox1 gene localization

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work, we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c₁ together with an up-regulation of l-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.     

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.     

Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues in addition to conserved cysteines in alpha-keto acid activation

Activity of the plant mitochondrial alternative oxidase (AOX) can be regulated by organic acids, notably pyruvate. To date, only two well-conserved cysteine residues have been implicated in this process. We report the functional expression of two AOX isozymes (Sauromatum guttatum Sg-AOX and Arabidopsis thaliana At-AOX1a) in Schizosaccharomyces pombe. Comparison of the response of these two isozymes to pyruvate in isolated yeast mitochondria and disrupted mitochondrial membranes reveals that in contrast to At-AOX1a, Sg-AOX activity is insensitive to pyruvate and appears to be in a constitutively active state. As both of these isozymes conserve the two cysteines, we propose that such contrasting behaviour must be a direct result of differences in their amino acid sequence and have subsequently identified novel candidate residues.     

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active “oxidized” form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the “reduced” active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.     

Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable

Cladosporiumfulvum is a mitosporic ascomycete pathogen of tomato. A study of fungal genes expressed during carbon starvation in vitro identified several genes that were up regulated during growth in planta. These included genes predicted to encode acetaldehyde dehydrogenase (Aldh1) and alcohol oxidase (Aox1). An Aldh1 deletion mutant was constructed. This mutant lacked all detectable ALDH activity, had lost the ability to grow with ethanol as a carbon source, but was unaffected in pathogenicity. Aox1 expression was induced by carbon starvation and during the later stages of infection. The alcohol oxidase enzyme activity has broadly similar properties (Km values, substrate specificity, pH, and heat stability) to yeast enzymes. Antibodies raised to Hansenula polymorpha alcohol oxidase (AOX) detected antigens in Western blots of starved C. fulvum mycelium and infected plant material. Antigen reacting with the antibodies was localized to organelles resembling peroxisomes in starved mycelium and infected plants. Disruption mutants of Aox1 lacked detectable AOX activity and had markedly reduced pathogenicity as assayed by two different measures of fungal growth. These results identify alcohol oxidase as a novel pathogenicity factor and are discussed in relation to peroxisomal metabolism of fungal pathogens during growth in planta.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Molecular localization of a redox-modulated process regulating plant mitochondrial electron transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.     

Alternative oxidases in Arabidopsis: a comparative analysis of differential expression in the gene family provides new insights into function of non-phosphorylating bypasses

The emergence of Arabidopsis as a model plant provides an opportunity to gain insights into the role of the alternative oxidase that cannot be as readily achieved in other plant species. The analysis of extensive mRNA expression data indicates that all five Aox genes (Aox1a, 1b, 1c, 1d and 2) are expressed, but organ and developmental regulation are evident, suggesting regulatory specialisation of Aox gene members. The stress-induced nature of the alternative pathway in a variety of plants is further supported in Arabidopsis as Aox1a and Aox1d are amongst the most stress responsive genes amongst the hundreds of known genes encoding mitochondrial proteins. Analysis of genes co-expressed with Aoxs from studies of responses to various treatments altering mitochondrial functions and/or from plants with altered Aox levels reveals that: (i) this gene set encodes more functions outside the mitochondrion than functions in mitochondria, (ii) several pathways for induction exist and there is a difference in the magnitude of the induction in each pathway, (iii) the magnitude of induction may depend on the endogenous levels of Aox, and (iv) induction of Aox can be oxidative stress-dependent or -independent depending on the gene member and the tissue analysed. An overall role for Aox in re-programming cellular metabolism in response to the ever changing environment encountered by plants is proposed.     

What couples glycolysis to mitochondrial signal generation in glucose-stimulated insulin secretion?

Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.     

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O₂ evolution and O₂ uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m^?2 s^?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO₃‐dependent O₂ evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O₂ uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO₃‐dependent O₂ evolution or p‐benzoquinone (BQ)‐dependent O₂ evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.     

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids.

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.