Salicylhydroxamic Acid-sensitive Respiration Research Articles

Photosynthetic performance and fertility are repressed in GmAOX2b antisense soybean.

The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function.

Cloning and functional expression of the mitochondrial alternative oxidase ofAspergillus fumigatusand its induction by oxidative stress

Aspergillus fumigatus possesses a branched mitochondrial electron transport chain, with both cyanide-sensitive and -insensitive oxygen-consumption activities. Mitochondrial reactive oxygen species mediate signaling for alternative oxidase (AOX) expression. A 1173 bp-long Afaox gene encoding a 40 kDa protein has been cloned and identified. Recombinant constructs containing the Afaox ORF were transformed into Escherichia coli and Saccharomyces cerevisiae for heterologous expression. In A. fumigatus, AOX activity and mRNA expression were both induced with menadione or paraquat, suggesting an important role of AOX under oxidative stress. Therefore, positive transformants showed a cyanide-resistant and salicylhydroxamic acid-sensitive respiration, whereas in control cells the oxygen uptake was completely inhibited after KCN addition.

Alteration of respiration capacity and transcript accumulation level of alternative oxidase genes in necrosis lines of common wheat

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

Overexpression of wheat alternative oxidase gene Waox1a alters respiration capacity and response to reactive oxygen species under low temperature in transgenic Arabidopsis

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.

Characterization of two non-homoeologous nuclear genes encoding mitochondrial alternative oxidase in common wheat.

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. We have isolated two non-homoeologous genes (Waox1a and Waox1c) encoding AOX proteins from common wheat (Triticum aestivum L.). These two genes were orthologous to rice AOX1a and AOX1c, and their exon/intron structure was conserved, as it is in most other plant AOX genes. Southern blot analysis indicated that both Waox1a and Waox1c were located in at least three homoeologous loci and that additional AOX genes with lower homology were present in the genome of common wheat. The Waox1a and Waox1c loci were respectively assigned to the homoeologous group 2 and 6 chromosomes. The steady-state level of Waox1a and Waox1c transcripts increased under cold stress, while only that of Waox1a was increased by cyanide treatment.

Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger

The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide- m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.

Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger.

The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide-m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.

Existence of Cyanide-Insensitive Respiration in the Yeast Pichia stipitis and Its Possible Influence on Product Formation during Xylose Utilization

A cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in the yeast Pichia stipitis in contrast to Candida utilis, Pachysolen tannophilus, and Saccharomyces cerevisiae. During xylose utilization in the presence of either salicyl hydroxamic acid or cyanide, P. stipitis formed xylitol, arabitol, and ribitol. The existence of CIR is discussed in terms of a redox sink preventing xylitol formation in P. stipitis.

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.

Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.

Respiration of Bloodstream Forms of the Parasite Trypanosoma brucei brucei Is Dependent on a Plant-like Alternative Oxidase

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.

Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO2

Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O(2) uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O(2) uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O(2) uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO(2), the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO(2), which induced a CO(2) concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.