The production of an inducible antisense alternative oxidase (Aox1a) plant

Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain. Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh. and the procedures used to determine the resulting alternative pathway activity. The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation. Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct. Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A. After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves. Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction. Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this. These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system. Received: 31 March 2000 / Accepted: 10 May 2000     

Visualization of alternative splicing in vivo

The analysis of sequences required for alternative splicing of mRNA has predominantly been performed using cell culture systems. However, the phenotype of cultured cells is almost invariably different from that of cells in the intact animal. It is therefore possible that there are significant differences in the regulation of specific splicing reactions in vivo compared to in cell culture. Here, we describe methods for the visualization and analysis of alternative splicing in vivo using transgenic mice. These methods allow for the analysis of the temporal and tissue-specific regulation of alternative splicing both visually and quantitatively.     

Regulation of alternative oxidase gene expression in soybean

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 °C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.     

Influence of ethanol on alternative oxidase in mitochondria from callus-forming potato tuber discs

Ethanol, when added to the incubation medium of callus-forming potato tuber discs, inhibits callus growth and causes an increase of the mitochondrial antimycin-A resistant respiration, expressed as a percentage of state III-respiration. This increase in resistance to antimycin-A is the result of a poor development of the cytochrome pathway in tissue discs treated with ethanol. The development of the antimycin-A resistant alternative oxidase sensitive to chelator is about the same for treated and untreated discs. The respiratory control (RC) ratio of the mitochondrial respiration increases after addition of a chelator, which inhibits the alternative pathway. The RC ratio of the uninhibited mitochondrial respiration appears to be inversely related to the capacity of the alternative pathway, when mitochondrial preparations with different capacities to transfer electrons via the alternative path are compared. From the experimentally observed relation between RC-ratio and alternative oxidase capacity, it was concluded that at least half of the capacity of the alternative path is used in uninhibited state IV respiration.     

Regulation of alternative oxidase activity in higher plants

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanideinsensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.     

The expression of alternative oxidase and alternative respiratory capacity in cytoplasmic male sterile common bean

Summary The alternative respiratory pathway is present in all plant species investigated to date. Yet, the role of the alternative pathway is not clear. Some evidence suggests an important role in pollen development. We undertook this study to investigate the expression of alternative oxidase, in comparison with expression of a component of cytochrome oxidase, during pollen formation in common bean (Phaseolus vulgaris L.). Expression was evaluated immunohistochemically. In addition, we compared both the alternative oxidase capacity in young seedling tissues and alternative oxidase expression in developing flower buds of isonuclear cytoplasmic male sterile and male fertile bean lines. We observed no evidence of an association between the abnormal pollen development of CMS bean and changes in alternative oxidase expression or capacity. We did observe a tissueand stage-specific pattern of expression of alternative oxidase, differing from the expression pattern of cytochrome oxidase subunit II, during anther development in normal bean lines. Although no association was evident between the cytoplasmic male sterility phenotype and differential expression of alternative oxidase, the regulated pattern of alternative oxidase expression in developing anthers does suggest that the alternative pathway may play a role in microgametogenesis and microsporogenesis.     

Origins, evolutionary history, and taxonomic distribution of alternative oxidase and plastoquinol terminal oxidase

Alternative oxidase (AOX) and plastoquinol terminal oxidase (PTOX) are related quinol oxidases associated with respiratory and photosynthetic electron transport chains, respectively. Contrary to previous belief, AOX is present in numerous animal phyla, as well as heterotrophic and marine phototrophic proteobacteria. PTOX appears limited to organisms capable of oxygenic photosynthesis, including cyanobacteria, algae and plants. We propose that both oxidases originated in prokaryotes from a common ancestral di-iron carboxylate protein that diversified to AOX within ancient proteobacteria and PTOX within ancient cyanobacteria. Each then entered the eukaryotic lineage separately; AOX by the endosymbiotic event that gave rise to mitochondria and later PTOX by the endosymbiotic event that gave rise to chloroplasts. Both oxidases then spread through the eukaryotic domain by vertical inheritance, as well as by secondary and potentially tertiary endosymbiotic events.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Role of the alternative oxidase in limiting superoxide production by plant mitochondria

Mitochondria isolated from the pericarp tissue of green bell pepper ( Capsicum annuum L.) fruit and purified on a Percoll gradient produced superoxide in buffers aerated with oxygen. ADP and uncouplers of the electron transport chain reduced superoxide production. Disulfiram, an inhibitor of the alternative oxidase, enhanced superoxide production. Inhibitors of complex III had little effect on superoxide production by mitochondria which were insensitive to cyanide. Less superoxide was produced when dithiothreitol was used to reduce the sulfhydryl groups of the alternative oxidase protein and the enzyme was activated with pyruvate than when the sulfhydryl groups were oxidized with diamide. A role for the alternative oxidase in limiting the level of reactive oxygen species produced in stressed and senescing plant tissues is suggested.     

Structure-function relationships of the alternative oxidase of plant mitochondria: A model of the active site

A major characteristic of plant mitochondria is the presence of a cyanide-insensitive alternative oxidase which catalyzes the reduction of oxygen to water. Current information on the properties of the oxidase is reviewed. Conserved amino acid motifs have been identified which suggest the presence of a hydroxo-bridged di-iron center in the active site of the alternative oxidase. On the basis of sequence comparison with other di-iron center proteins, a structural model for the active site of the alternative oxidase has been developed that has strong similarity to that of methane monoxygenase. Evidence is presented to suggest that the alternative oxidase of plant mitochondria is the newest member of the class II group of di-iron center proteins.     

Exploring the molecular nature of alternative oxidase regulation and catalysis

Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein-derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.     

Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a GA change at 291 in the first putative exon, resulting in a ValMet substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this GA change at the equivalent position (5751) within exon 10.     

Utilization of a chloroplast membrane sulfolipid as a major internal sulfur source for protein synthesis in the early phase of sulfur starvation in Chlamydomonas reinhardtii

Information is limited on sulfur (S)-sources inside plant cells for synthesis of the proteins for acclimation to S-starvation. We found that a green alga, Chlamydomonas reinhardtii, when transferred to S-starved conditions, degrades 85% of a chloroplast membrane lipid, sulfoquinovosyl diacylglycerol (SQDG), to redistribute its S to a large part of protein fraction as early as by 6h. Furthermore, the degradation of SQDG preceded that of proteins such as ribulose bisphosphate carboxylase/oxygenase, the candidates of internal S-sources. SQDG was thus demonstrated to yield a major internal S-source for protein synthesis during the early phase of acclimation process to S-starvation.     

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.     

Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation.

The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.