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.     

Analysis of the alternative oxidase promoters from soybean

Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive beta-glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.     

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.     

Chemically induced virus resistance in Arabidopsis thaliana is independent of pathogenesis-related protein expression and the NPR1 gene

Salicylic acid (SA) treatment triggers inhibition of replication or movement of several positive-sense RNA plant viruses in tobacco. This resistance can also be stimulated by nonlethal concentrations of cyanide and antimycin A (AA) without triggering induction of pathogenesis-related PR-1 protein genes. In two ecotypes of Arabidopsis thaliana (Columbia and N?ssen), SA-induced resistance to a tobamovirus, Turnip vein clearing virus (TVCV), was also induced by nonlethal concentrations of cyanide and AA without concomitant induction of PR-1 gene expression. Furthermore, chemically induced resistance to TVCV, as well as the induction of the plant mitochondrial alternative oxidase (a potential target for the chemicals), was independent of NPR1, a gene that plays a key role downstream of SA in the induction of PR proteins. The chemically induced resistance to TVCV appeared to be due to inhibition of replication at the site of inoculation. Taken together, these results show that in Arabidopsis, as in tobacco, resistance to viruses can be induced via a distinct branch of the defensive signal transduction pathway. This suggests that the existence of this virus-specific branch may be widespread among plants.     

Mitochondrial electron transport regulation of nuclear gene expression. Studies with the alternative oxidase gene of tobacco.

We have isolated a cDNA representing the tobacco (Nicotiana tabacum L. cv Bright Yellow) nuclear gene Aox1, which encodes the alternative oxidase of plant mitochondria. The clone contains the complete coding region (1059 base pairs) of a precursor protein of 353 amino acids with a calculated molecular mass of 39.8 kD. A putative transit peptide contains common signals believed to be important for import and processing of mitochondrially localized proteins. We have studied changes in Aox1 gene expression in tobacco in response to changes in cytochrome pathway activity. Inhibition of the cytochrome pathway by antimycin A resulted in a rapid and dramatic accumulation of Aox1 mRNA, whereas the level of mRNAs encoding two proteins of the cytochrome pathway did not change appreciably. This was accompanied by a dramatic increase in alternative pathway capacity and engagement in whole cells. Respiration under these conditions was unaffected by the uncoupler p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, levels of Aox1 mRNA returned to control levels, alternative pathway capacity and engagement declined, and respiration could once again be stimulated by FCCP. The results show that a mechanism involving changes in Aox1 gene expression exists whereby the capacity of the alternative pathway can be adjusted in response to changes in the activity of the cytochrome pathway.     

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.     

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     

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.     

nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.     

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.     

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.     

The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.