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.     

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.     

Genetic modification of respiratory capacity in potato.

Mitochondrial respiration was altered in transgenic potato (Solanum tuberosum) lines by overexpression of the alternative oxidase Aox1 gene. Overexpressing lines showed higher levels of Aox1 mRNA, increased levels of alternative oxidase protein(s), and an unusual higher molecular weight polypeptide, which may be a normal processing/modification intermediate. Evidence suggests that the alternative oxidase protein is further processed/modified beyond removal of the transit peptide. Addition of pyruvate to mitochondria oxidizing succinate or NADH increased the alternative pathway capacity but did not eliminate the difference in the capacity between these two substrates. Induction of alternative pathway capacity by aging of tubers appeared to be more dependent on increased levels of alternative oxidase protein than changes in its oxidation state. In leaf and tuber mitochondria, overexpressing lines possessed higher alternative pathway capacity than the control line, which suggests that changing the alternative oxidase protein level by genetic engineering can effectively change alternative pathway capacity.     

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.     

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.     

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.     

Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated tobacco plants

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O(3)). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O(3) response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O(3) fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O(3)-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.     

Effects of water stress on development, operation and gene expression of cyanide-resistant respiratory pathway in wheat

Osmotic dehydration of wheat seedlings in-0.5 MPa polyethylene glycol (PEG) solutions for 24, 48 and 72 h resulted in mild, moderate and severe water stress respectively in leaves, but only caused mild water stress in roots as reflected by the changes in relative water content (RWC). In response to the above water stress conditions, leaf total respiratory rate (V_t) decreased progressively, and the alternative pathway (AP) capacity (V_(alt)) and its actual operation activity (ρV_(alt)) decreased more severely. Water stress also led to continuous reduction in cytochrome pathway (CP) activity ((ρ' V_(cyt)) and different changes in the contribution of ρV_(alt) and ρ' V_(cyt) to V_t in leaves, with ρV_(alt)/V_t decreasing and ρ' V_(cyt)/V_t increasing. The change pattern of root V_t was similar to that of its RWC, while root V_(alt) and ρV_(alt) were found to decrease during the first 24 h of stress and thereafter recover to a level close to that of the control (O h). These data indicate that the alt     

The expression of alternative oxidase and uncoupling protein during fruit ripening in mango

The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of the single gene for the Ucp peaked at the turning stage and the protein abundance peaked at the ripe stage. Proteins of the cytochrome chain peaked at the mature stage of ripening. The pattern of protein accumulation suggested that increases in cytochrome chain components played an important role in facilitating the climacteric burst of respiration and that the Aox and Ucp may play a role in post-climacteric senescent processes. Because both message and protein for the Aox and Ucp increased in a similar pattern, it suggests that their expression is not controlled in a reciprocal manner but may be active simultaneously.     

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.     

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.     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

Inactivation of the petE gene for plastocyanin lowers photosynthetic capacity and exacerbates chilling-induced photoinhibition in the cyanobacterium Synechococcus.

We describe the identification and expression of a petE gene in Synechococcus sp. PCC 7942, a cyanobacterium previously thought to lack plastocyanin. The petE gene is a 420-bp open reading frame that encodes a protein 70 to 75% similar to plastocyanins from other cyanobacteria. Synechococcus possesses a single genomic copy of petE located immediately upstream of the clpB gene. It is transcribed as a single mRNA (550 bases) and, in contrast to most other photobionts, the level of petE expression in Synechococcus is unaffected by variable copper concentrations during acclimated growth. Inactivation of petE does not prevent photoautotrophic growth, but does induce a dramatic increase in mRNA for the alternative electron carrier cytochrome C6. Despite this adjustment, loss of plastocyanin results in slower growth, lower photosystem I content, and a decreased maximum capacity for photosynthetic electron transport. The mutant is also more susceptible to chilling-induced photoinhibition during a shift from 37 to 25 degrees C, at which temperature its inherently lower photosynthetic capacity exacerbates the normal slowing of electron transfer reactions at low temperatures. Under similar conditions, the amount of petE message in the wild type decreases by 50% in the 1st h, but then increases dramatically to almost three times the 37 degrees C level by 9 h.     

Rapid induction by wounding and bacterial infection of an S gene family receptor-like kinase gene in Brassica oleracea.

A receptor-like kinase, SRK, has been implicated in the autoincompatible response that leads to the rejection of self-pollen in Brassica plants. SRK is encoded by one member of a multigene family, which includes several receptor-like kinase genes with patterns of expression very different from that of SRK but of unknown function. Here, we report the characterization of a novel member of the Brassica S gene family, SFR2. RNA gel blot analysis demonstrated that SFR2 mRNA accumulated rapidly in response both to wounding and to infiltration with either of two bacteria: Xanthomonas campestris, a pathogen, and Escherichia coli, a saprophyte. SFR2 mRNA also accumulated rapidly after treatment with salicylic acid, a molecule that has been implicated in plant defense response signaling pathways. A SFR2 promoter and reporter gene fusion was introduced into tobacco and was shown to be induced by bacteria of another genus, Ralstonia (Pseudomonas) solanacearum. The accumulation of SFR2 mRNA in response to wounding and pathogen invasion is typical of a gene involved in the defense responses of the plant. The rapidity of SFR2 mRNA accumulation is consistent with SFR2 playing a role in the signal transduction pathway that leads to induction of plant defense proteins, such as pathogenesis-related proteins or enzymes of phenylpropanoid metabolism.