Expression of uncoupling protein and alternative oxidase depends on lipid or carbohydrate substrates in thermogenic plants

Thermogenesis, in which cellular respiratory activity is considerably stimulated, requires mitochondrial uncoupling protein (UCP) in mammals and an alternative oxidase (AOX) in plants. Here, we show that the genes for both proteins are expressed in thermogenic plants, but the type correlates with the respiratory substrate. A novel gene termed PsUCPa encoding a variant of UCP was specifically expressed in thermogenic flowers of Philodendron selloum, which uses lipids as substrates. However, a gene termed DvAOX encoding for AOX protein was expressed in thermogenic flowers of Dracunculus vulgaris, which presumably uses carbohydrates as substrates. These findings suggest that cellular metabolism is a major determinant in selective expression of appropriate thermogenic genes in plants.     

Branched mitochondrial electron transport in the Animalia: presence of alternative oxidase in several animal phyla

The mitochondrion of most eukaryotes has multiple electron transport components that increase the points of entry and/or exit of electrons, thus giving a branched nature to the respiratory chain. In plants and many other organisms, a prominent example is alternative oxidase, a non-energy conserving branch in the respiratory chain and an additional terminal oxidase for the exit of electrons. Our genome database searches have now revealed the presence of alternative oxidase in four animal species from three different phyla (Mollusca, Nematoda and Chordata), consistent with frequent reports of cyanide-resistant respiration in the Animalia. In Ciona intestinalis and Crassostrea gigas, alternative oxidase is expressed in several different tissues. Phylogenetic analysis is consistent with the animal proteins having originated by vertical inheritance. We hypothesize that alternative oxidase is likely widespread in the Animalia and discuss some of the potential role(s) for such a branched respiratory chain.     

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis

Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membranebound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants’ seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants.     

Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase

The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.     

Supramolecular structure of the OXPHOS system in highly thermogenic tissue of Arum maculatum

The protein complexes of the mitochondrial respiratory chain associate in defined ways forming supramolecular structures called respiratory supercomplexes or respirasomes. In plants, additional oxidoreductases participate in respiratory electron transport, e.g. the so-called “alternative NAD(P)H dehydrogenases” or an extra terminal oxidase called “alternative oxidase” (AOX). These additional enzymes were previously reported not to form part of respiratory supercomplexes. However, formation of respiratory supercomplexes might indirectly affect “alternative respiration” because electrons can be channeled within the supercomplexes which reduces access of the alternative enzymes towards their electron donating substrates. Here we report an investigation on the supramolecular organization of the respiratory chain in thermogenic Arum maculatum appendix mitochondria, which are known to have a highly active AOX for heat production. Investigations based on mild membrane solubilization by digitonin and protein separation by blue native PAGE revealed a very special organization of the respiratory chain in A. maculatum, which strikingly differs to the one described for the model plant Arabidopsis thaliana: (i) complex I is not present in monomeric form but exclusively forms part of a I + III₂ supercomplex, (ii) the III₂ + IV and I + III₂ + IV supercomplexes are detectable but of low abundance, (iii) complex II has fewer subunits than in A. thaliana, and (iv) complex IV is mainly present as a monomer in a larger form termed “complex IVa”. Since thermogenic tissue of A. maculatum at the same time has high AOX and I + III₂ supercomplex abundance and activity, negative regulation of the alternative oxidase by supercomplex formation seems not to occur. Functional implications are discussed.     

Mutants of the phytopathogenic fungus magnaporthe grisea deficient in alternative, cyanide-resistant, respiration

The phytopathogenic fungus Magnaporthe grisea has a cyanide-resistant respiratory pathway. The fungicide SSF-126 ((E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl) acetamide) blocks the cytochrome electron transport of M. grisea and induces the alternative respiratory pathway. Twelve mutants of M. grisea more susceptible to SSF-126 than wild type were identified after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Five mutants retained a reduced alternative respiration activity, and seven mutants lacked alternative pathway activity. A monoclonal antibody against the maize alternative oxidase cross-reacted against a 40-kDa mitochondrial protein of M. grisea, indicating that the 40-kDa protein is an alternative oxidase. Immunoblot analysis indicated that the seven completely deficient mutants grouped into two classes: four mutants produced the 40-kDa proteins while the other three mutants failed to produce the functional protein. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP(+). In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90-95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP(+). Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP(+) photoreduction.     

The plant mitochondrial proteome and the challenge of defining the posttranslational modifications responsible for signalling and stress effects on respiratory functions

The mitochondrion is the principle organelle in plant aerobic respiration, where the oxidation of organic acids to CO₂ and H₂O, combined with the coupling of electron transfer to O₂ via the respiratory electron transport chain to adenosine triphosphate synthesis, takes place. Plant mitochondria also have important secondary roles, such as the synthesis of nucleotides, amino acids, lipids, prosthetic groups and vitamins. They also interact with chloroplasts and peroxisomes through a series of primary metabolic pathways. By using proteomic tools such as polyacrylamide gel-based and mass spectrometry-based methods, over 400 proteins, including 30 proteins from the tricarboxylic acid cycle, 78 proteins from the electron transport chain and more than 20 proteins from amino acid metabolism pathways have been identified in mitochondria of the model plant, Arabidopsis thaliana . Beyond the mitochondrial proteome, there is growing evidence for reversible protein phosphorylation and oxidative posttranslational modifications (PTMs) that could affect functions of individual plant mitochondrial proteins or protein complexes. This review will discuss the progress in defining the PTMs that have the potential to regulate plant mitochondrial functions, with references to studies in plants, yeast and mammalian mitochondria and the development of various proteomic and affinity purification methods to study them.     

Cyanide resistant respiration and the alternative oxidase pathway: A journey from plants to mammals

In a large number of organisms covering all phyla, the mitochondrial respiratory chain harbors, in addition to the conventional elements, auxiliary proteins that confer adaptive metabolic plasticity. The alternative oxidase (AOX) represents one of the most studied auxiliary proteins, initially identified in plants. In contrast to the standard respiratory chain, the AOX mediates a thermogenic cyanide-resistant respiration; a phenomenon that has been of great interest for over 2 centuries in that energy is not conserved when electrons flow through it. Here we summarize centuries of studies starting from the early observations of thermogenicity in plants and the identification of cyanide resistant respiration, to the fascinating discovery of the AOX and its current applications in animals under normal and pathological conditions.     

Enzymology and bioenergetics of respiratory nitrite ammonification

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.     

Effects of light on respiration and oxygen isotope fractionation in soybean cotyledons

Light effects on electron flow through the cyanide-resistant respiratory pathway, oxygen isotope fractionation and total respiration were studied in soybean (Glycine max L.) cotyledons. During the first 12 h of illumination there was an increase in both electron partitioning through the alternative pathway and oxygen isotope fractionation by the alternative oxidase. The latter probably indicates a change in the properties of the alternative oxidase. There was no engagement of the alternative oxidase in darkness and its fractionation was 27‰. In green cotyledons 60% of the respiration flux was through the alternative pathway and the alternative oxidase fractionation was 32‰. Exposing previously illuminated tissue to continuous darkness induced a decrease in the electron partitioning through the alternative pathway. However, this decrease was not directly linked with the low cellular sugar concentration resulting from the lack of light because 5 min of light every 12 h was sufficient to keep the alternative pathway engaged to the same extent as plants grown under control conditions.     

The Effect of Growth and Measurement Temperature on the Activity of the Alternative Respiratory Pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19°C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28°C but was unchanged in soybean cotyledons grown at 14°C compared with plants grown at 28°C. When the short-term response of tissue respiration was measured over the temperature range of 35°C to 9°C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19°C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.     

Chitinase III in pomegranate seeds (Punica granatum Linn.): a high-capacity calcium-binding protein in amyloplasts

Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.