Analysis of the sodium chloride-dependent respiratory kinetics of wheat mitochondria reveals differential effects on phosphorylating and non-phosphorylating electron transport pathways.

A number of previous studies have documented the gross response of mitochondrial respiration to salinity treatment, but it is unclear how NaCl directly affects the kinetics of plant phosphorylating and non-phosphorylating electron transport pathways. This study investigates the direct effects of NaCl upon different respiratory pathways in wheat, by measuring rates of isolated mitochondrial oxygen consumption across different substrate oxidation pathways in saline media. We also profile the abundance of respiratory proteins by using targeted selected reaction monitoring (SRM) mass spectrometry of mitochondria isolated from control and salt-treated wheat plants. We show that all pathways of electron transport were inhibited by NaCl concentrations above 400 mM; however electron transfer chains showed divergent responses to NaCl concentrations between 0 and 200 mM. Stimulation of oxygen consumption was measured in response to NaCl in scenarios where exogenous NADH was provided as substrate and electron flow was coupled to the generation of a proton gradient across the inner membrane. Protein abundance measurements show that several enzymes with activities less affected by NaCl are induced by salinity, whereas enzymes with activities inhibited by NaCl are depleted. These data deepen our understanding of how plant respiration responds to NaCl, offering new mechanistic explanations for the divergent salinity responses of whole-plant respiratory rate in the literature.

Proteomics in evolutionary ecology.

Evolutionary ecologists are traditionally gene-focused, as genes propagate phenotypic traits across generations and mutations and recombination in the DNA generate genetic diversity required for evolutionary processes. As a consequence, the inheritance of changed DNA provides a molecular explanation for the functional changes associated with natural selection. A direct focus on proteins on the other hand, the actual molecular agents responsible for the expression of a phenotypic trait, receives far less interest from ecologists and evolutionary biologists. This is partially due to the central dogma of molecular biology that appears to define proteins as the 'dead-end of molecular information flow' as well as technical limitations in identifying and studying proteins and their diversity in the field and in many of the more exotic genera often favored in ecological studies. Here we provide an overview of a newly forming field of research that we refer to as 'Evolutionary Proteomics'. We point out that the origins of cellular function are related to the properties of polypeptide and RNA and their interactions with the environment, rather than DNA descent, and that the critical role of horizontal gene transfer in evolution is more about coopting new proteins to impact cellular processes than it is about modifying gene function. Furthermore, post-transcriptional and post-translational processes generate a remarkable diversity of mature proteins from a single gene, and the properties of these mature proteins can also influence inheritance through genetic and perhaps epigenetic mechanisms. The influence of post-transcriptional diversification on evolutionary processes could provide a novel mechanistic underpinning for elements of rapid, directed evolutionary changes and adaptations as observed for a variety of evolutionary processes. Modern state-of the art technologies based on mass spectrometry are now available to identify and quantify peptides, proteins, protein modifications and protein interactions of interest with high accuracy and assess protein diversity and function. Therefore, proteomic technologies can be viewed as providing evolutionary biologist with exciting novel opportunities to understand very early events in functional variation of cellular molecular machinery that are acting as part of evolutionary processes.

Sperm and seminal fluid proteomes of the field cricket Teleogryllus oceanicus: identification of novel proteins transferred to females at mating.

Reproductive proteins are amongst the most evolutionarily divergent proteins known, and research on genetically well-characterized species suggests that postcopulatory sexual selection might be important in their evolution; however, we lack the taxonomic breadth of information on reproductive proteins that is required to determine the general importance of sexual selection for their evolution. We used transcriptome sequencing and proteomics to characterize the sperm and seminal fluid proteins of a cricket, Teleogryllus oceanicus, that has been widely used in the study of postcopulatory sexual selection. We identified 57 proteins from the sperm of these crickets. Many of these had predicted function in glycolysis and metabolism, or were structural, and had sequence similarity to sperm proteins found across taxa ranging from flies to humans. We identified 21 seminal fluid proteins, some of which resemble those found to be involved in postmating changes to female reproduction in other species. Some 27% of sperm proteins and 48% of seminal fluid proteins were of unknown function. The characterization of seminal fluid proteins in this species will allow us to explore their adaptive significance, and to contribute comparative data that will facilitate a general appreciation of the evolution of reproductive proteins within and among animal taxa.

The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase.

The F(O) portion of the mitochondrial ATP synthase contains a range of different subunits in bacteria, yeast and mammals. A search of the Arabidopsis genome identified sequence orthologs for only some of these subunits. Blue native polyacrylamide gel electrophoresis separation of Arabidopsis mitochondrial respiratory chain complexes revealed intact F(1)F(O), and separated F(1) and F(O) components. The subunits of each complex were analysed by mass spectrometry and matched to Arabidopsis genes. In the F(1)F(O) complex a series of nine known subunits were identified along with two additional proteins matching the predicted products of the mitochondrial encoded orfB and orf25 genes. The F(1) complex contained the five well-characterised F(1) subunits, while four subunits in the F(O) complex were identified: subunit 9, d subunit, and the orfB and orf25 products. Previously, orfB has been suggested as the plant equivalent of subunit 8 based on structural and sequence similarity. We propose that orf25 is the plant b subunit based on structural similarity and its presence in the F(O) complex. Chimerics of orf25, orfB, subunit 9 and subunit 6 have been associated with cytoplasmic male sterility in a variety of plant species, our additional findings now place all these proteins in the same protein complex.

Proteomic identification of divalent metal cation binding proteins in plant mitochondria.

Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identified a total of 23 distinct protein spots as the products of at least 11 different Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included: succinyl CoA ligase beta subunit, Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c(1) complex. A further set of four proteins of known function but without known divalent binding properties were also identified: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.

The impact of oxidative stress on Arabidopsis mitochondria.

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.

Analysis of the Arabidopsis mitochondrial proteome.

The complete set of nuclear genes that encode proteins targeted to mitochondria in plants is currently undefined and thus the full range of mitochondrial functions in plants is unknown. Analysis of two-dimensional gel separations of Arabidopsis cell culture mitochondrial protein revealed approximately 100 abundant proteins and 250 low-abundance proteins. Comparison of subfractions of mitochondrial protein on two-dimensional gels provided information on the soluble, membrane, or integral membrane locations of this protein set. A total of 170 protein spots were excised, trypsin-digested, and matrix-assisted laser desorption ionization/time of flight mass spectrometry spectra obtained. Using this dataset, 91 of the proteins were identified by searching translated Arabidopsis genomic databases. Of this set, 81 have defined functions based on sequence comparison. These functions include respiratory electron transport, tricarboxylic acid cycle metabolism, amino acid metabolism, protein import, processing, and assembly, transcription, membrane transport, and antioxidant defense. A total of 10 spectra were matched to Arabidopsis putative open reading frames for which no specific function has been determined. A total of 64 spectra did not match to an identified open reading frame. Analysis of full-length putative protein sequences using bioinformatic tools to predict subcellular targeting (TargetP, Psort, and MitoProt) revealed significant variation in predictions, and also a lack of mitochondrial targeting prediction for several characterized mitochondrial proteins.

Nucleoside diphosphate kinase III is localized to the inter-membrane space in plant mitochondria.

Three types of nucleoside diphosphate kinases (NDPKs) are found in plants but the intra-cellular compartmentation of these proteins is not certain, especially the location of the recently identified type III proteins. Through the fractionation of plant mitochondria from potato and Arabidopsis, display of protein profiles by 2D gel electrophoresis, and identification by mass spectrometry, we present the first direct evidence that type III proteins are localized in the inter-membrane space of plant mitochondria. The possible metabolic functions of NDPK III are discussed in light of its sub-cellular localization.

NAD malic enzyme and the control of carbohydrate metabolism in potato tubers.

Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations.

Mitochondrial biogenesis during germination in maize embryos.

Mitochondrial biogenesis and metabolism were investigated during maize (Zea mays) seed germination. Mitochondria from dry and imbibed seed exhibited NADH-dependent O(2) uptake that was completely inhibited by KCN and antimycin A. Mitochondria in the dry seed had a lower rate of succinate-dependent O(2) uptake relative to that measured in imbibed and germinated seed. The activities of the tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, NAD-malic enzyme, and citrate synthase, are similarly low in mitochondria from dry seed and this correlates with a lower relative abundance of the mitochondrial matrix-located citrate synthase and pyruvate dehydrogenase complex E1alpha-subunit polypeptides. Electron microscopy revealed that mitochondria in the dry seed have a poorly developed internal membrane structure with few cristae; following 24 h of germination the mitochondria developed a more normal structure with more developed cristae. The mitochondria from maize embryos could be fractionated into two subpopulations by Suc density gradient centrifugation: one subpopulation of buoyant density equivalent to 22% to 28% (w/w) Suc; the other equivalent to 37% to 42% (w/w) Suc. These two subpopulations had different activities of specific mitochondrial enzymes and contained different amounts of specific mitochondrial proteins as revealed by western-blot analysis. Both subpopulations from the dry embryo were comprised of poorly developed mitochondria. However, during imbibition mitochondria in the heavy fraction (37%-42% [w/w] Suc) progressively acquired characteristics of fully functional mitochondria found in the germinated seedling in terms of structure, enzymic activity, and protein complement. In contrast, mitochondria in the light fraction (22% to 28% [w/w] Suc) show no significant structural change during imbibition and the amounts of specific mitochondrial proteins decreased significantly during germination.

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria.

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.

Plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterization in potato.

The 2-oxoglutarate dehydrogenase complex (OGDC) in potato (Solanum tuberosum cv. Romano) tuber mitochondria is largely associated with the membrane fraction of osmotically ruptured organelles, whereas most of the other tricarboxylic acid cycle enzymes are found in the soluble matrix fraction. The purification of OGDC from either membrane or soluble matrix fractions resulted in the increasing dependence of its activity on the addition of dihydrolipoamide dehydrogenase (E3). A 30-fold purification of OGDC to apparent homogeneity and with a specific activity of 4.6 micromol/min per mg of protein in the presence of exogenously added E3 was obtained. SDS/PAGE revealed that the purified complex consisted of three major polypeptides with apparent molecular masses of 48, 50 and 105 kDa. Before the gel-filtration purification step, E3 polypeptides of 57 and 58 kDa were identified by immunoreaction as minor proteins associated with OGDC. The N-terminal sequence of the 57 kDa protein was identical with that previously purified as the E3 component of the pyruvate dehydrogenase complex from potato. The 105 kDa protein was identified as the 2-oxoglutarate dehydrogenase subunit of OGDC by N-terminal sequencing. The N-terminal sequences of the 50 and 48 kDa proteins shared 90-95% identity over 20 residues and were identified by sequence similarity as dihydrolipoamide succinyltransferases (OGDC-E2). The incubation of OGDC with [U-(14)C]2-oxoglutarate resulted in the reversible succinylation of both the 48 and the 50 kDa protein bands. Proteins previously reported as subunits of complex I of the respiratory chain from Vicia faba and Solanum tuberosum are proposed to be OGDC-E2 and the possible basis of this association is discussed.

Characterization of the dihydrolipoamide acetyltransferase of the mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar enzymes in diverse plant species.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.

Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase.

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.

Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plant mitochondria.

Oxygen consumption via the cytochrome pathway in isolated soybean (Glycine max [L.] Merr.) cotyledon mitochondria was inhibited by nitric oxide (NO) while respiration via the cyanide-insensitive alternative oxidase was not significantly affected. Inhibition of cytochrome pathway activity was rapidly reversible upon depletion of the added NO. NO production was also detected in solutions of NaNO2 plus ascorbate and the extent of cytochrome pathway inhibition was dependent on the NO2- concentration. Little inhibition of alternative pathway respiration was observed under similar conditions. The alternative oxidase may play a role in nitric oxide tolerance in higher plants and in organisms such as trypanosomes which contain a plant-like alternative oxidase.

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria.

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.

The alternative oxidase is encoded in a multigene family in soybean.

The copy number of the alternative oxidase gene, Aox, was investigated in soybean (Glycine max L.) using a Polymerase chain reaction (PCR) approach to amplify fragments from soybean genomic DNA. The primers used were based on absolutely conserved regions of Aox cDNA clones from a variety of plant species and the yeast Hansenula anomala. After subcloning of the 170-bp PCR products, 12 individual colonies were sequenced. Eleven plasmids yielded inserts representing three sequences in the ratio 4:3:4 (Aox1-3). The sequence of Aox1 was 100% identical at the nucleic acid level to the published full-length cDNA from soybean. The other two sequences were 60-75% identical to Aox1 and to each other at the nucleic acid and protein levels. Similar analysis of Nicotiana tabacum L. revealed an additional gene copy with high homology to the soybean Aox2 sequence. Genomic DNA from soybean cut with Hind III and probed with the full-length Aox1 yielded a single positive band of 6.5 kb; when the same genomic blot was probed with a mixture of all three PCR fragments, three bands of 9 kb, 6.5 kb and 3 kb were detected. Reverse transcription-PCR performed on total RNA from various soybean tissues, followed by hybridisation with the three Aox sequences individually, revealed differential expression of the Aox genes between cotyledons and leaves. It is suggested that soybean contains a multigene Aox family. The implication of this for the understanding of alternative oxidase expression and regulation in plant tissues is discussed.