Biomedical subjects

David A Day

Publications and source records attributed to David A Day.

At least 19 recordsLinked to original sources

PubMed2005

Characterization of mitochondrial alternative NAD(P)H dehydrogenases in Arabidopsis: intraorganelle location and expression.

The intramitochondrial location of putative type II NAD(P)H dehydrogenases (NDs) in Arabidopsis was investigated by measuring the ability of isolated mitochondria to take up precursor proteins generated from cDNAs using an in vitro translation system. The mature proteins of NDA1, NDA2 and NDC1 were judged to be located on the inside of the inner membrane because they were protected from protease added after the mitochondrial outer membrane had been ruptured. In contrast, NDB1, NDB2 and NDB4 were not protected from protease digestion in mitochondria with ruptured outer membranes and were deemed to be located on the outside of the inner membrane. Expression of all ND genes was measured using quantitative reverse transcription-PCR (RT-PCR) to determine transcript abundance, and compared with expression of alternative oxidase, uncoupler proteins and selected components of the oxidative phosphorylation complexes. NDA1 and NDB2 were the most prominently expressed members in a variety of tissues, and were up-regulated in the early daytime in a diurnal manner. Analysis of array data suggested that NDA1 clustered closest to the gene encoding the P-subunit of glycine decarboxylase. Taken together with the diurnal regulation of NDA1 observed here and in other studies, this suggests that NDA1 plays a role in integrating metabolic activities of chloroplasts and mitochondria. NDA2, NDB2 and Aox1a were up-regulated in a coordinated manner under various treatments, potentially forming a complete respiratory chain capable of oxidizing matrix and cytosolic NAD(P)H. NDB1 and NDC1 were down-regulated under the same conditions and may be regarded as housekeeping genes.

Arabidopsis↗

PubMed2005

Effects of water stress on respiration in soybean leaves.

The effect of water stress on respiration and mitochondrial electron transport has been studied in soybean (Glycine max) leaves, using the oxygen-isotope-fractionation technique. Treatments with three levels of water stress were applied by irrigation to replace 100%, 50%, and 0% of daily water use by transpiration. The levels of water stress were characterized in terms of light-saturated stomatal conductance (g(s)): well irrigated (g(s) > 0.2 mol H(2)O m(-2) s(-1)), mildly water stressed (g(s) between 0.1 and 0.2 mol H(2)O m(-2) s(-1)), and severely water stressed (g(s) < 0.1 mol H(2)O m(-2) s(-1)). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (V(t)) was not significantly different at any water-stress level. However, severe water stress caused a significant shift of electrons from the cytochrome to the alternative pathway. The electron partitioning through the alternative pathway increased from 10% to 12% under well-watered or mild water-stress conditions to near 40% under severe water stress. Consequently, the calculated rate of mitochondrial ATP synthesis decreased by 32% under severe water stress. Unlike many other stresses, water stress did not affect the levels of mitochondrial alternative oxidase protein. This suggests a biochemical regulation (other than protein synthesis) that causes this mitochondrial electron shift.

Adenosine Triphosphate↗

PubMed2005

Differential impact of environmental stresses on the pea mitochondrial proteome.

Exposure to adverse environmental conditions causes oxidative stress in many organisms, leading either to disease and debilitation or to response and tolerance. Mitochondria are a key site of oxidative stress and of cellular response and play important roles in cell survival. We analyzed the response of mitochondria in pea (Pisum sativum) plants to the common stresses associated with drought, cold, and herbicides. These treatments all altered photosynthetic and respiratory rates of pea leaves to various extents, but only herbicides significantly increased lipid peroxidation product accumulation. Mitochondria isolated from the stressed pea plants maintained their electron transport chain activity, but changes were evident in the abundance of uncoupling proteins, non-phosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties on mitochondrial proteins. These data suggest that herbicide treatment placed a severe oxidative stress on mitochondria, whereas chilling and particularly drought were milder stresses. Detailed analysis of the soluble proteome of mitochondria by gel electrophoresis and mass spectrometry revealed differential degradation of key matrix enzymes during treatments with chilling being significantly more damaging than drought. Differential induction of heat shock proteins and specific losses of other proteins illustrated the diversity of response to these stresses at the protein level. Cross-species matching was required for mass spectrometry identification of nine proteins because only a limited number of pea cDNAs have been sequenced, and the full pea genome is not available. Blue-native separation of intact respiratory chain complexes revealed little if any change in response to environmental stresses. Together these data suggest that although many of the molecular events identified by chemical stresses of mitochondria from a range of model eukaryotes are also apparent during environmental stress of plants, their extent and significance can vary substantially.

Cold Temperature↗

PubMed2005

Sensitivity of plant mitochondrial terminal oxidases to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

We have investigated the effect of the lipid peroxidation product, HNE (4-hydroxy-2-nonenal), on plant mitochondrial electron transport. In mitochondria isolated from Arabidopsis thaliana cell cultures, HNE inhibited succinate-dependent oxygen consumption via the Aox (alternative oxidase), but had minimal effect on respiration via Cox (cytochrome c oxidase). Maximal Cox activity, measured with reduced cytochrome c as substrate, was only slightly inhibited by high concentrations of HNE, at which Aox was completely inhibited. Incubation with HNE prevented dimerization of the Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerization and activation of this enzyme (Cys(I)). However, a naturally occurring isoform of Aox lacking Cys(I) and unable to be dimerized, LeAox1b from tomato (Lycopersicon esculentum), was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE. The presence of HNE in vivo in Arabidopsis cell cultures was also investigated. Induction of oxidative stress in the cell cultures by the addition of hydrogen peroxide, antimycin A or menadione, caused a significant increase in hydroxyalkenals (of which HNE is the most prominent). Western blotting of mitochondrial proteins with antibodies against HNE adducts, demonstrated significant modification of proteins during these treatments. The implications of these results for the response of plants to reactive oxygen species are discussed.

Aldehydes↗

PubMed2005

Stress-induced co-expression of alternative respiratory chain components in Arabidopsis thaliana.

Plant mitochondria contain non-phosphorylating bypasses of the respiratory chain, catalysed by the alternative oxidase (AOX) and alternative NADH dehydrogenases (NDH), as well as uncoupling (UCP) protein. Each of these components either circumvents or short-circuits proton translocation pathways, and each is encoded by a small gene family in Arabidopsis. Whole genome microarray experiments were performed with suspension cell cultures to examine the effects of various 3 h treatments designed to induce abiotic stress. The expression of over 60 genes encoding components of the classical, phosphorylating respiratory chain and tricarboxylic acid cycle remained largely constant when cells were subjected to a broad range of abiotic stresses, but expression of the alternative components responded differentially to the various treatments. In detailed time-course quantitative PCR analysis, specific members of both AOX and NDH gene families displayed coordinated responses to treatments. In particular, the co-expression of AOX1a and NDB2 observed under a number of treatments suggested co-regulation that may be directed by common sequence elements arranged hierarchically in the upstream promoter regions of these genes. A series of treatment sets were identified, representing the response of specific AOX and NDH genes to mitochondrial inhibition, plastid inhibition and abiotic stresses. These treatment sets emphasise the multiplicity of pathways affecting alternative electron transport components in plants.

Anaerobiosis↗

PubMed2004

Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase.

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.

Carbon Dioxide↗

PubMed2004

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis.

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2004

Mitochondrial permeability transition induced by dinuclear gold(I)-carbene complexes: potential new antimitochondrial antitumour agents.

Seven dinuclear gold(I) complexes of bidentate N-heterocyclic carbene ligands have been evaluated for their ability to induce mitochondrial membrane permeabilisation (MMP) in isolated rat liver mitochondria. Six of the compounds, at concentrations of 10 microM, induced Ca(2+)-sensitive MMP as evidenced by mitochondrial swelling. In the absence of low concentrations of exogenous Ca(2+), the compounds were either inactive or their activity was significantly decreased. The mitochondrial swelling was completely blocked by the addition of cyclosporin A, a well established inhibitor of the mitochondrial permeability transition pore (MPT) that is believed to be responsible for MMP. The rates and levels of uptake of these compounds into mitochondria were estimated by measuring mitochondrial Au levels using inductively coupled plasma optical emission spectroscopy. Significant differences were found in the levels at which the different compounds accumulated in the mitochondria, but these differences did not correlate with the rate at which they induced mitochondrial swelling. These results suggest that the mechanism by which MMP is induced by these lipophilic cationic Au(I)-carbene complexes is not purely a function of the level of compound accumulation. Instead, a more specific mechanism, possibly involving disruption of the function of a particular enzyme, or interaction with a MPT component, appears to be more likely.

Animals↗

PubMed2004

Maintenance of growth rate at low temperature in rice and wheat cultivars with a high degree of respiratory homeostasis is associated with a high efficiency of respiratory ATP production.

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature) when grown at different temperatures. This phenomenon is referred to as respiratory homeostasis. Using wheat and rice cultivars with different degrees of respiratory homeostasis (H), we previously demonstrated that high-H cultivars maintained shoot and root growth at low temperature [Kurimoto et al. (2004) Plant Cell Environ., 27: 853]. Here, we assess the relationship between respiratory homeostasis and the efficiency of respiratory ATP production, by measuring the levels of alternative oxidase (AOX) and uncoupling protein (UCP), which have the potential to decrease respiratory ATP production per unit of oxygen consumed. We also measured SHAM- and CN-resistant respiration of intact roots, and the capacity of the cytochrome pathway (CP) and AOX in isolated mitochondria. Irrespective of H, SHAM-resistant respiration of intact roots and CP capacity of isolated root mitochondria were larger when plants were grown at low temperature, and the maximal activity and relative amounts of cytochrome c oxidase showed a similar trend. In contrast, CN-resistant respiration of intact roots and relative amounts of AOX protein in mitochondria isolated from those roots, were lower in high-H plants grown at low temperature. In the roots of low-H cultivars, relative amounts of AOX protein were higher at low growth temperature. Relative amounts of UCP protein showed similar trends to AOX. We conclude that maintenance of growth rate in high-H plants grown at low temperature is associated with both respiratory homeostasis and a high efficiency of respiratory ATP production.

Adenosine Triphosphate↗

PubMed2003

Salicylic acid is an uncoupler and inhibitor of mitochondrial electron transport.

The effect of salicylic acid (SA) on respiration and mitochondrial function was examined in tobacco (Nicotiana tabacum) suspension cell cultures in the range of 0.01 to 5 mm. Cells rapidly accumulated SA up to 10-fold of the externally applied concentrations. At the lower concentrations, SA accumulation was transitory. When applied at 0.1 mm or less, SA stimulated respiration of whole cells and isolated mitochondria in the absence of added ADP, indicating uncoupling of respiration. However, at higher concentrations, respiration was severely inhibited. Measurements of ubiquinone redox poise in isolated mitochondria suggested that SA blocked electron flow from the substrate dehydrogenases to the ubiquinone pool. This inhibition could be at least partially reversed by re-isolating the mitochondria. Two active analogs of SA, benzoic acid and acetyl-SA, had the same effect as SA on isolated tobacco mitochondria, whereas the inactive p-hydroxybenzoic acid was without effect at the same concentration. SA induced an increase in Aox protein levels in cell suspensions, and this was correlated with an increase in Aox1 transcript abundance. However, when applied at 0.1 mM, this induction was transient and disappeared as SA levels in the cells declined. SA at 0.1 mM also increased the expression of other SA-responsive genes, and this induction was dependent on active mitochondria. The results indicate that SA is both an uncoupler and an inhibitor of mitochondrial electron transport and suggest that this underlies the induction of some genes by SA. The possible implications of this for the interpretation of SA action in plants are discussed.

Base Sequence↗

PubMed2003

Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins.

A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography-tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered. Nearly 20% of the experimentally identified proteins are of unknown function, suggesting a wealth of undiscovered mitochondrial functions in plants. Only approximately half of the experimental set is predicted to be mitochondrial by targeting prediction programs, allowing an assessment of the benefits and limitations of these programs in determining plant mitochondrial proteomes. Maps of putative orthology networks between yeast, human, and Arabidopsis mitochondrial proteomes and the Rickettsia prowazekii proteome provide detailed insights into the divergence of the plant mitochondrial proteome from those of other eukaryotes. These show a clear set of putative cross-species orthologs in the core metabolic functions of mitochondria, whereas considerable diversity exists in many signaling and regulatory functions.

Arabidopsis↗

PubMed2003

Targets of stress-induced oxidative damage in plant mitochondria and their impact on cell carbon/nitrogen metabolism.

Plant mitochondria link the cellular processes of carbon and nitrogen metabolism through the tricarboxylic acid cycle and the photorespiratory cycle. Environmental stresses lead to damage of specific mitochondrial targets through the direct action of reactive oxygen species and indirect action of lipid peroxidation products. Uncovering the extent of this damage, the exact sites of damage and the mechanisms of avoidance and/or repair remains a largely unresearched challenge for plant scientists. Damage to Fe-S centres and proteins containing lipoic acid moieties appear to predominate in current reports. Substantial evidence that both TCA cycle and photorespiratory capacity of mitochondria are sensitive sites for damage is highlighted and the implications for mitochondrial-dependent carbon and nitrogen metabolism are discussed.

Carbon↗

PubMed2003

Identification of AtNDI1, an internal non-phosphorylating NAD(P)H dehydrogenase in Arabidopsis mitochondria.

Plant mitochondria contain non-phosphorylating NAD(P)H dehydrogenases (DHs) that are not found in animal mitochondria. The physiological function, substrate specificity, and location of enzymes within this family have yet to be conclusively determined. We have linked genome sequence information to protein and biochemical data to identify that At1g07180 (SwissProt Q8GWA1) from the Arabidopsis Genome Initiative database encodes AtNDI1, an internal NAD(P)H DH in Arabidopsis mitochondria. Three lines of evidence are presented: (a). The predicted protein sequence of AtNDI1 has high homology with other designated NAD(P)H DHs from microorganisms, (b). the capacity for matrix NAD(P)H oxidation via the rotenone-insensitive pathway is significantly reduced in the Atndi1 mutant plant line, and (c). the in vitro translation product of AtNDI1 is imported into isolated mitochondria and located on the inside of the inner membrane.

Amino Acid Sequence↗

PubMed2003

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.

Arabidopsis↗

PubMed2003

A tomato alternative oxidase protein with altered regulatory properties.

We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L. Mill cv. Sweetie) after storage of green fruit at 4 degrees C. Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit. The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein. The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue. LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate. This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform. The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism.

Amino Acid Sequence↗

PubMed2003

Environmental stresses inhibit and stimulate different protein import pathways in plant mitochondria.

The impact of various environmental stresses (drought, chilling or herbicide treatment) on the capacity of plant mitochondria to import precursor proteins was investigated. Drought treatment stimulated import and processing of various precursor proteins via the general import pathway. The stimulatory effect of drought on the general import pathway was due to an increased rate of import, was accompanied by an increased rate of processing, and could be attributed to the presequence of the precursor protein. Interestingly, drought decreased the import of the F(A)d subunit of ATP synthase suggesting a bypass of the point of stimulation during import of this precursor. Both chilling and herbicide treatment of plants, on the other hand, caused inhibition of import with all precursors tested. No decrease in processing of imported proteins was observed by these stress treatments. Western analysis of several mitochondrial proteins indicated that the steady-state level of several mitochondrial components, including the TOM20 receptor and the core subunits of the cytochrome bc(1) complex responsible for processing, remained largely unchanged. Thus environmental stresses differentially affect import of precursor proteins in a complicated manner dependent on the import pathway utilised.

Chloroplasts↗

PubMed2003

What makes a mitochondrion?

Experimental analyses of the proteins found in the mitochondria of yeast, humans and Arabidopsis have confirmed some expectations but given some surprises and some insights into the evolutionary origins of mitochondrial proteins.

Evolution, Molecular↗

PubMed2003

Respiratory gene expression in soybean cotyledons during post-germinative development.

Gene expression for nuclear- and mitochondrial-encoded subunits of respiratory chain components was measured in developing soybean cotyledons and compared to the abundance of the relevant proteins. Overall respiratory gene expression peaked at day 16, close to the peak in cytochrome chain and TCA cycle activities from day 10 to 15. Protein abundance followed transcript abundance for all components examined with the exception of the F1beta subunit of ATP synthase. A dramatic peak in F1beta transcript levels early in development (day 5 to 7) was not mirrored by an increase in protein suggesting translational or post-translational control. Mitochondrial-encoded transcripts were at least 10-fold more abundant than nuclear-encoded transcripts. The pattern of transcript and protein abundance for uncoupling proteins displayed a trend similar to other respiratory proteins examined, implicating similar control mechanisms. The expression of alternative oxidase differed, increasing throughout development with protein peaking at day 20, perhaps suggesting a role in senescence. Overall, this study indicated that respiratory gene expression and protein abundance is co-ordinated with respiratory activity for most components but that some components, such as the F1beta subunit may be under discrete forms of regulation.

Cell Nucleus↗