Nicolas L. Taylor

The SOL Genomics Network. A Comparative Resource for Solanaceae Biology and Beyond

The SOL Genomics Network (SGN; http://sgn.cornell.edu) is a rapidly evolving comparative resource for the plants of the Solanaceae family, which includes important crop and model plants such as potato (Solanum tuberosum), eggplant (Solanum melongena), pepper (Capsicum annuum), and tomato (Solanum lycopersicum). The aim of SGN is to relate these species to one another using a comparative genomics approach and to tie them to the other dicots through the fully sequenced genome of Arabidopsis (Arabidopsis thaliana). SGN currently houses map and marker data for Solanaceae species, a large expressed sequence tag collection with computationally derived unigene sets, an extensive database of phenotypic information for a mutagenized tomato population, and associated tools such as real-time quantitative trait loci. Recently, the International Solanaceae Project (SOL) was formed as an umbrella organization for Solanaceae research in over 30 countries to address important questions in plant biology. The first cornerstone of the SOL project is the sequencing of the entire euchromatic portion of the tomato genome. SGN is collaborating with other bioinformatics centers in building the bioinformatics infrastructure for the tomato sequencing project and implementing the bioinformatics strategy of the larger SOL project. The overarching goal of SGN is to make information available in an intuitive comparative format, thereby facilitating a systems approach to investigations into the basis of adaptation and phenotypic diversity in the Solanaceae family, other species in the Asterid clade such as coffee (Coffea arabica), Rubiaciae, and beyond.

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.

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 (gs): well irrigated (gs > 0.2 mol H2O m−2 s−1), mildly water stressed (gs between 0.1 and 0.2 mol H2O m−2 s−1), and severely water stressed (gs < 0.1 mol H2O m−2 s−1). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (Vt) 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.

The Pentatricopeptide Repeat Gene OTP43 Is Required for trans-Splicing of the Mitochondrial nad1 Intron 1 in Arabidopsis thaliana

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a large protein complex formed from both nuclearly and mitochondrially encoded subunits. Subunit ND1 is encoded by a mitochondrial gene comprising five exons, and the mature transcript requires four RNA splicing events, two of which involve trans-splicing independently transcribed RNAs. We have identified a nuclear gene (OTP43) absolutely required for trans-splicing of intron 1 (and only intron 1) of Arabidopsis thaliana nad1 transcripts. This gene encodes a previously uncharacterized pentatricopeptide repeat protein. Mutant Arabidopsis plants with a disrupted OTP43 gene do not present detectable mitochondrial Complex I activity and show severe defects in seed development, germination, and to a lesser extent in plant growth. The alternative respiratory pathway involving alternative oxidase is significantly induced in the mutant.

Mitochondrial uncoupling protein is required for efficient photosynthesis

Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane and dissipate the proton gradient across this membrane that is normally used for ATP synthesis. Although the catalytic function and regulation of plant UCPs have been described, the physiological purpose of UCP in plants has not been established. Here, biochemical and physiological analyses of an insertional knockout of one of the Arabidopsis UCP genes (AtUCP1) are presented that resolve this issue. Absence of UCP1 results in localized oxidative stress but does not impair the ability of the plant to withstand a wide range of abiotic stresses. However, absence of UCP1 results in a photosynthetic phenotype. Specifically there is a restriction in photorespiration with a decrease in the rate of oxidation of photorespiratory glycine in the mitochondrion. This change leads to an associated reduced photosynthetic carbon assimilation rate. Collectively, these results suggest that the main physiological role of UCP1 in Arabidopsis leaves is related to maintaining the redox poise of the mitochondrial electron transport chain to facilitate photosynthetic metabolism.

Novel Proteins, Putative Membrane Transporters, and an Integrated Metabolic Network Are Revealed by Quantitative Proteomic Analysis of Arabidopsis Cell Culture Peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, β-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.

Divalent Metal Ions in Plant Mitochondria and Their Role in Interactions with Proteins and Oxidative Stress-Induced Damage to Respiratory Function

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu2+, Zn2+, and Co2+ was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.

Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis

Lipoic acid-dependent pathways of α-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 α-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 α-keto acids. Pyruvate- and α-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 α-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and α-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived α-keto acid, α-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.

Abiotic environmental stress induced changes in the Arabidopsis thaliana chloroplast, mitochondria and peroxisome proteomes

Exposure to adverse abiotic environmental conditions causes oxidative stress in plants, leading to debilitation and death or to response and tolerance. The subcellular energy organelles (chloroplast, mitochondria and peroxisomes) in plants are responsible for major metabolic processes including photosynthesis, photorespiration, oxidative phosphorylation, beta-oxidation and the tricarboxylic acid cycle. Here we analyze data and review a collection of both whole tissue and organellar proteomic studies that have investigated the effects of environmental stress in the model plant Arabidopsis thaliana. We assess these data from an organellar perspective to begin to build an understanding of the changes in protein abundance within these organelles during environmental stresses. We found 279 claims of proteins that change in abundance that could be assigned to protein components of the energy organelles. These could be placed into eight different functional categories and nearly 80% of the specific protein isoforms detected were only reported to change in a single environmental stress. We propose primary and secondary mechanisms in organelles by which the protein changes observed could be mediated in order to begin developing an integrated and mechanistic understanding of environmental stress response.

Experimental Analysis of the Rice Mitochondrial Proteome, Its Biogenesis, and Heterogeneity

Mitochondria in rice (Oryza sativa) are vital in expanding our understanding of the cellular response to reoxygenation of tissues after anaerobiosis, the crossroads of carbon and nitrogen metabolism, and the role of respiratory energy generation in cytoplasmic male sterility. We have combined density gradient and surface charge purification techniques with proteomics to provide an in-depth proteome of rice shoot mitochondria covering both soluble and integral membrane proteins. Quantitative comparisons of mitochondria purified by density gradients and after further surface charge purification have been used to ensure that the proteins identified copurify with mitochondria and to remove contaminants from the analysis. This rigorous approach to defining a subcellular proteome has yielded 322 nonredundant rice proteins and highlighted contaminants in previously reported rice mitochondrial proteomes. Comparative analysis with the Arabidopsis (Arabidopsis thaliana) mitochondrial proteome reveals conservation of a broad range of known and unknown function proteins in plant mitochondria, with only approximately 20% not having a clear homolog in the Arabidopsis mitochondrial proteome. As in Arabidopsis, only approximately 60% of the rice mitochondrial proteome is predictable using current organelle-targeting prediction tools. Use of the rice protein data set to explore rice transcript data provided insights into rice mitochondrial biogenesis during seed germination, leaf development, and heterogeneity in the expression of nucleus-encoded mitochondrial components in different rice tissues. Highlights include the identification of components involved in thiamine synthesis, evidence for coexpressed and unregulated expression of specific components of protein complexes, a selective anther-enhanced subclass of the decarboxylating segment of the tricarboxylic acid cycle, the differential expression of DNA and RNA replication components, and enhanced expression of specific metabolic components in photosynthetic tissues.