Richard P. Jacoby

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.

The role of mitochondrial respiration in salinity tolerance

NaCl is the most abundant salt in salinity-affected land. The ability of plants to sift the water table, limit NaCl uptake, compartmentalise Na⁺/Cl⁻ ions and prevent negative ionic and osmotic effects on cell function, are the foundations of salinity tolerance mechanisms. In this review, we show that although the quantitative response of respiratory rate to changes in salt concentration is complex, the properties of respiratory processes are crucial for tolerance during ion exclusion and tissue tolerance. We consider whole-plant gas exchange and carbon balance analysis alongside the salt responses of mitochondrial properties and genetic studies manipulating respiratory processes. We showcase the importance of efficient ATP generation, dampened reactive oxygen species and mitochondrial osmolytes for salinity tolerance in plants.

Wheat Mitochondrial Proteomes Provide New Links between Antioxidant Defense and Plant Salinity Tolerance

The mitochondrial proteome and differences associated with salt tolerance have been investigated in Australian commercial varieties of wheat. Mitochondria isolated from shoots were used to generate a wheat mitochondrial reference map; 68 unique wheat mitochondrial proteins were identified from 192 gel spots using 2D PAGE and LC-MS/MS. This analysis also provided MS/MS spectra for 199 proteotypic peptides as a foundation for the development of targeted proteomics to study the respiratory apparatus in wheat. Using this reference map and 2D DIGE, we have found quantitative differences in the shoot mitochondrial proteomes of v. Wyalkatchem and v. Janz, two commercially important wheat varieties that are known from a range of experiments to differ in salinity tolerance. These proteins included Mn-superoxide dismutase (Mn-SOD), cysteine synthase, nucleotide diphosphate kinase, and the voltage dependent anion channel (VDAC). Antibodies to the mitochondrial alternative oxidase (AOX), previously linked to reduced ROS formation from the electron transport chain and salt tolerance in Arabidopsis, also showed a commensurate higher abundance in v. Wyakatchem in both control and salt-treated conditions. Together, the data presented here suggest that differences in mitochondrial ROS defense pathways in the mitochondrial proteomes of key Australian wheat varieties correlate with whole-plant salinity tolerance.

Mitochondrial Composition, Function and Stress Response in Plants†

The primary function of mitochondria is respiration, where catabolism of substrates is coupled to ATP synthesis via oxidative phosphorylation. In plants, mitochondrial composition is relatively complex and flexible and has specific pathways to support photosynthetic processes in illuminated leaves. This review begins with outlining current models of mitochondrial composition in plant cells, with an emphasis upon the assembly of the complexes of the classical electron transport chain (ETC). Next, we focus upon the comparative analysis of mitochondrial function from different tissue types. A prominent theme in the plant mitochondrial literature involves linking mitochondrial composition to environmental stress responses, and this review then gives a detailed outline of how oxidative stress impacts upon the plant mitochondrial proteome with particular attention to the role of transition metals. This is followed by an analysis of the signaling capacity of mitochondrial reactive oxygen species, which studies the transcriptional changes of stress responsive genes as a framework to define specific signals emanating from the mitochondrion. Finally, specific mitochondrial roles during exposure to harsh environments are outlined, with attention paid to mitochondrial delivery of energy and intermediates, mitochondrial support for photosynthesis, and mitochondrial processes operating within root cells that mediate tolerance to anoxia and unfavorable soil chemistries. [Formula: see text] [ A. Harvey Millar (Corresponding author)].

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

Proteins turn over at different rates in plant tissues, and these have been quantified using stable isotope labeling of nitrogen and peptide mass spectrometry of leaf tissue from hydroponically grown barley. Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

Degradation Rate of Mitochondrial Proteins in Arabidopsis thaliana Cells

The turnover of the proteomes of organelles in plant cells are known to be governed by both whole cell and organelle-specific processes. However, the rate and specificity of this protein turnover has not been explored in depth to understand how it affects different organellar processes. Here we have used progressive ¹⁵N labeling of Arabidopsis cells, and focused on the turnover rate of proteins in mitochondria. We provide estimates of degradation rate (K(d)) for 224 mitochondrial proteins, showing a range of over 50-fold in K(d). Protein complexes, most notably the respiratory chain complexes, had K(d) values that were generally coordinated and we have interpreted these measurements to outline how protein K(d) differs within protein complexes and between functional categories. The fastest turnover rates were reported for DNA/RNA metabolism enzymes, chaperones, and proteases.

Investigating the Role of Respiration in Plant Salinity Tolerance by Analyzing Mitochondrial Proteomes from Wheat and a Salinity-Tolerant Amphiploid (Wheat × Lophopyrum elongatum)

The effect of salinity on mitochondrial properties was investigated by comparing the reference wheat variety Chinese Spring (CS) to a salt-tolerant amphiploid (AMP). The octoploid AMP genotype was previously generated by combining hexaploid bread wheat (CS) with the diploid wild wheatgrass adapted to salt marshes, Lophopyrum elongatum. Here we used a combination of physiological, biochemical, and proteomic analyses to explore the mitochondrial and respiratory response to salinity in these two genotypes. The AMP showed greater growth tolerance to salinity treatments and altered respiration rate in both roots and shoots. A proteomic workflow of 2D-DIGE and MALDI TOF/TOF mass spectrometry was used to compare the protein composition of isolated mitochondrial samples from roots and shoots of both genotypes, following control or salt treatment. A large set of mitochondrial proteins were identified as responsive to salinity in both genotypes, notably enzymes involved in detoxification of reactive oxygen species. Genotypic differences in mitochondrial composition were also identified, with AMP exhibiting a higher abundance of manganese superoxide dismutase, serine hydroxymethyltransferase, aconitase, malate dehydrogenase, and β-cyanoalanine synthase compared to CS. We present peptide fragmentation spectra derived from some of these AMP-specific protein spots, which could serve as biomarkers to track superior protein variants.

Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL)

Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges.

Plant Mitochondrial Proteomics

Mitochondria are responsible for a number of major biochemical processes in plant cells including oxidative phosphorylation and photorespiration. Traditionally their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons to O2. More recently its role in the synthesis of many metabolites such as amino acids, lipids, and vitamins has been revealed. They also contain large number of transporters including members of the mitochondrial carrier substrate family (MCSF) that allow the exchange of metabolites with the cytosol. Mitochondria also contain their own genome and actively transcribe and translate a set of proteins that are coordinated with proteins encoded by the nuclear genome to produce large multisubunit enzymes. To reveal the full diversity of metabolism carried out by mitochondria significant efforts have sought to uncover the protein profile of mitochondria from both crops and model plants. Successful proteomic analysis depends on the preparation of high-quality isolated mitochondria, coupled to high-resolution proteomic techniques for identification, quantitation, and assessment of the degree of contamination by other organelles and cellular compartments. Here we outline a mitochondrial isolation protocol that can be applied to a range of plant tissues, and detail methods of assessing the quality and purity of the resultant sample, including calculations of respiratory control ratio, marker enzyme assays, differential in-gel electrophoresis, and quantitative gel-free mass spectrometry.

Application of selected reaction monitoring mass spectrometry to field-grown crop plants to allow dissection of the molecular mechanisms of abiotic stress tolerance

One major constraint upon the application of molecular crop breeding approaches is the small number of genes linked to agronomically desirable traits through defined biochemical mechanisms. Proteomic investigations of crop plants under abiotic stress treatments have identified many proteins that differ in control versus stress comparisons, however, this broad profiling of cell physiology is poorly suited to ranking the effects and identifying the specific proteins that are causative in agronomically relevant traits. Here we will reason that insights into a protein’s function, its biochemical process and links to stress tolerance are more likely to arise through approaches that evaluate these differential abundances of proteins and include varietal comparisons, precise discrimination of protein isoforms, enrichment of functionally related proteins, and integration of proteomic datasets with physiological measurements of both lab and field-grown plants. We will briefly explain how applying the emerging proteomic technology of multiplexed selective reaction monitoring mass spectrometry with its accuracy and throughput can facilitate and enhance these approaches and provide a clear means to rank the growing cohort of stress responsive proteins. We will also highlight the benefit of integrating proteomic analyses with cultivar-specific genetic databases and physiological assessments of cultivar performance in relevant field environments for revealing deeper insights into molecular crop improvement.