ORCID Profile
0000-0002-2080-3826
Current Organisation
University of Melbourne
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Plant Biology | Plant Cell and Molecular Biology | Plant Biochemistry And Physiology | Protein Targeting And Signal Transduction | Colloid And Surface Chemistry | Physical Chemistry (Incl. Structural) | Plant Improvement (Selection, Breeding And Genetic Engineering) | Protein Trafficking | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Plant Developmental and Reproductive Biology | Plant Physiology | Analytical Biochemistry | Membrane Biology | Population And Ecological Genetics | Crop and Pasture Improvement (Selection and Breeding) | Crop and Pasture Biomass and Bioproducts | Analytical Biochemistry | Bioinformatics | Biological Adaptation
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Plant Production and Plant Primary Products not elsewhere classified | Field crops | Biological sciences | Oilseeds | Barley | Wheat | Nutraceuticals and Functional foods | Immune system and allergy | Higher education | Biofuel (Biomass) Energy | Primary plant products not elsewhere classified |
Related Links
Publications
The elaborate route for UDP-arabinose delivery into the Golgi of plants
Publisher: Proceedings of the National Academy of Sciences
Date: 03-04-2017
Abstract: Nucleotide sugars, the activated sugar donors essential for processes such as cell wall biosynthesis and protein and lipid glycosylation are predominantly made in the cytosol. However, a highly erse range of glycosyltransferases that are located within the Golgi lumen, mediate the above-mentioned glycosylation reactions. Thus, transport of nucleotide sugars across the Golgi membrane into the lumen is crucial for growth and development of many species including microorganisms, plants, and humans. In this study, we identify and functionally characterize four UDP-arabinofuranose transporters from Arabidopsis that are responsible for the delivery of activated arabinose, a critical sugar of plant cell walls, glycoproteins, and signaling peptides.
Differential distributions of trafficking and signaling proteins of the maize ER-Golgi apparatus
Publisher: Informa UK Limited
Date: 28-09-2019
Towards an Analysis of the Rice Mitochondrial Proteome
Publisher: Oxford University Press (OUP)
Date: 05-2003
Abstract: Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing olyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = −1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0–12.5), and proteins over a large molecular mass range (6.7–252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.
Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis
Publisher: Oxford University Press (OUP)
Date: 19-03-2012
Abstract: The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.
Engineering temporal accumulation of a low recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls
Publisher: Wiley
Date: 14-01-2015
DOI: 10.1111/PBI.12326
Abstract: Reduced cell wall recalcitrance and increased C6 monosaccharide content are desirable traits for future biofuel crops, as long as these biomass modifications do not significantly alter normal growth and development. Mixed-linkage glucan (MLG), a cell wall polysaccharide only present in grasses and related species among flowering plants, is comprised of glucose monomers linked by both β-1,3 and β-1,4 bonds. Previous data have shown that constitutive production of MLG in barley (Hordeum vulgare) severely compromises growth and development. Here, we used spatio-temporal strategies to engineer Arabidopsis thaliana plants to accumulate significant amounts of MLG in the cell wall by expressing the rice CslF6 MLG synthase using secondary cell wall and senescence-associated promoters. Results using secondary wall promoters were suboptimal. When the rice MLG synthase was expressed under the control of a senescence-associated promoter, we obtained up to four times more glucose in the matrix cell wall fraction and up to a 42% increase in saccharification compared to control lines. Importantly, these plants grew and developed normally. The induction of MLG deposition at senescence correlated with an increase of gluconic acid in cell wall extracts of transgenic plants in contrast to the other approaches presented in this study. MLG produced in Arabidopsis has an altered structure compared to the grass glucan, which likely affects its solubility, while its molecular size is unaffected. The induction of cell wall polysaccharide biosynthesis in senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops.
The Green Proteome: Challenges in Plant Proteomics
Publisher: Frontiers Media SA
Date: 2011
SUBA: the Arabidopsis subcellular database
Publisher: Oxford University Press (OUP)
Date: 28-10-2007
DOI: 10.1093/NAR/GKL863
AtMYB32 is required for normal pollen development in Arabidopsis thaliana
Publisher: Wiley
Date: 12-2004
Plant Mitochondrial Carriers: Molecular Gatekeepers That Help to Regulate Plant Central Carbon Metabolism
Publisher: MDPI AG
Date: 17-01-2020
Abstract: The evolution of membrane-bound organelles among eukaryotes led to a highly compartmentalized metabolism. As a compartment of the central carbon metabolism, mitochondria must be connected to the cytosol by molecular gates that facilitate a myriad of cellular processes. Members of the mitochondrial carrier family function to mediate the transport of metabolites across the impermeable inner mitochondrial membrane and, thus, are potentially crucial for metabolic control and regulation. Here, we focus on members of this family that might impact intracellular central plant carbon metabolism. We summarize and review what is currently known about these transporters from in vitro transport assays and in planta physiological functions, whenever available. From the biochemical and molecular data, we hypothesize how these relevant transporters might play a role in the shuttling of organic acids in the various flux modes of the TCA cycle. Furthermore, we also review relevant mitochondrial carriers that may be vital in mitochondrial oxidative phosphorylation. Lastly, we survey novel experimental approaches that could possibly extend and/or complement the widely accepted proteoliposome reconstitution approach.
Analysis of theArabidopsisCytosolic Proteome Highlights Subcellular Partitioning of Central Plant Metabolism
Publisher: American Chemical Society (ACS)
Date: 04-2011
DOI: 10.1021/PR1009433
Abstract: The plant cell cytosol is a dynamic and complex intracellular matrix that, by definition, contains no compartmentalization. Nonetheless, it maintains a wide variety of biochemical networks and often links metabolic pathways across multiple organelles. There have been numerous detailed proteomic studies of organelles in the model plant Arabidopsis thaliana, although no such analysis has been undertaken on the cytosol. The cytosolic protein fraction from cell suspensions of Arabidopsis thaliana was isolated and analyzed using offline strong cation exchange liquid chromatography and LC-MS/MS. This generated a robust set of 1071 cytosolic proteins. Functional annotation of this set revealed major activities in protein synthesis and degradation, RNA metabolism and basic sugar metabolism. This included an array of important cytosol-related functions, specifically the ribosome, the set of tRNA catabolic enzymes, the ubiquitin-proteasome pathway, glycolysis and associated sugar metabolism pathways, phenylpropanoid biosynthesis, vitamin metabolism, nucleotide metabolism, an array of signaling and stress-responsive molecules, and NDP-sugar biosynthesis. This set of cytosolic proteins provides for the first time an extensive analysis of enzymes responsible for the myriad of reactions in the Arabidopsis cytosol and defines an experimental set of plant protein sequences that are not targeted to subcellular locations following translation and folding in the cytosol.
The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis
Publisher: Proceedings of the National Academy of Sciences
Date: 22-07-2014
Abstract: Delivery of nucleotide sugar substrates into the Golgi apparatus and endoplasmic reticulum for processes such as cell wall biosynthesis and protein glycosylation is critical for plant growth and development. Plant genomes encode large families of uncharacterized nucleotide sugar transporters that are specifically presumed to deliver the erse array of nucleotide sugars found in plants. This study has developed a novel approach that enabled functional characterization of six bifunctional UDP-rhamnose (Rha)/UDP-galactose (Gal) transporters from Arabidopsis . An analysis of loss-of-function and overexpression lines for two of these transporters identified biochemical alterations supporting their roles in the biosynthesis of Rha- and Gal-containing polysaccharides. Thus, cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is likely also regulated by substrate transport mechanisms.
Proteomic dissection of the Arabidopsis Golgi and trans-Golgi network
Publisher: Frontiers Media SA
Date: 2013
Arabidopsis Mitochondrial Proteomics
Publisher: Humana Press
Date: 2007
DOI: 10.1007/978-1-59745-365-3_38
Abstract: Significant efforts have sought to uncover the protein profile of Arabidopsis mitochondria to act as a model for the mitochondrial proteome from plants. A combination of techniques have been undertaken to achieve this goal. We outline a basic two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation of mitochondrial proteins, in-gel trypsination techniques, complex protein lysate digestions, and the identification of proteins by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry.
Mitochondrial cytochrome c oxidase and succinate dehydrogenase complexes contain plant specific subunits
Publisher: Springer Science and Business Media LLC
Date: 09-2004
DOI: 10.1007/S11103-004-2316-2
Abstract: Respiratory oxidative phosphorylation represents a central functionality in plant metabolism, but the subunit composition of the respiratory complexes in plants is still being defined. Most notably, complex II (succinate dehydrogenase) and complex IV (cytochrome c oxidase) are the least defined in plant mitochondria. Using Arabidopsis mitochondrial s les and 2D Blue-native/SDS-PAGE, we have separated complex II and IV from each other and displayed their in idual subunits for analysis by tandem mass spectrometry and Edman sequencing. Complex II can be discretely separated from other complexes on Blue-native gels and consists of eight protein bands. It contains the four classical SDH subunits as well as four subunits unknown in mitochondria from other eukaryotes. Five of these proteins have previously been identified, while three are newly identified in this study. Complex IV consists of 9-10 protein bands, however, it is more diffuse in Blue-native gels and co-migrates in part with the translocase of the outer membrane (TOM) complex. Differential analysis of TOM and complex IV reveals that complex IV probably contains eight subunits with similarity to known complex IV subunits from other eukaryotes and a further six putative subunits which all represent proteins of unknown function in Arabidopsis . Comparison of the Arabidopsis data with Blue-native/SDS-PAGE separation of potato and bean mitochondria confirmed the protein band complexity of these two respiratory complexes in plants. Two-dimensional Blue-native/Blue-native PAGE, using digitonin followed by dodecylmaltoside in successive dimensions, separated a diffusely staining complex containing both TOM and complex IV. This suggests that the very similar mass of these complexes will likely prevent high purity separations based on size. The documented roles of several of the putative complex IV subunits in hypoxia response and ozone stress, and similarity between new complex II subunits and recently identified plant specific subunits of complex I, suggest novel biological insights can be gained from respiratory complex composition analysis.
Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls
Publisher: Frontiers Media SA
Date: 18-08-2015
Defective Pollen Wall 2 (DPW2) Encodes an Acyl Transferase Required for Rice Pollen Development.
Publisher: Oxford University Press (OUP)
Date: 11-07-2020
DOI: 10.1104/PP.16.00095
We Are Not Alone: The iMOP Initiative and Its Roles in a Biology- and Disease-Driven Human Proteome Project
Publisher: American Chemical Society (ACS)
Date: 31-10-2017
DOI: 10.1021/ACS.JPROTEOME.7B00408
Abstract: Mapping of the human proteome has advanced significantly in recent years and will provide a knowledge base to accelerate our understanding of how proteins and protein networks can affect human health and disease. However, providing solutions to human health challenges will likely fail if insights are exclusively based on studies of human s les and human proteomes. In recent years, it has become evident that human health depends on an integrated understanding of the many species that make human life possible. These include the commensal microorganisms that are essential to human life, pathogens, and food species as well as the classic model organisms that enable studies of biological mechanisms. The Human Proteome Organization (HUPO) initiative on multiorganism proteomes (iMOP) works to support proteome research undertaken on nonhuman species that remain widely under-studied compared with the progress in human proteome research. This perspective argues the need for further research on multiple species that impact human life. We also present an update on recent progress in model organisms, microbiota, and food species, address the emerging problem of antibiotics resistance, and outline how iMOP activities could lead to a more inclusive approach for the human proteome project (HPP) to better support proteome research aimed at improving human health and furthering knowledge on human biology.
N-linked Glycan Micro-heterogeneity in Glycoproteins of Arabidopsis
Publisher: Elsevier BV
Date: 03-2018
Multiple marker abundance profiling: combining selected reaction monitoring and data‐dependent acquisition for rapid estimation of organelle abundance in subcellular samples
Publisher: Wiley
Date: 20-11-2017
DOI: 10.1111/TPJ.13743
Isolation of the plant cytosolic fraction for proteomic analysis
Publisher: Humana Press
Date: 22-08-2014
DOI: 10.1007/978-1-62703-631-3_31
Abstract: The cytosol is the fluid portion of the cell that is not partitioned by membranes. It contains a highly erse collection of substances and is central to many essential cellular processes ranging from signal transduction, metabolite production and transport, protein biosynthesis and degradation to stress response and defense. Despite its importance, only a few proteomic studies have been performed on the plant cytosol. This is largely due to difficulties in isolating relatively pure s les from plant material free of disrupted organelle material. In this chapter we outline methods for isolating the cytosolic fraction from Arabidopsis cell cultures and seedlings and provide guidance on assessing purity for analysis by mass spectrometry.
The seminal fluid proteome of the honeybee Apis mellifera
Publisher: Wiley
Date: 04-2009
Abstract: Ejaculates contain sperm but also seminal fluid, which is increasingly recognized to be of central importance for reproductive success. However, a detailed biochemical composition and physiological understanding of seminal fluid is still elusive. We have used MS to identify the 57 most abundant proteins within the ejaculated seminal fluid of the honeybee Apis mellifera. Their amino acid sequences revealed the presence of erse functional categories of enzymes, regulators and structural proteins. A number have known or predicted roles in maintaining sperm viability, protecting sperm from microbial infections or interacting with the physiology of the female. A range of putative glycoproteins or glycosylation enzymes were detected among the 57, subsequent fluorescent staining of glycolysation revealed several prominent glycoproteins in seminal fluid, while no glycoproteins were detected in sperm s les. Many of the abundant proteins that accumulate in the seminal fluid did not contain predictable tags for secretion for the cell. Comparison of the honeybee seminal fluid proteins with Drosophila seminal fluid proteins (including secreted accessory gland proteins known as ACPs), and with the human seminal fluid proteome revealed the bee protein set contains a range of newly identified seminal fluid proteins and we noted more similarity of the bee protein set with the current human seminal fluid protein set than with the known Drosophila seminal fluid proteins. The honeybee seminal fluid proteome thus represents an important addition to available data for comparative studies of seminal fluid proteomes in insects.
Editorial: International Plant Proteomics Organization (INPPO) World Congress 2014
Publisher: Frontiers Media SA
Date: 05-08-2016
Proteomic identification of extracellular proteins regulated by the Gna1 Gα subunit in Stagonospora nodorum
Publisher: Elsevier BV
Date: 05-2009
DOI: 10.1016/J.MYCRES.2009.01.004
Abstract: The fungus Stagonospora nodorum is the causal agent of stagonospora nodorum blotch (syn. leaf and glume blotch) disease of wheat. The Gna1-encoded Galpha protein is an important signal transduction component in the fungus, which is required for full pathogenicity, sporulation and extracellular depolymerase production. In this study, we sought to gain a better understanding of defects associated with the gna1 mutant by using two-dimensional gel electrophoresis to analyse the extracellular proteome for differences to the wildtype. Mass spectrometry analysis of altered abundant protein spots and peptide matching to the Stagonospora nodorum genome database have led to the identification of genes implicated in cell wall degradation, proteolysis, RNA hydrolysis and aromatic compound metabolism. In addition, quantitative RT-PCR has demonstrated that some of the encoding genes showed differential expression throughout host infection. Implications of these proteins and their corresponding genes in fungal virulence are discussed.
PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update
Publisher: Oxford University Press (OUP)
Date: 30-10-2009
DOI: 10.1093/NAR/GKP810
Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.YMBEN.2018.03.001
Abstract: Cyanobacteria fix atmospheric CO
UDP-Glucuronic Acid Transport Is Required for Virulence of Cryptococcus neoformans.
Publisher: American Society for Microbiology
Date: 07-03-2018
Abstract: Glycans play erse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. In Cryptococcus neoformans , an opportunistic pathogen of humans, the acidic monosaccharide glucuronic acid (GlcA) is a critical component of multiple essential glycoconjugates. One of these glycoconjugates is the polysaccharide capsule, a major virulence factor that enables this yeast to modulate the host immune response and resist antimicrobial defenses. This allows cryptococci to colonize the lung and brain, leading to hundreds of thousands of deaths each year worldwide. Synthesis of most glycans, including capsule polysaccharides, occurs in the secretory pathway. However, the activated precursors for this process, nucleotide sugars, are made primarily in the cytosol. This topological problem is resolved by the action of nucleotide sugar transporters (NSTs). We discovered that Uut1 is the sole UDP-GlcA transporter in C. neoformans and is unique among NSTs for its narrow substrate range and high affinity for UDP-GlcA. Mutant cells with UUT1 deleted lack capsule polysaccharides and are highly sensitive to environmental stress. As a result, the deletion mutant is internalized and cleared by phagocytes more readily than wild-type cells are and is completely avirulent in mice. These findings expand our understanding of the requirements for capsule synthesis and cryptococcal virulence and elucidate a critical protein family. IMPORTANCE Cryptococcus neoformans causes lethal meningitis in almost two hundred thousand immunocompromised patients each year. Much of this fungal pathogen’s ability to resist host defenses and cause disease is mediated by carbohydrate structures, including a complex polysaccharide capsule around the cell. Like most eukaryotic glycoconjugates, capsule polysaccharides are made within the secretory pathway, although their precursors are generated in the cytosol. Specific transporters are therefore required to convey these raw materials to the site of synthesis. One precursor of particular interest is UDP-glucuronic acid, which donates glucuronic acid to growing capsule polysaccharides. We discovered a highly specific, high-affinity transporter for this molecule. Deletion of the gene encoding this unusual protein abolishes capsule synthesis, alters stress resistance, and eliminates fungal virulence. In this work, we have identified a novel transporter, elucidated capsule synthesis and thereby aspects of fungal pathogenesis, and opened directions for potential antifungal therapy.
Xylose donor transport is critical for fungal virulence.
Publisher: Public Library of Science (PLoS)
Date: 18-01-2201
Role of UDP-Glucuronic Acid Decarboxylase in Xylan Biosynthesis in Arabidopsis
Publisher: Elsevier BV
Date: 08-2016
DOI: 10.1016/J.MOLP.2016.04.013
Abstract: UDP-xylose (UDP-Xyl) is the Xyl donor used in the synthesis of major plant cell-wall polysaccharides such as xylan (as a backbone-chain monosaccharide) and xyloglucan (as a branching monosaccharide). The biosynthesis of UDP-Xyl from UDP-glucuronic acid (UDP-GlcA) is irreversibly catalyzed by UDP-glucuronic acid decarboxylase (UXS). Until now, little has been known about the physiological roles of UXS in plants. Here, we report that AtUXS1, AtUXS2, and AtUXS4 are located in the Golgi apparatus whereas AtUXS3, AtUXS5, and AtUXS6 are located in the cytosol. Although all six single AtUXS T-DNA mutants and the uxs1 usx2 uxs4 triple mutant show no obvious phenotype, the uxs3 uxs5 uxs6 triple mutant has an irregular xylem phenotype. Monosaccharide analysis showed that Xyl levels decreased in uxs3 uxs5 uxs6 and linkage analysis confirmed that the xylan content in uxs3 xus5 uxs6 declined, indicating that UDP-Xyl from cytosol AtUXS participates in xylan synthesis. Gel-permeation chromatography showed that the molecular weight of non-cellulosic polysaccharides in the triple mutants, mainly composed of xylans, is lower than that in the wild type, suggesting an effect on the elongation of the xylan backbone. Upon saccharification treatment stems of the uxs3 uxs5 uxs6 triple mutants released monosaccharides with a higher efficiency than those of the wild type. Taken together, our results indicate that the cytosol UXS plays a more important role than the Golgi-localized UXS in xylan biosynthesis.
Changes in the mitochondrial proteome during the anoxia to air transition in rice focus around cytochrome-containing respiratory complexes
Publisher: Elsevier BV
Date: 09-2004
A Transcriptomic and Proteomic Characterization of the Arabidopsis Mitochondrial Protein Import Apparatus and Its Response to Mitochondrial Dysfunction
Publisher: Oxford University Press (OUP)
Date: 02-2004
Abstract: Mitochondria import hundreds of cytosolically synthesized proteins via the mitochondrial protein import apparatus. Expression analysis in various organs of 19 components of the Arabidopsis mitochondrial protein import apparatus encoded by 31 genes showed that although many were present in small multigene families, often only one member was prominently expressed. This was supported by comparison of real-time reverse transcriptase-polymerase chain reaction and microarray experimental data with expressed sequence tag numbers and massive parallel signature sequence data. Mass spectrometric analysis of purified mitochondria identified 17 import components, their mitochondrial sub-compartment, and verified the presence of TIM8, TIM13, TIM17, TIM23, TIM44, TIM50, and METAXIN proteins for the first time, to our knowledge. Mass spectrometry-detected isoforms correlated with the most abundant gene transcript measured by expression data. Treatment of Arabidopsis cell culture with mitochondrial electron transport chain inhibitors rotenone and antimycin A resulted in a significant increase in transcript levels of import components, with a greater increase observed for the minor isoforms. The increase was observed 12 h after treatment, indicating that it was likely a secondary response. Microarray analysis of rotenone-treated cells indicated the up-regulation of gene sets involved in mitochondrial chaperone activity, protein degradation, respiratory chain assembly, and ision. The rate of protein import into isolated mitochondria from rotenone-treated cells was halved, even though rotenone had no direct effect on protein import when added to mitochondria isolated from untreated cells. These findings suggest that transcription of import component genes is induced when mitochondrial function is limited and that minor gene isoforms display a greater response than the predominant isoforms.
Mitochondrial acyl carrier proteins in Arabidopsis thaliana are predominantly soluble matrix proteins and none can be confirmed as subunits of respiratory Complex I
Publisher: Springer Science and Business Media LLC
Date: 04-04-2007
DOI: 10.1007/S11103-007-9156-9
Abstract: Arabidopsis mitochondria are predicted to contain three acyl carrier proteins (ACPs). These small proteins are involved in fatty acid and lipoic acid synthesis in other organisms and have been previously reported to be subunits of respiratory Complex I in mitochondria in mammals, fungi and plants. Recently, the mammalian mitochondrial ACP (mtACP) has been shown to be largely a soluble matrix protein but also to be minimally associated with Complex I (Cronan et al. 2005), consistent with its involvement in synthesis of lipoic acid for TCA cycle decarboxylating dehydrogenases in the matrix but contrary to earlier claims it was primarily a Complex I subunit. We have investigated the localization of the ACPs in Arabidopsis mitochondria. Evidence is presented that mtACP1 and mtACP2 dominate the ACP composition in Arabidopsis mitochondria, and both are present in the mitochondrial matrix rather than in the membrane. No significant amounts of mtACPs were detected in Complex I isolated by blue native gel electrophoresis, rather mtACPs were detected at low molecular mass in the soluble fraction, showing that in A. thaliana mtACPs are predominately free soluble matrix proteins.
A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor
Publisher: Wiley
Date: 16-12-2003
DOI: 10.1016/S0014-5793(03)01457-1
Abstract: We have identified a novel protein on the outer membrane of Arabidopsis thaliana mitochondria. This protein displays 67% sequence identity with the 64 kDa translocase of the outer envelope membrane of chloroplasts (Toc). A mitochondrial localisation for this protein was determined by (i). its presence in the proteome of highly purified Arabidopsis mitochondria, (ii). Western blot analysis with antibodies to Toc64 from pea that indicate its presence in Arabidopsis and pea mitochondria, (iii). green fluorescent protein fusion proteins that indicate an exclusive mitochondrial localisation for this protein, and (iv). expression profiles in various tissue types and during development that are more similar to translocase of the outer mitochondrial membrane components than to chloroplastic Toc components. Thus Arabidopsis mitochondria contain a protein with high sequence identity to a plastid protein import receptor.
Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings
Publisher: American Chemical Society (ACS)
Date: 04-02-2016
DOI: 10.1021/ACS.JPROTEOME.5B00876
Abstract: The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the ersity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.
Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques
Publisher: Frontiers Media SA
Date: 28-08-2015
A survey of the Arabidopsis thaliana mitochondrial phosphoproteome
Publisher: Wiley
Date: 31-08-2009
Abstract: Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2-DE separations of Arabidopsis mitochondrial proteins and affinity-enriched phosphoproteins using the Pro-Q Diamond phospho-specific in-gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.
Three UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis.
Publisher: Oxford University Press (OUP)
Date: 28-12-2018
DOI: 10.1093/JXB/ERX448
Glycome and Proteome Components of Golgi Membranes Are Common between Two Angiosperms with Distinct Cell-Wall Structures
Publisher: Oxford University Press (OUP)
Date: 26-03-2019
DOI: 10.1105/TPC.18.00755
The Arabidopsis thaliana 2-D gel mitochondrial proteome: Refining the value of reference maps for assessing protein abundance, contaminants and post-translational modifications
Publisher: Wiley
Date: 07-04-2011
Abstract: Mitochondria undertake the process of oxidative phosphorylation yielding ATP for plant cell maintenance and growth. The principles of isolation and fractionation of plant mitochondrial proteins have been improved over decades, and surveys of the mitochondrial proteome in a number of plants species have been performed. Over time, many quantitative analyses of changes in the plant mitochondrial proteome have been performed by 2-D gel analyses revealing the induction, degradation and modification of mitochondrial proteins in responses to mutation, stress and development. Here, we present a saturating MS analysis of 2-D gel separable protein spots from a typical purification of Arabidopsis mitochondria identifying 264 proteins, alongside an LC-MS/MS survey by non-gel methods identifying 220 proteins. This allowed us to characterise the major mitochondrial proteins that are not observed on 2-D gels, the common contaminants and the abundance of the protein machinery of key mitochondrial biochemical pathways, and consider the impact of N-terminal pre-sequence cleavage and phosphorylation as explanations of multiple protein spots and the co-ordinates of proteins on 2-D gels.
The impact of oxidative stress on Arabidopsis mitochondria
Publisher: Wiley
Date: 12-2002
DOI: 10.1046/J.1365-313X.2002.01474.X
Abstract: 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 s les. 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.
The plant mitochondrial proteome and the challenge of defining the posttranslational modifications responsible for signalling and stress effects on respiratory functions
Publisher: Wiley
Date: 11-2007
Proteomic Characterization of Golgi Membranes Enriched from Arabidopsis Suspension Cell Cultures.
Publisher: Springer New York
Date: 2016
DOI: 10.1007/978-1-4939-6463-5_8
Abstract: The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.
AtAPY1 and AtAPY2 Function as Golgi-Localized Nucleoside Diphosphatases in Arabidopsis thaliana
Publisher: Oxford University Press (OUP)
Date: 03-10-2012
DOI: 10.1093/PCP/PCS131
Abstract: Nucleoside triphosphate diphosphohydrolases (NTPDases apyrases) (EC 3.6.1.5) hydrolyze di- and triphosphate nucleotides, but not monophosphate nucleotides. They are categorized as E-type ATPases, have a broad alent cation (Mg(2+), Ca(2+)) requirement for activation and are insensitive to inhibitors of F-type, P-type and V-type ATPases. Among the seven NTPDases identified in Arabidopsis, only APYRASE 1 (AtAPY1) and APYRASE 2 (AtAPY2) have been previously characterized. In this work, either AtAPY1 or AtAPY2 tagged with C-terminal green fluorescent protein (GFP) driven by their respective native promoter can rescue the apy1 apy2 double knockout (apy1 apy2 dKO) successfully, and confocal microscopy reveals that these two Arabidopsis apyrases reside in the Golgi apparatus. In Saccharomyces cerevisiae, both AtAPY1 and AtAPY2 can complement the Golgi-localized GDA1 mutant, rescuing its aberrant protein glycosylation phenotype. In Arabidopsis, microsomes of the wild type show higher substrate preferences toward UDP compared with other NDP substrates. Loss-of-function Arabidopsis AtAPY1 mutants exhibit reduced microsomal UDPase activity, and this activity is even more significantly reduced in the loss-of-function AtAPY2 mutant and in the AtAPY1/AtAPY2 RNA interference (RNAi) technology repressor lines. Microsomes from wild-type plants also have detectable GDPase activity, which is significantly reduced in apy2 but not apy1 mutants. The GFP-tagged AtAPY1 or AtAPY2 constructs in the apy1 apy2 dKO plants can restore microsomal UDP/GDPase activity, confirming that they both also have functional competency. The cell walls of apy1, apy2 and the RNAi-silenced lines all have an increased composition of galactose, but the transport efficiency of UDP-galactose across microsomal membranes was not altered. Taken together, these results reveal that AtAPY1 and AtAPY2 are Golgi-localized nucleotide diphosphatases and are likely to have roles in regulating UDP/GDP concentrations in the Golgi lumen.
Isolation and Subfractionation of Plant Mitochondria for Proteomic Analysis
Publisher: Humana Press
Date: 2007
Abstract: Mitochondria carry out a variety of biochemical processes in plant cells. Their primary role is the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons from reduced NAD+ to O2 via the electron transport chain. However, they also perform many important secondary functions such as synthesis of nucleotides, amino acids, lipids, and vitamins, they contain their own genome and undertake transcription and translation by some unique mechanisms, they actively import proteins and metabolites from the cytosol by a complex set of carriers and membrane channels, they influence programmed cell death of plants, and they respond to cellular signals such as oxidative stress. To understand the full range of mitochondrial functions in plants, the mechanisms that govern their biogenesis, and the way in which mitochondrial activity is perceived by the nucleus requires precise information about the protein components of these organelles. Isolation of mitochondria to identify their proteomes and the changes in these proteomes during development and environmental stress treatments is already under way. In this chapter we provide methods for isolating mitochondria from different plant tissue types, advice on assessing purity and storage of mitochondrial s les, and approaches to fractionate mitochondria to separate their membranes and soluble compartments from each other for proteome analysis.
Proteome coverage of the model plant Arabidopsis thaliana: Implications for shotgun proteomic studies
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.JPROT.2012.12.009
Abstract: The recent aggregation of matched proteomics data for the model plant Arabidopsis has enabled the assessment of a erse array of large scale shotgun proteomics data. A collection of over nine million matched peptides was used to assess proteome coverage and experimental parameters when compared to the theoretical tryptic peptide population. The analysis indicated that the experimentally identified median peptide mass was significantly higher than the theoretical median tryptic peptide in Arabidopsis. This finding led to a critical examination of precursor scan ranges currently being employed by shotgun proteomic studies. The analysis revealed diminishing returns at the high end scan range and opportunities for greater coverage and identifications at the low mass range. Based on these findings, a recommended basic scan range of 300 to 1200m/z would suitably capture the peptide population in shotgun proteomic analyses in Arabidopsis.
Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses
Publisher: Cold Spring Harbor Laboratory
Date: 11-06-2014
DOI: 10.1101/006155
Abstract: Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.
Managing the green proteomes for the next decade of plant research
Publisher: Frontiers Media SA
Date: 2013
BOTRYOID POLLEN 1 regulates ROS-triggered PCD and pollen wall development by controlling UDP-sugar homeostasis in rice
Publisher: Oxford University Press (OUP)
Date: 23-06-2023
Abstract: Uridine diphosphate (UDP)-sugars are important metabolites involved in the biosynthesis of polysaccharides and may be important signaling molecules. UDP-glucose 4-epimerase (UGE) catalyzes the interconversion between UDP-Glc and UDP-Gal, whose biological function in rice (Oryza sativa) fertility is poorly understood. Here, we identify and characterize the botryoid pollen 1 (bp1) mutant and show that BP1 encodes a UGE that regulates UDP-sugar homeostasis, thereby controlling the development of rice anthers. The loss of BP1 function led to massive accumulation of UDP-Glc and imbalance of other UDP-sugars. We determined that the higher levels of UDP-Glc and its derivatives in bp1 may induce the expression of NADPH oxidase genes, resulting in a premature accumulation of reactive oxygen species (ROS), thereby advancing programmed cell death (PCD) of anther walls but delaying the end of tapetal degradation. The accumulation of UDP-Glc as metabolites resulted in an abnormal degradation of callose, producing an adhesive microspore. Furthermore, the UDP-sugar metabolism pathway is not only involved in the formation of intine but also in the formation of the initial framework for extine. Our results reveal how UDP-sugars regulate anther development and provide new clues for cellular ROS accumulation and PCD triggered by UDP-Glc as a signaling molecule.
What makes a mitochondrion?
Publisher: Springer Science and Business Media LLC
Date: 2003
Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis
Publisher: Wiley
Date: 11-2007
Abstract: Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.
Untangling multi-gene families in plants by integrating proteomics into functional genomics
Publisher: Elsevier BV
Date: 06-2004
Absolute Quantitation of In Vitro Expressed Plant Membrane Proteins by Targeted Proteomics (MRM) for the Determination of Kinetic Parameters.
Publisher: Springer New York
Date: 31-10-2018
DOI: 10.1007/978-1-4939-7411-5_15
Abstract: The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination of the turnover number of an enzyme-substrate complex (k
Analysis of plant nucleotide sugars by hydrophilic interaction liquid chromatography and tandem mass spectrometry
Publisher: Elsevier BV
Date: 03-2014
Abstract: Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in their cell wall sugar compositions and indicated significant differences in the levels of 6 out of 12 nucleotide sugars. The plant nucleotide sugar extraction procedure was demonstrated to have good recovery rates with minimal matrix effects. The approach results in a significant improvement in sensitivity when applied to plant s les over currently employed techniques.
Novel Proteins, Putative Membrane Transporters, and an Integrated Metabolic Network Are Revealed by Quantitative Proteomic Analysis of Arabidopsis Cell Culture Peroxisomes
Publisher: Oxford University Press (OUP)
Date: 17-10-2008
Abstract: 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.
GDP-L-fucose transport in plants: The missing piece.
Publisher: Informa UK Limited
Date: 02-09-2017
Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses
Publisher: Oxford University Press (OUP)
Date: 10-2003
Transgenic Expression of the Dicotyledonous Pattern Recognition Receptor EFR in Rice Leads to Ligand-Dependent Activation of Defense Responses
Publisher: Public Library of Science (PLoS)
Date: 30-03-2015
Plant Proteomics: Challenges and Resources
Publisher: Blackwell Publishing Ltd
Date: 2007
Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signaling and Regulatory Components, Provides Assessment of Targeting Prediction Programs, and Indicates Plant-Specific Mitoc
Publisher: Oxford University Press (OUP)
Date: 2004
DOI: 10.1105/TPC.016055
Abstract: 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 ergence 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 ersity exists in many signaling and regulatory functions.
A Pipeline towards the Biochemical Characterization of the Arabidopsis GT14 Family
Publisher: MDPI AG
Date: 29-01-2021
DOI: 10.3390/IJMS22031360
Abstract: Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The Arabidopsis thaliana genome is predicted to encode over 573 GTs that are currently classified into 42 erse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI Arabidopsis GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (Nicotiana benthamiana) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal4) and β-1,3-galactopentose (β-1,3-Gal5) to microsomes expressing in idual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated Arabidopsis GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.
The Three Members of the Arabidopsis thaliana Glycosyltransferase Family 92 are Functional β-1,4-Galactan Synthases.
Publisher: Oxford University Press (OUP)
Date: 09-2018
DOI: 10.1093/PCP/PCY180
Abstract: Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.
Structural and Chemical Characterization of Hardwood from Tree Species with Applications as Bioenergy Feedstocks
Publisher: Public Library of Science (PLoS)
Date: 28-12-2012
An Integrative Approach to the Identification of Arabidopsis and Rice Genes Involved in Xylan and Secondary Wall Development
Publisher: Public Library of Science (PLoS)
Date: 23-11-2010
UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex
Publisher: Wiley
Date: 30-07-2020
DOI: 10.1111/TPJ.14921
The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development.
Publisher: Springer Science and Business Media LLC
Date: 06-07-2016
DOI: 10.1038/NCOMMS12119
Abstract: Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP- L -fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP- L -fucose over other nucleotide sugars in vitro , while GFT1 -silenced plants are almost devoid of L -fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L -fucose content in N -glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP- L -fucose into the Golgi and is required for proper plant growth and development.
The cytotoxic lipid peroxidation product 4-hydroxy-2-nonenal covalently modifies a selective range of proteins linked to respiratory function in plant mitochondria
Publisher: Elsevier BV
Date: 12-2007
Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast
Publisher: Springer Science and Business Media LLC
Date: 21-11-2016
Analysis of the Rice Mitochondrial Carrier Family Reveals Anaerobic Accumulation of a Basic Amino Acid Carrier Involved in Arginine Metabolism during Seed Germination
Publisher: Oxford University Press (OUP)
Date: 18-08-2010
Abstract: Given the substantial changes in mitochondrial gene expression, the mitochondrial proteome, and respiratory function during rice (Oryza sativa) germination under anaerobic and aerobic conditions, we have attempted to identify changes in mitochondrial membrane transport capacity during these processes. We have assembled a preliminary rice mitochondrial carrier gene family of 50 members, defined its orthology to carriers of known function, and observed significant changes in microarray expression data for these rice genes during germination under aerobic and anaerobic conditions and across rice development. To determine if these transcript changes reflect alteration of the carrier profile itself and to determine which members of the family encode the major mitochondrial carrier proteins, we analyzed mitochondrial integral membrane protein preparations using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass spectrometry, identifying seven distinct carrier proteins. We have used mass spectrometry-based quantitative approaches to compare the abundance of these carriers between mitochondria from dry seeds and those from aerobic- or anaerobic-germinated seeds. We highlight an anaerobic-enhanced basic amino acid carrier and show concomitant increases in mitochondrial arginase and the abundance of arginine and ornithine in anaerobic-germinated seeds, consistent with an anaerobic role of this mitochondria carrier. The potential role of this carrier in facilitating mitochondrial involvement in arginine metabolism and the plant urea cycle during the growth of rice coleoptiles and early seed nitrate assimilation under anaerobic conditions are discussed.
UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage.
Publisher: Oxford University Press (OUP)
Date: 2017
DOI: 10.1105/TPC.16.00465
Genomic and proteomic analysis of mitochondrial carrier proteins in Arabidopsis
Publisher: Oxford University Press (OUP)
Date: 02-2003
DOI: 10.1104/PP.009985
Abstract: Plant mitochondria maintain metabolic communication with the cytosol through a family of carrier proteins. In Arabidopsis, a subset of 45 putative genes encoding members of this family have been identified based on generalized mitochondrial carrier features. No gene clusters are apparent and few of the predicted protein products have mitochondrial targeting sequences recognized by bioinformatic predictors. Only nine genes are currently represented by more than 10 expressed sequence tags at The Institute for Genomic Research. Analyses of public microarray experiments reveal differential expression profiles of the more highly expressed members of this gene family in different plant organs and in response to plant hormone application and environmental stresses. A comparison of this Arabidopsis carrier subset (45) to the yeast gene family (35) reveals 10 orthologous groups between the two species. Recent surveys of the Arabidopsis mitochondrial proteome by two-dimensional gel separations have not identified any of these carrier proteins, presumably because of their hydrophobicity and basicity. Isolating integral membrane proteins from Arabidopsis mitochondria, using one-dimensional electrophoresis for protein separation and tandem mass spectrometry-based sequencing of doubly charged peptides, we have unequivocally identified specific carrier gene products located in mitochondria. This approach has identified six of the nine carriers represented highly in expressed sequence tag databases: adenine nucleotide translocator (At3g8580 and At5g13490), dicarboxylate/tricarboxylate carrier (At5g19760), phosphate carrier (At5g14040), uncoupling protein (At3g54110), and a carrier gene of unknown function (At4g01100). Overall, the combined transcript and protein expression data indicates that only a small subset of the carrier family of genes provide the majority of carrier proteins of Arabidopsis mitochondria.
Combining experimental and predicted datasets for determination of the subcellular location of proteins in arabidopsis
Publisher: Oxford University Press (OUP)
Date: 10-2005
Abstract: Substantial experimental datasets defining the subcellular location of Arabidopsis (Arabidopsis thaliana) proteins have been reported in the literature in the form of organelle proteomes built from mass spectrometry data (approximately 2,500 proteins). Subcellular location for specific proteins has also been published based on imaging of chimeric fluorescent fusion proteins in intact cells (approximately 900 proteins). Further, the more erse history of biochemical determination of subcellular location is stored in the entries of the Swiss-Prot database for the products of many Arabidopsis genes (approximately 1,800 proteins). Combined with the range of bioinformatic targeting prediction tools and comparative genomic analysis, these experimental datasets provide a powerful basis for defining the final location of proteins within the wide variety of subcellular structures present inside Arabidopsis cells. We have analyzed these published experimental and prediction data to answer a range of substantial questions facing researchers about the veracity of these approaches to determining protein location and their interrelatedness. We have merged these data to form the subcellular location database for Arabidopsis proteins (SUBA), providing an integrated understanding of protein location, encompassing the plastid, mitochondrion, peroxisome, nucleus, plasma membrane, endoplasmic reticulum, vacuole, Golgi, cytoskeleton structures, and cytosol (www.suba.bcs.uwa.edu.au). This includes data on more than 4,400 nonredundant Arabidopsis protein sequences. We also provide researchers with an online resource that may be used to query protein sets or protein families and determine whether predicted or experimental location data exist to analyze the nature of contamination between published proteome sets and/or for building theoretical subcellular proteomes in Arabidopsis using the latest experimental data.
1001 Proteomes: a functional proteomics portal for the analysis of Arabidopsis thaliana accessions
Publisher: Oxford University Press (OUP)
Date: 25-03-2012
DOI: 10.1093/BIOINFORMATICS/BTS133
Abstract: Motivation: The sequencing of over a thousand natural strains of the model plant Arabidopsis thaliana is producing unparalleled information at the genetic level for plant researchers. To enable the rapid exploitation of these data for functional proteomics studies, we have created a resource for the visualization of protein information and proteomic datasets for sequenced natural strains of A. thaliana. Results: The 1001 Proteomes portal can be used to visualize amino acid substitutions or non-synonymous single-nucleotide polymorphisms in in idual proteins of A. thaliana based on the reference genome Col-0. We have used the available processed sequence information to analyze the conservation of known residues subject to protein phosphorylation among these natural strains. The substitution of amino acids in A. thaliana natural strains is heavily constrained and is likely a result of the conservation of functional attributes within proteins. At a practical level, we demonstrate that this information can be used to clarify ambiguously defined phosphorylation sites from phosphoproteomic studies. Protein sets of available natural variants are available for download to enable proteomic studies on these accessions. Together this information can be used to uncover the possible roles of specific amino acids in determining the structure and function of proteins in the model plant A. thaliana. An online portal to enable the community to exploit these data can be accessed at 1001proteomes.masc-proteomics.org/ Contact: jlheazlewood@lbl.gov Supplementary information: Supplementary data are available at Bioinformatics online.
Standard Flow Liquid Chromatography for Shotgun Proteomics in Bioenergy Research
Publisher: Frontiers Media SA
Date: 04-2015
Golgi Enrichment and Proteomic Analysis of Developing Pinus radiata Xylem by Free-Flow Electrophoresis
Publisher: Public Library of Science (PLoS)
Date: 26-12-2013
Multi-Organism Proteomes (iMOP): Advancing our Understanding of Human Biology
Publisher: Wiley
Date: 09-2015
The plant glycosyltransferase clone collection for functional genomics
Publisher: Wiley
Date: 09-07-2014
DOI: 10.1111/TPJ.12577
Abstract: The glycosyltransferases (GTs) are an important and functionally erse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website
Beyond the Western front: targeted proteomics and organelle abundance profiling
Publisher: Frontiers Media SA
Date: 05-05-2015
Boosting the Globalization of Plant Proteomics through INPPO: Current Developments and Future Prospects
Publisher: Wiley
Date: 30-01-2012
Abstract: The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific).
The plant mitochondrial proteome
Publisher: Elsevier BV
Date: 2005
DOI: 10.1016/J.TPLANTS.2004.12.002
Abstract: The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid chromatography linked on-line with tandem mass spectrometry, have identified >400 mitochondrial proteins, including subunits of mitochondrial respiratory complexes, supercomplexes, phosphorylated proteins and oxidized proteins. The results also highlight a range of new mitochondrial proteins, new mitochondrial functions and possible new mechanisms for regulating mitochondrial metabolism. More than 70 identified proteins in Arabidopsis mitochondrial s les lack similarity to any protein of known function. In some cases, unknown proteins were found to form part of protein complexes, which allows a functional context to be defined for them. There are indications that some of these proteins add novel activities to mitochondrial protein complexes in plants.
Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum)
Publisher: Wiley
Date: 16-03-2015
Abstract: The cost-effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 (ataset/PXD001351).
Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis.
Publisher: Oxford University Press (OUP)
Date: 24-03-2015
Abstract: Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.
MASCP gator: an overview of the Arabidopsis proteomic aggregation portal
Publisher: Frontiers Media SA
Date: 2013
The Arabidopsis cytosolic proteome: the metabolic heart of the cell
Publisher: Frontiers Media SA
Date: 2014
Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization
Publisher: Oxford University Press (OUP)
Date: 02-07-2019
DOI: 10.1105/TPC.19.00081
Mitochondrial complex I from Arabidopsis and rice: Orthologs of mammalian and fungal components coupled with plant-specific subunits
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S0005-2728(03)00045-8
Abstract: The NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain is a large multisubunit complex in eukaryotes containing 30-40 different subunits. Analysis of this complex using blue-native gel electrophoresis coupled to tandem mass spectrometry (MS) has identified a series of 30 different proteins from the model dicot plant, Arabidopsis, and 24 different proteins from the model monocot plant, rice. These proteins have been linked back to genes from plant genome sequencing and comparison of this dataset made with predicted orthologs of complex I components in these plants. This analysis reveals that plants contain the series of 14 highly conserved complex I subunits found in other eukaryotic and related prokaryotic enzymes and a small set of 9 proteins widely found in eukaryotic complexes. A significant number of the proteins present in bovine complex I but absent from fungal complex I are also absent from plant complex I and are not encoded in plant genomes. A series of plant-specific nuclear-encoded complex I associated subunits were identified, including a series of ferripyochelin-binding protein-like subunits and a range of small proteins of unknown function. This represents a post-genomic and large-scale analysis of complex I composition in higher plants.
Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix
Publisher: American Chemical Society (ACS)
Date: 31-10-2006
DOI: 10.1021/PR060403J
Abstract: The interactions of ATP inside plant mitochondria were investigated by identifying the soluble nucleotide binding proteome captured using immobilized ATP. Selected proteins were separated by 1D SDS-PAGE and 2D IEF-SDS-PAGE and identified by ESI-Q-TOF MS/MS. A range of highly enriched proteins were identified from the mitochondrial proteome, including 14-3-3 proteins and RNA binding proteins, as well as proteins known to contain nucleotide binding domains and/or to be inhibited or stimulated by ATP.
Identification of a Sphingolipid α-Glucuronosyltransferase That Is Essential for Pollen Function inArabidopsis
Publisher: Oxford University Press (OUP)
Date: 08-2014
Abstract: Glycosyl inositol phosphorylceramide (GIPC) sphingolipids are a major class of lipids in fungi, protozoans, and plants. GIPCs are abundant in the plasma membrane in plants, comprising around a quarter of the total lipids in these membranes. Plant GIPCs contain unique glycan decorations that include a conserved glucuronic acid (GlcA) residue and various additional sugars however, no proteins responsible for glycosylating GIPCs have been identified to date. Here, we show that the Arabidopsis thaliana protein INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE1 (IPUT1) transfers GlcA from UDP-GlcA to GIPCs. To demonstrate IPUT1 activity, we introduced the IPUT1 gene together with genes for a UDP-glucose dehydrogenase from Arabidopsis and a human UDP-GlcA transporter into a yeast mutant deficient in the endogenous inositol phosphorylceramide (IPC) mannosyltransferase. In this engineered yeast strain, IPUT1 transferred GlcA to IPC. Overexpression or silencing of IPUT1 in Nicotiana benthamiana resulted in an increase or a decrease, respectively, in IPC glucuronosyltransferase activity in vitro. Plants in which IPUT1 was silenced accumulated IPC, the immediate precursor, as well as ceramides and glucosylceramides. Plants overexpressing IPUT1 showed an increased content of GIPCs. Mutations in IPUT1 are not transmitted through pollen, indicating that these sphingolipids are essential in plants.
Erratum: Analysis of the arabidopsis cytosolic proteome highlights subcellular partitioning of central plant metabolism (Journal of Proteome Research (2011) 10 (1571-1582) DOI: 10.1021/pr1009433)
Publisher: American Chemical Society (ACS)
Date: 20-05-2011
DOI: 10.1021/PR200437V
Integrated plant proteomics - Putting the green genomes to work
Publisher: CSIRO Publishing
Date: 2003
DOI: 10.1071/FP03036
Abstract: Protein analysis has been at the heart of plant science for many years, but with new questions emerging from an abundance of genomic information and further improvements in technology, there are now new opportunities to undertake large-scale analyses and to move to more complex systems than has been possible previously. This explosion of interest and data is often referred to simply as proteomics, which is the study of the complete set of proteins expressed at a given time and place, the proteome. As its name suggests proteomics is intricately linked to allied technologies such as genomics, transcriptomics and metabolomics. In this review of plant proteomics we outline a series of issues that face the practical user, particularly the largest problem that currently faces researchers, the myriad of options to choose from. The choices, problems and pitfalls of entering into gel-based and non-gel-based arraying techniques are discussed together with advances in pre-fractionation of s les, liquid chromatography separations and subcellular analyses. Issues relating to mass spectrometry analysis and the eventual protein identification are outlined, and the dilemmas of data storage and analysis are highlighted. During this tour we provide a series of references to the literature — experimental, theoretical and technical — to illustrate the breadth of current investigations using these techniques.
The Multinational Arabidopsis Steering Subcommittee for Proteomics Assembles the Largest Proteome Database Resource for Plant Systems Biology
Publisher: American Chemical Society (ACS)
Date: 12-09-2008
DOI: 10.1021/PR800480U
Expansin-like molecules: Novel functions derived from common domains
Publisher: Springer Science and Business Media LLC
Date: 05-2002
DOI: 10.1007/S00239-001-0055-4
Abstract: An Arabidopsis thaliana transcript ( AtPNP-A) encoding an immunoreactant plant natriuretic peptide (irPNP) analog was identified and isolated. The encoded protein shows similarity to CjBAp12, a functionally undefined protein from citrus that is induced in response to blight infection. CjBAp12 shows significant sequence identity to domains found in the cell wall loosening expansins but has tested negative for cell wall loosening activity. We have thus undertaken to establish the evolutionary and functional relationships of irPNP-like molecules within the superfamily of expansins, pollen allergens, and distantly related molecules such as endoglucanases. We show that irPNP-like molecules are related to expansins and fall in two groups one includes CjBAp12 and the other AtPNP-A. Members of both groups share distinct sequence motifs (K[VI]VD and [LM]SxxAFxxI) but do not contain the tryptophan and tyrosine rich C-terminal putative polysaccharide-binding domain typical of expansins or bacterial cellulases and hemicellulases. We argue that both irPNP-like molecules and expansin have evolved from primitive/ancestral glucanase-like molecules that hydrolysed the cell wall. Importantly, we have previously demonstrated that irPNPs act on protoplasts, that is plant cells without cell walls as well as microsomes, indicating that these novel proteins specifically interact with the plasma membrane. It follows that the cell wall cannot be an obligatory substrate for irPNPs. Thus, both irPNP function and domain structure point to these molecules having a systemic role in H2O and solute homeostasis.
PhosPhAt: a database of phosphorylation sites in Arabidopsis thaliana and a plant-specific phosphorylation site predictor
Publisher: Oxford University Press (OUP)
Date: 23-12-2007
DOI: 10.1093/NAR/GKM812
Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings.
Publisher: Springer New York
Date: 12-10-2017
DOI: 10.1007/978-1-4939-6533-5_11
Abstract: The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.
An XA21-Associated Kinase (OsSERK2) Regulates Immunity Mediated by the XA21 and XA3 Immune Receptors
Publisher: Elsevier BV
Date: 05-2014
DOI: 10.1093/MP/SSU003
Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier Transform Near Infrared spectroscopy
Publisher: Springer Science and Business Media LLC
Date: 18-08-2011
Abstract: We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.
Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification
Publisher: Elsevier BV
Date: 02-2008
Secretome analysis of the thermophilic xylanase hyper-producer Thermomyces lanuginosus SSBP cultivated on corn cobs
Publisher: Oxford University Press (OUP)
Date: 11-2014
DOI: 10.1007/S10295-014-1509-1
Abstract: Thermomyces lanuginosus is a thermophilic fungus known for its ability to produce industrially important enzymes including large amounts of xylanase, the key enzyme in hemicellulose hydrolysis. The secretome of T. lanuginosus SSBP was profiled by shotgun proteomics to elucidate important enzymes involved in hemicellulose saccharification and to characterise the presence of other industrially interesting enzymes. This study reproducibly identified a total of 74 proteins in the supernatant following growth on corn cobs. An analysis of proteins revealed nine glycoside hydrolase (GH) enzymes including xylanase GH11, β-xylosidase GH43, β-glucosidase GH3, α-galactosidase GH36 and trehalose hydrolase GH65. Two commercially produced Thermomyces enzymes, lipase and amylase, were also identified. In addition, other industrially relevant enzymes not currently explored in Thermomyces were identified including glutaminase, fructose-bisphosphate aldolase and cyanate hydratase. Overall, these data provide insight into the novel ability of a cellulase-free fungus to utilise lignocellulosic material, ultimately producing a number of enzymes important to various industrial processes.
Enrichment of the Plant Cytosolic Fraction
Publisher: Springer New York
Date: 12-10-2017
DOI: 10.1007/978-1-4939-6533-5_17
Abstract: The cytosol is at the core of cellular metabolism and contains many important metabolic pathways, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. Despite the importance of this matrix, few attempts have sought to specifically enrich this compartment from plants. Although a variety of biochemical pathways and signaling cascades pass through the cytosol, much of the focus has usually been targeted at the reactions that occur within membrane-bound organelles of the plant cell. In this chapter, we outline a method for the enrichment of the cytosol from rice suspension cell cultures which includes s le preparation and enrichment as well as validation using immunoblotting and fluorescence-tagged proteins.
Proteomic identification of divalent metal cation binding proteins in plant mitochondria
Publisher: Wiley
Date: 07-02-2003
DOI: 10.1016/S0014-5793(03)00101-7
Abstract: Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of alent 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 alent cation-binding proteins, or to catalyse alent 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 alent 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 alent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.
Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis
Publisher: Oxford University Press (OUP)
Date: 02-2004
Abstract: 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.
Biochemical characterization of Arabidopsis APYRASE family reveals their roles in regulating endomembrane NDP/NMP homoeostasis
Publisher: Portland Press Ltd.
Date: 30-10-2015
DOI: 10.1042/BJ20150235
Abstract: Plant apyrases are nucleoside triphosphate (NTP) diphosphohydrolases (NTPDases) and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound apyrases (AtAPY1–7) that comprise three distinct clades, all of which contain the five conserved apyrase domains. With the exception of AtAPY1 and AtAPY2, the biochemical and the sub-cellular characterization of the other members are currently unavailable. In this research, we have shown all seven Arabidopsis apyrases localize to internal membranes comprising the cis-Golgi, endoplasmic reticulum (ER) and endosome, indicating an endo-apyrase classification for the entire family. In addition, all members, with the exception of AtAPY7, can function as endo-apyrases by complementing a yeast double mutant (Δynd1Δgda1) which lacks apyrase activity. Interestingly, complementation of the mutant yeast using well characterized human apyrases could only be accomplished by using a functional ER endo-apyrase (NTPDase6), but not the ecto-apyrase (NTPDase1). Furthermore, the substrate specificity analysis for the Arabidopsis apyrases AtAPY1–6 indicated that each member has a distinct set of preferred substrates covering various NDPs (nucleoside diphosphates) and NTPs. Combining the biochemical analysis and sub-cellular localization of the Arabidopsis apyrases family, the data suggest their possible roles in regulating endomembrane NDP/NMP (nucleoside monophosphate) homoeostasis.
AMPDB: The Arabidopsis Mitochondrial Protein Database
Publisher: Oxford University Press (OUP)
Date: 17-12-2005
DOI: 10.1093/NAR/GKI048
A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans
Publisher: Springer Science and Business Media LLC
Date: 18-04-2016
A hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia
Publisher: Oxford University Press (OUP)
Date: 19-08-2019
DOI: 10.1093/HMG/DDZ200
Abstract: We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a ‘Schneckenbecken-like dysplasia.’ The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.
A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids
Publisher: Springer Science and Business Media LLC
Date: 17-09-2018
DOI: 10.1038/S41477-018-0235-5
Abstract: Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cytosolic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphorylceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids.
Differential impact of environmental stresses on the pea mitochondrial proteome
Publisher: Elsevier BV
Date: 08-2005
The relationship between sleep problems and working memory in children born very preterm
Publisher: Informa UK Limited
Date: 19-10-2016
DOI: 10.1080/09297049.2016.1235144
Abstract: In two studies, the relationship between sleep and working memory performance was investigated in children born very preterm (i.e., gestation less than 32 weeks) and the possible mechanisms underlying this relationship. In Study 1, parent-reported measures of snoring, night-time sleep quality, and daytime sleepiness were collected on 89 children born very preterm aged 6 to 7 years. The children completed a verbal working memory task, as well as measures of processing speed and verbal storage capacity. Night-time sleep quality was found to be associated with verbal working memory performance over and above the variance associated with in idual differences in processing speed and storage capacity, suggesting that poor sleep may have an impact on the executive component of working memory. Snoring and daytime sleepiness were not found to be associated with working memory performance. Study 2 introduced a direct measure of executive functioning and examined whether sleep problems would differentially impact the executive functioning of children born very preterm relative to children born to term. Parent-reported sleep problems were collected on 43 children born very preterm and 48 children born to term (aged 6 to 9 years). Problematic sleep was found to adversely impact executive functioning in the very preterm group, while no effect of sleep was found in the control group. These findings implicate executive dysfunction as a possible mechanism by which problematic sleep adversely impacts upon cognition in children born very preterm, and suggest that sleep problems can increase the cognitive vulnerability already experienced by many of these children.
A signaling-regulated, short-chain dehydrogenase of Stagonospora nodorum regulates asexual development
Publisher: American Society for Microbiology
Date: 11-2008
DOI: 10.1128/EC.00237-08
Abstract: The fungus Stagonospora nodorum is a causal agent of leaf and glume blotch disease of wheat. It has been previously shown that inactivation of heterotrimeric G protein signaling in Stagonospora nodorum caused development defects and reduced pathogenicity [P. S. Solomon et al., Mol. Plant-Microbe Interact. 17:456-466, 2004]. In this study, we sought to identify targets of the signaling pathway that may have contributed to phenotypic defects of the signaling mutants. A comparative analysis of Stagonospora nodorum wild-type and Gα-defective mutant ( gna1 ) intracellular proteomes was performed via two-dimensional polyacrylamide gel electrophoresis. Several proteins showed significantly altered abundances when comparing the two strains. One such protein, the short-chain dehydrogenase Sch1, was 18-fold less abundant in the gna1 strain, implying that it is positively regulated by Gα signaling. Gene expression and transcriptional enhanced green fluorescent protein fusion analyses of Sch1 indicates strong expression during asexual development. Mutant strains of Stagonospora nodorum lacking Sch1 demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Detailed histological experiments on sch1 pycnidia revealed that the gene is required for the differentiation of the subparietal layers of asexual pycnidia resulting in a significant reduction in both pycnidiospore size and numbers.
The products of the mitochondrial orf25 and orfB genes are FO components in the plant F1FO ATP synthase
Publisher: Wiley
Date: 20-03-2003
DOI: 10.1016/S0014-5793(03)00264-3
Abstract: 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.
The Role of Proteomics in the Development of Cellulosic Biofuels
Publisher: Bentham Science Publishers Ltd.
Date: 07-2010
Can Social Media-Based Self-testing Be a New Paradigm for Human Immunodeficiency Virus Testing in China?
Publisher: Oxford University Press (OUP)
Date: 25-06-2020
DOI: 10.1093/CID/CIAA817
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