Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques

This study presents a comparative proteomic analysis of the membrane subproteome of whole Arabidopsis seedlings using 2% Brij-58 or 60% methanol to enrich and solubilize membrane proteins for strong cation exchange fractionation and reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 441 proteins were identified by our Brij-58 method, and 300 proteins were detected by our methanol-based solubilization approach. Although the total number of proteins obtained using the nonionic detergent was higher than the total obtained by organic solvent, the ratio of predicted membrane proteins to total proteins identified indicates up to an 18.6% greater enrichment efficiency using methanol. Using two different bioinformatics approaches, between 31.0 and 40.0% of the total proteins identified by the methanol-based method were classified as containing at least one putative transmembrane domain as compared to 22.0-23.4% for Brij-58. In terms of protein hydrophobicity as determined by the GRAVY index, it was revealed that methanol was more effective than Brij-58 for solubilizing membrane proteins ranging from -0.4 (hydrophilic) to +0.4 (hydrophobic). Methanol was also approximately 3-fold more effective than Brij-58 in identifying leucine-rich repeat receptor-like kinases. The ability of methanol to effectively solubilize and denature both hydrophobic and hydrophilic proteins was demonstrated using bacteriorhodopsin and cytochrome c, respectively, where both proteins were identified with at least 82% sequence coverage from a single reversed-phase LC-MS/MS analysis. Overall, our data show that methanol is a better alternative for identifying a wider range of membrane proteins than the nonionic detergent Brij-58.     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC–MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC–MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.     

Functional analysis of the tubulin-folding cofactor C in Arabidopsis thaliana

The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

Characterization of microstructure and cell wall components of Arabidopsis thaliana overexpressing cyclin-dependent kinase inhibitor 2

Overexpression of a cyclin-dependent kinase inhibitor (KRP2) caused changes in the general morphology in the leaves of Arabidopsis thaliana. The wild type plant had obovate leaves with entire margins whereas the transgenic line had leaves with denticulate margins. The epidermal cells and stomata of the adult transgenic leaves were significantly larger than those of the wild-type plants and the number of stomata was in proportion to the number of epidermal cells. No apparent differences in thickness and structure of cell walls of the mesophyll cells between the two samples were observed. The smaller amount of cell wall material in the transgenic leaves caused by the larger cell size was also apparent in the lower dry weight of the transgenic leaves. The chemical analysis revealed the main differences to be in pectin and neutral sugar contents, and especially in the amounts of glucose, all being higher in the leaves of the KRP2 transgenic plants. p-Coumaric acid content varied more in the transgenic leaf material than in the control one reflecting possibly fewer cross-links in the cell walls of transgenic plants.     

Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis

In a recent bioinformatic analysis, we predicted the presence of multiple families of cell surface glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) in Arabidopsis (G.H.H. Borner, D.J. Sherrier, T.J. Stevens, I.T. Arkin, P. Dupree [2002] Plant Physiol 129: 486-499). A number of publications have since demonstrated the importance of predicted GAPs in diverse physiological processes including root development, cell wall integrity, and adhesion. However, direct experimental evidence for their GPI anchoring is mostly lacking. Here, we present the first, to our knowledge, large-scale proteomic identification of plant GAPs. Triton X-114 phase partitioning and sensitivity to phosphatidylinositol-specific phospholipase C were used to prepare GAP-rich fractions from Arabidopsis callus cells. Two-dimensional fluorescence difference gel electrophoresis and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of a large number of phospholipase C-sensitive Arabidopsis proteins. Using liquid chromatography-tandem mass spectrometry, 30 GAPs were identified, including six beta-1,3 glucanases, five phytocyanins, four fasciclin-like arabinogalactan proteins, four receptor-like proteins, two Hedgehog-interacting-like proteins, two putative glycerophosphodiesterases, a lipid transfer-like protein, a COBRA-like protein, SKU5, and SKS1. These results validate our previous bioinformatic analysis of the Arabidopsis protein database. Using the confirmed GAPs from the proteomic analysis to train the search algorithm, as well as improved genomic annotation, an updated in silico screen yielded 64 new candidates, raising the total to 248 predicted GAPs in Arabidopsis.     

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H(2)O(2) content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.     

Identification and structural analysis of SINE elements in the Arabidopsis thaliana genome

An insertion sequence was found in a Mu homologue in the genome of Arabidopsis thaliana. The insertion sequence had poly(A) at the 3' end, and promoter motifs (A- and B-boxes) recognized by RNA polymerase III. The sequence was flanked by direct repeats of a 15-bp sequence of the Mu homologue, which appears to be a target-site sequence duplicated upon insertion. These findings indicate that the insertion sequence is a retroposon SINE, and it was therefore named AtSN (A. thaliana SINE). Many members of the AtSN family were identified through a computer-aided homology search of databases and classified into two subfamilies, AtSN1 and AtSN2, having consensus sequences 159 and 149 bp in length, respectively. These had no homology to SINEs in other organisms. About half of AtSN members were truncated through loss of a region at either end of the element. Most of them were truncated at the 5' end, and had a duplication of the target-site sequence. This suggests that the ones with 5' truncation retroposed by the same mechanism as those without truncation. Members of the AtSN1 or AtSN2 subfamilies had many base substitutions when compared with the consensus sequence. All of the members examined were present in three different ecotypes of A. thaliana (Columbia, Landsberg erecta, and Wassilewskija). These findings suggest that AtSN members had proliferatedbefore the A. thaliana ecotype strains diverged.     

Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in

Identification of two cell-cycle-controlling cdc2 gene homologs in Arabidopsis thaliana

The cdc2 gene product (p34cdc2) has been thought to play a central role in control of the mitotic cell cycle of yeasts and animals. To approach an understanding of the cell-cycle-control system in higher plants, we isolated, from an Arabidopsis thaliana cDNA library, two clones (CDC2a and CDC2b) similar to the Schizosaccharomyces pombe cdc2 gene. Genomic Southern-blot analysis with the CDC2a and CDC2b cDNA probes suggested that the A. thaliana genome contains several additional cdc2-like genes, which together with the CDC2a and CDC2b genes may constitute a CDC2 gene family. The CDC2a cDNA expressed in Sc. pombe corrected the elongated morphology, caused by the temperature-sensitive cdc2-33 mutation, to the normal shapes, indicating that the A. thaliana CDC2a gene product resembles Sc. pombe p34cdc2 functionally as well as structurally. These results support the view that the cell cycle of higher plants is controlled by an analogue of a p34cdc2-centered regulatory system like that of yeasts and animals.     

Identification of miRNAs in C. roseus and their potential targets

MicroRNAs are small (20-22 nucleotides) none coding, regulatory RNAs, whose pivotal role in gene expression has been associated in number of diseases, therefore prediction of miRNA is an essential yet challenging field. In this study miRNAs of C. roseus are predicted along with their possible target genes. A total of 19,899 ESTs were downloaded from dbEST database and processed and trimmed through SeqClean. Nine sequences were trashed and 31 sequences were trimmed by the program and the resulting sequences were submitted to Repeatmasker and TGICL for clustering and assembly. This contig database was now used to find the putative miRNAs by performing a local BLAST with the miRNAs of B. rapa retrieved from miRBase. The targets were scanned by hybridizing screened ESTs with the UTRs of human using miRanda software. Finally, 7 putative miRNAs were found to hybridize with the various targets of signal transduction and apoptosis that may play significant role in preventing diseases like Leukemia, Arthritis and Alzheimer.     

Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology

A proteome study based on 2-D gel electrophoresis was performed in order to analyse the cold-stress response of Arabidopsis plants. The emphasis was to monitor the overall changes in the protein complement after prolonged exposure rather than short-term responses. Two different temperature regimes were used (6 degrees C and 10 degrees C) and plants were exposed to cold-stress exposure for 1 week. Protein patterns were also monitored after re-shifting plants to control conditions for a further week. To monitor gradual changes in the response to the two cold-stress conditions, the analysis was performed with DIGE technology with the inclusion of an internal standard. In the experiments using 6 degrees C, 22 spots with at least 2-fold altered expression were found; among them 18 were increased and four were decreased. When plants were exposed to 10 degrees C, 18 of these 22 spots still showed a 2-fold change; however, the alterations were, in general, more moderate than observed under 6 degrees C. Spot identification was performed by MALDI-TOF and ESI-MS/MS. Many of the proteins identified have previously been described in the context of cold-stress responses, indicating the validity of this proteome approach for further in-depth studies.     

Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.     

Identification of new minisatellites loci in Arabidopsis thaliana

In order to characterize new CG-rich minisatellites present in theArabidopsis thaliana genome, a genomic library wasscreened at low stringency with a probe containing nine repeated-units of aminisatellite (CMs1) previously identified. Both minisatellites andminisatellite-like elements were identified. The minisatellites, with atandemly-repeated structure, all contain the Arabidopsisthaliana-core sequence previously defined (Tourmenteet al., 1994). Both minisatellite andminisatellite-like sequences occur in the Arabidopsisgenome in low copy and are weakly polymorphic between ecotypes. The geneticmapping of these markers has shown that they are dispersed on the genome.YACs clones of the CIC library carrying these minisatellites andminisatellite-like sequences were identified.Key words:Arabidopsis thaliana, minisatellites, polymorphism     

Identification of new celiac disease autoantigens using proteomic analysis

Celiac disease is an autoimmune disorder in which gluten peptides presented by specific HLA-DQ2- and HLA-DQ8-positive antigen presenting cells elicit immune response in connective tissue of lamina propria. Immunoglobulin A (IgA) antiendomysial antibodies are specific for celiac disease and are used for screening, diagnosis and follow-up of this disease with an almost 100% sensitivity and specificity. The major target antigen of IgA antiendomysial antibodies was identified as tissue transglutaminase; nevertheless, the existence of the additional unique celiac disease-specific autoantigens is anticipated. In this study we have utilized a proteomic approach in order to search out new autoantigens recognized by serum antibodies of patients with active celiac disease. We report the detection of 11 proteins that were immunorecognized with various frequencies by sera of patients with celiac disease. Four autoantigens were identified by mass fingerprinting approach as actin, ATP synthase beta chain and two charge variants of enolase alpha. While production of IgA antibodies against actin molecules were described earlier, the existence of autoantibodies to ATP synthase beta chain and enolase alpha species in sera collected from patients with active celiac disease are described for the first time. These results are suggestive of the existence of additional celiac disease autoantigens with possible diagnostic utility.