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• W2255979074 abstract "Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell–cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which had no known function hitherto, is a negative regulator of cell death and regulates pathogen-induced symptom development in Arabidopsis. Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell–cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which had no known function hitherto, is a negative regulator of cell death and regulates pathogen-induced symptom development in Arabidopsis. Programmed cell death (pcd) 1The abbreviations used are:pcdprogrammed cell death2D-DiGE2 dimensional difference gel electrophoresisCFUcolony forming unitsDIAPDrosophila inhibitor of apoptosiseATPextracellular ATPFB1fumonisin B1GOGene OntologyLCBslong chain basesLerLandsberg erectaNo-0Nossen-0SAsalicylic acidT-DNAtransfer-DNA. 1The abbreviations used are:pcdprogrammed cell death2D-DiGE2 dimensional difference gel electrophoresisCFUcolony forming unitsDIAPDrosophila inhibitor of apoptosiseATPextracellular ATPFB1fumonisin B1GOGene OntologyLCBslong chain basesLerLandsberg erectaNo-0Nossen-0SAsalicylic acidT-DNAtransfer-DNA. is a genetically controlled dismantling of cells, which is indispensable for plant development and stress-adaptive responses. In development, pcd is invoked to facilitate xylem tracheary element differentiation, to remodel leaf shape, and to delete ephemeral cells and organs such as embryonic suspensor cells (1Greenberg J.T. Programmed cell death: a way of life for plants.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 12094-12097Crossref PubMed Scopus (498) Google Scholar, 2Beers E.P. Programmed cell death during plant growth and development.Cell Death Differ. 1997; 4: 649-661Crossref PubMed Scopus (131) Google Scholar, 3Gunawardena A.H.L.A.N. Greenwood J.S. Dengler N.D. Programmed cell death remodels lace plant shape during development.Plant Cell. 2004; 16: 60-73Crossref PubMed Scopus (166) Google Scholar). In response to drought stress, pcd is used to break root apical meristem dominance in order to remodel root system architecture as an adaptive response to water deficit (4Duan Y.F. Zhang W.S. Li B. Wang Y.N. Li K.X. Sodmergen Han C. Zhang Y. Li X. An endoplasmic reticulum response pathway mediates programmed cell death of root tip induced by water stress in Arabidopsis.New Phytol. 2010; 186: 681-695Crossref PubMed Scopus (145) Google Scholar). Additionally, a specialized form of pcd known as the hypersensitive response kills plant cells at the epicenter of attack by certain pathogens, which activate the effector-triggered immune response (5Dodds P.N. Rathjen J.P. Plant immunity: towards an integrated view of plant-pathogen interactions.Nat. Rev. Genet. 2010; 11: 539-548Crossref PubMed Scopus (2032) Google Scholar, 6Spoel S.H. Dong X. How do plants achieve immunity? Defence without specialized immune cells.Nat. Rev. Immunol. 2012; 12: 89-100Crossref PubMed Scopus (683) Google Scholar). A detailed understanding of the signal transduction pathways that trigger, propagate, and terminate plant pcd requires identification of the key components of the underlying protein networks. Our group has been using Arabidopsis cell death induced by fumonisin B1 (FB1) as an experimental system to study plant pcd and identify the key regulatory proteins (6Spoel S.H. Dong X. How do plants achieve immunity? Defence without specialized immune cells.Nat. Rev. Immunol. 2012; 12: 89-100Crossref PubMed Scopus (683) Google Scholar). programmed cell death 2 dimensional difference gel electrophoresis colony forming units Drosophila inhibitor of apoptosis extracellular ATP fumonisin B1 Gene Ontology long chain bases Landsberg erecta Nossen-0 salicylic acid transfer-DNA. programmed cell death 2 dimensional difference gel electrophoresis colony forming units Drosophila inhibitor of apoptosis extracellular ATP fumonisin B1 Gene Ontology long chain bases Landsberg erecta Nossen-0 salicylic acid transfer-DNA. FB1, a mycotoxin that triggers cell death in both animal and plant cells (8Huang C. Dickman M. Henderson G. Jones C. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen.Cancer Res. 1995; 55: 1655-1659PubMed Google Scholar, 9Gilchrist D.G. Ward B. Moussatos V. Mirocha C.J. Genetic and physiological response to fumonisins and AAL-toxin by intact tissue of a higher plant.Mycopathologia. 1992; 117: 57-64Crossref Scopus (54) Google Scholar), disrupts sphingolipid biosynthesis via inhibition of ceramide synthase (10Merrill Jr., A.H. Wang E. Gilchrist D.G. Riley R.T. Fumonisins and other inhibitors of de novo sphingolipid biosynthesis.Adv. Lipid Res. 1993; 26: 215-234PubMed Google Scholar). Several proteins directly involved in sphingolipid biosynthesis and metabolism have been shown to regulate FB1-induced plant pcd because of their influence on levels of metabolic intermediates, such as long chain bases (LCBs), which act as second messengers of plant cell death. For example, activity of serine palmitoyltransferase, the enzyme catalyzing the first rate-limiting step in sphingolipid biosynthesis, strongly controls Arabidopsis sensitivity to FB1 (11Kimberlin A.N. Majumder S. Han G. Chen M. Cahoon R.E. Stone J.M. Dunn T.M. Cahoon E.B. Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity.Plant Cell. 2013; 25: 4627-4639Crossref PubMed Scopus (44) Google Scholar). Serine palmitoyltransferase has two subunits – LCB1 and LCB2. Resistance to FB1-induced death is manifested in Arabidopsis loss-of-function mutants of LCB1 (12Shi L. Bielawski J. Mu J. Dong H. Teng C. Zhang J. Yang X. Tomishige N. Hanada K. Hannun Y.A. Zuo J. Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis.Cell Res. 2007; 17: 1030-1040Crossref PubMed Scopus (166) Google Scholar) and LCB2a (13Saucedo-García M. Guevara-García A. González-Solís A. Cruz-García F. Vázquez-Santana S. Markham J.E. Lozano-Rosas M.G. Dietrich C.R. Ramos-Vega M. Cahoon E.B. Gavilanes-Ruíz M. MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis.New Phytol. 2011; 191: 943-957Crossref PubMed Scopus (97) Google Scholar) genes. Overexpression of endogenous Arabidopsis 56 amino acid polypeptides that interact with and stimulate serine palmitoyltransferase activity increases sensitivity to FB1, whereas RNA interference lines have reduced sensitivity to the mycotoxin (11Kimberlin A.N. Majumder S. Han G. Chen M. Cahoon R.E. Stone J.M. Dunn T.M. Cahoon E.B. Arabidopsis 56-amino acid serine palmitoyltransferase-interacting proteins stimulate sphingolipid synthesis, are essential, and affect mycotoxin sensitivity.Plant Cell. 2013; 25: 4627-4639Crossref PubMed Scopus (44) Google Scholar). Although exogenous ceramide can suppress FB1-induced death in animal cells (14Harel R. Futerman A.H. Inhibition of sphingolipid synthesis affects axonal outgrowth in cultured hippocampal neurons.J. Biol. Chem. 1993; 268: 14476-14481Abstract Full Text PDF PubMed Google Scholar), it fails to block cell death in Arabidopsis (15Stone J.M. Heard J.E. Asai T. Ausubel F.M. Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants.Plant Cell. 2000; 12: 1811-1822Crossref PubMed Scopus (188) Google Scholar), indicating that other factors work in concert with ceramide depletion in pcd induction in Arabidopsis. Identification of these factors is essential to the understanding of general pcd regulation in plants, given that Arabidopsis responses to FB1 share common features with the pathogen-induced hypersensitive response (15Stone J.M. Heard J.E. Asai T. Ausubel F.M. Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants.Plant Cell. 2000; 12: 1811-1822Crossref PubMed Scopus (188) Google Scholar). Clues that may lead to mechanistic details of pcd could arise from focusing on known regulatory signals that control FB1-mediated responses. FB1-induced cell death is regulated by extracellular ATP (eATP) (16Chivasa S. Ndimba B.K. Simon W.J. Lindsey K. Slabas A.R. Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability.Plant Cell. 2005; 17: 3019-3034Crossref PubMed Scopus (151) Google Scholar) and the plant defense hormone, salicylic acid (SA) (17Asai T. Stone J.M. Heard J.E. Kovtun Y. Yorgey P. Sheen J. Ausubel F.M. Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways.Plant Cell. 2000; 12: 1823-1835Crossref PubMed Scopus (292) Google Scholar). NahG transgenic plants, which degrade SA, are resistant to FB1 as are pad4–1 mutants, which have an impaired SA amplification mechanism (17Asai T. Stone J.M. Heard J.E. Kovtun Y. Yorgey P. Sheen J. Ausubel F.M. Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways.Plant Cell. 2000; 12: 1823-1835Crossref PubMed Scopus (292) Google Scholar). Mutants that constitutively accumulate greater amounts of SA, cpr1 and cpr6, manifest increased susceptibility to FB1 (17Asai T. Stone J.M. Heard J.E. Kovtun Y. Yorgey P. Sheen J. Ausubel F.M. Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways.Plant Cell. 2000; 12: 1823-1835Crossref PubMed Scopus (292) Google Scholar). Thus, SA functions as a positive regulator of FB1-induced pcd. In contrast, eATP is a negative regulator of FB1-triggered pcd in Arabidopsis. Accordingly, FB1 activates eATP depletion prior to onset of death and addition of exogenous ATP to FB1-treated Arabidopsis cell suspension cultures blocks pcd (16Chivasa S. Ndimba B.K. Simon W.J. Lindsey K. Slabas A.R. Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability.Plant Cell. 2005; 17: 3019-3034Crossref PubMed Scopus (151) Google Scholar). This suggests that SA- and eATP-mediated signaling converge onto the signal transduction cascade activated by FB1 to promote or inhibit pcd, respectively. We have developed an experimental system, which harnesses the effects of exogenous ATP and SA on FB1-induced death, to identify important proteins that regulate Arabidopsis pcd. It utilizes Arabidopsis cell suspension cultures treated with these compounds and proteomic analyses restricted to the mobile phase of the extracellular matrix. The extracellular matrix proteome consists of cell surface proteins fully or partially embedded in the plasma membrane, proteins immobilized in the cell wall, and soluble mobile proteins in the apoplastic fluid – the mobile phase. The rationale for this is predicated on the hypothesis that cells constantly communicate with their neighbors by releasing and sensing signal molecules in the mobile phase (18Chivasa S. Slabas A.R. Plant extracellular ATP signaling: new insight from proteomics.Mol. Biosyst. 2012; 8: 445-452Crossref PubMed Scopus (18) Google Scholar). Arabidopsis has more than 600 plasma membrane receptor kinases (19Shiu S.H. Bleecker A.B. Plant receptor-like kinase gene family: diversity, function, and signaling.Science's STKE. 2001; 2001: RE22PubMed Google Scholar) and ∼400 G-protein-coupled receptors (20Moriyama E.N. Strope P.K. Opiya S.O. Chen Z. Jones A.M. Mining the Arabidopsis genome for highly divergent seven transmembrane receptors.Genome Biol. 2006; 7: R96Crossref PubMed Scopus (68) Google Scholar, 21Tuteja N. Signaling through G protein coupled receptors.Plant Signal. Behav. 2009; 4: 942-947Crossref PubMed Scopus (137) Google Scholar), which sense extracellular signals at the cell surface and activate a cytoplasmic response. We hypothesize that upon receiving an exogenous chemical, cell–cell signaling is activated either by directly binding the chemical if it has a cell surface receptor, or by modulating signal regulatory proteins in the mobile phase to reset the communication and transmit new signals. Therefore, in this study, we used ATP and SA treatments to identify pcd regulatory proteins in the mobile phase of the Arabidopsis extracellular matrix. We provide a novel extracellular matrix putative cell death regulatory protein network and present evidence validating the role of CYCLASE1 in FB1- and pathogen-induced pcd and the control of disease symptoms. The T-DNA insertion mutant line of Arabidopsis from the JIC SM collection (GT_5_42439) (22Tissier A.F. Marillonnet S. Klimyuk V. Patel K. Torres M.A. Murphy G. Jones J.D.G. Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.Plant Cell. 1999; 11: 1841-1852Crossref PubMed Scopus (328) Google Scholar) was obtained from the Nottingham Arabidopsis Seed Stock Centre (Nottingham, UK). An Arabidopsis transposon-tagged line (RATM13_3839_1) (23Ito T. Motohashi R. Kuromori T. Mizukado S. Sakurai T. Kanahara H. Seki. M. Shinozaki K. A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome.Plant Physiol. 2002; 129: 1695-1699Crossref PubMed Scopus (84) Google Scholar, 24Kuromori T. Hirayama T. Kiyosue Y. Takabe H. Mizukado S. Sakurai T. Akiyama K. Kamiya A. Ito T. Shinozaki K. A collection of 11,800 single-copy Ds transposon insertion lines in Arabidopsis.Plant J. 2004; 37: 897-905Crossref PubMed Scopus (180) Google Scholar), developed by the plant genome project of RIKEN Genomic Sciences Centre, was ordered from RIKEN (Tsukuba, Japan). Plants were grown at 23 °C with a 16 h photoperiod at ∼150 μmolm−2sec−1 under cool white fluorescent lights. Cell suspension cultures of Arabidopsis thaliana derived from tissue of ecotype Landsberg erecta were maintained as described previously (25Hamilton J.M.U. Simpson D.J. Hyman S. Ndimba B.K. Slabas A.R. Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity, and tissue localization.Biochem. J. 2003; 370: 57-67Crossref PubMed Scopus (57) Google Scholar). All chemicals and growth media were purchased from Sigma (http://www.sigmaaldrich.com). Stock solutions of ATP and salicylic acid were prepared fresh in water and adjusted to pH 6.7 prior to application. FB1 stock solutions were prepared in 70% methanol. Cell cultures were treated by adding the appropriate volume of chemical into the growth medium, whereas leaves were infiltrated with the solutions into the apoplast using a syringe without a needle. All plants were used for experiments 4–5 weeks after sowing, whereas 30 ml cell cultures at 3 days post-subculturing were adjusted to a density of 5% (w/v) and treated by addition of appropriate solutions into the growth medium. RNeasy Plant Kit (Qiagen, Crawley, UK) with on-column DNase treatment was used to extract total RNA from Arabidopsis leaf tissues according to the manufacturer's instructions. A previously described protocol (26Chivasa S. Hamilton J.M. Pringle R.S. Ndimba B.K. Simon W.J. Lindsey K. Slabas A.R. Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures.J. Exp. Bot. 2006; 57: 1553-1562Crossref PubMed Scopus (91) Google Scholar) was used for first strand cDNA synthesis using 2 μg RNA template, oligo-(dT)15 (Promega, Southampton, UK), and SuperScript III reverse transcriptase (Invitrogen, Paisley, UK). For PCR reactions, the following primer pairs were used: CYCLASE1 (At4g34180) 5′-AACATCCAACACCGACAAGCGGC-3′ and 5′- AACATCCAACACCGACAAGCGGC-3′; ACTIN2 (At3g18780) 5′-GGATCGGTGGTTCCATTCTTG-3′; and 5′-AGAGTTGTCACACACAAGTG-3′. Pseudomonas syringae pv. tomato strain DC3000, and the derivative DC3000-hrcC and DC3000-avrRpm1 strains, were grown overnight at 28 °C on King's B agar supplemented with rifampicin. Colonies from agar plates were resuspended in water to an inoculum density of 106 colony forming units/ml. Three leaves per plant were syringe-infiltrated with the inoculum. Triplicate plants of each genotype were inoculated in this way and the bacterial titer in the leaf tissues assayed 3 days postinoculation for DC3000 and DC3000-avrRpm1, or 6 days post-inoculation for DC3000-hrcC. To determine bacterial titer, a pooled sample of 3 mm-diameter leaf discs, one from each of three replicate plants, was homogenized in sterile water and 10-fold dilutions of the homogenate plated out on agar plates with rifampicin. The number of colony forming units per square centimeter of infected leaf was calculated, converted to log scale, and the averages analyzed by Student's t test. Leaf discs of 8 mm diameter were cored from 4-week-old Arabidopsis plants and floated on 9 ml of 5 μm FB1 solution in a Petri-dish. A total of five-replicate Petri-dishes per genotype were generated, with each dish containing eight leaf discs originating from 10 different plants. The dishes were incubated for 48 h in the dark and transferred to a 16-hour photoperiod thereafter. Cell death progression was monitored by measuring conductivity of the FB1 solution in each dish every 24 h from 48 h onwards. To monitor pathogen-induced cell death, leaves were infiltrated with ∼107 colony forming units/ml Pseudomonas syringae pv. tomato strain DC3000-avrRpm1. Discs of 8 mm diameter were immediately cored from the inoculated leaves and floated on 9 ml deionized water in a Petri-dish. Five-replicate dishes per genotype were generated, with each dish containing 10 discs from 10 different plants. Conductivity of the deionized water was measured every hour until 4 h and every 2 h from then until 12 h. Cell cultures were treated with 200 μm SA or a combination of 200 μm SA + 200 μm ATP. Controls were treated with an equivalent volume of sterile water. After 48 h, the cells were separated from the growth medium by filtration using a Mira cloth. Proteins secreted into the growth medium were recovered by precipitation in 80% acetone at −20 °C. The precipitates were resolubilized in a solution containing 9 m urea/2 M thiourea/4% (w/v) CHAPS. Each of the treatments and the control had three biological replicates, giving rise to a total of nine samples. Differential protein expression was analyzed using isobaric tags for relative and absolute quantitation (iTRAQ), and 2D-DiGE was used as a tool to confirm quantitative data from iTRAQ on a few selected proteins. Protein samples were acetone-precipitated and resuspended in 50 mm triethylammonium bicarbonate buffer containing 0.1% SDS. Prior to digestion, 75 μg of each protein sample were sequentially reduced and alkylated with tris(2-carboxyethylphosphine) (TCEP) and methyl-methane-thiol-sulfonate (MMTS), respectively. Protein digestion was at a 1:10 trypsin ration. The digested peptides were vacuum-dried, resuspended in triethylammonium bicarbonate buffer (pH 8.5), and labeled with 4-plex iTRAQ reagent kits (Applied Biosystems, Foster City, USA) for 1 h at room temperature as previously described (27Rowland J.G. Simon W.J. Nishiyama Y. Slabas A.R. Differential proteomic analysis using iTRAQ reveals changes in thylakoids associated with Photosystem II-acquired thermotolerance in Synechocystis sp. PCC 6803.Proteomics. 2010; 10: 1917-1929Crossref PubMed Scopus (28) Google Scholar). Control, SA, and ATP+SA samples were labeled with the 114, 115, and 116 iTRAQ tags, respectively. The three samples of each individual replicate experiment were pooled, vacuum-dried, and processed separately from the other replicates. The pooled sample was resuspended in 3 ml of buffer A (10 mm K2HPO4/25% acetonitrile, pH 2.8) and separated on the Poly-LC strong cation exchange column (200 × 2.1 mm) at 200 μl/min on an Ettan LC (GE Healthcare) HPLC system. Peptide separation was performed using a biphasic gradient of: 0–150 mm KCl over 11.25 column volumes and 150–500 mm KCl in buffer A over 3.25 column volumes. A total of 52 × 200 μl fractions were collected over the gradient, but some were pooled to give a final total of 30 fractions that were dried down and resuspended in 90 μl of 2% acetonitrile/0.1% formic acid. Aliquots of 20 μl from each fraction were analyzed by LC-MS/MS using a nano-flow Ettan MDLC system (GE Healthcare) attached to a hybrid quadrapole-TOF mass spectrometer (QStar Pulsar i, Applied Biosystems, Foster City) coupled to a nanospray source (Protana) and a PicoTip silica emitter (New Objective, Woburn, MA). Samples were loaded and washed on a Zorbax 300SB-C18, 5 mm, 5 × 0.3 mm trap column (Agilent, Stockport, UK) and online chromatographic separation performed over 2 h on a Zorbax 300SB-C18 capillary column (3.5 × 75 μm) with a linear gradient of 0–40% acetonitrile, 0.1% formic acid at a flow rate of 200 nl/minute. Applied Biosystems Analyst software version 1.1 was used acquire all MS and MS/MS data switching between the survey scan (1 × 1 s MS) and three product ion scans (3 × 3 s MS/MS) every 10 s. Ions in the range of 2+ to 4+ charge state and with TIC > 10 counts selected for fragmentation. Protein Pilot software version 2.0.1 (Applied Biosystems) was used to process all MS/MS data files using the Arabidopsis TIGR.fas database containing 27,855 protein sequences (downloaded in August 2007). MS and MS/MS tolerances were set to 0.15 and 0.1Da, respectively, and analysis and search parameters were set as: iTRAQ 4-plex labeling, trypsin digestion with allowance for a single missed cleavage, and only two amino acid modifications viz. MMTS-alkylated cysteine and oxidized methionine. Quantitative data were obtained from the iTRAQ tags within the mass spectra. In order to reduce protein redundancy and determine protein identification confidence scores (ProtScores) from the ProID output for each fraction, the data from all fractions were combined, analyzed, and reported using ProGroup software (Applied Biosystems, Foster City, USA). A protein identification threshold of 1.3, which retains only proteins identified with a 95% confidence, was applied to the data sets. Systematic errors arising from possible unequal mixing of labeled peptides were excluded by applying a bias correction factor. Its calculation is based on the assumption that most proteins do not change, so the software identifies the median average protein ratio and corrects it to unity, and then applies this factor to all quantitation results. In order to measure the false discovery rate the peptide mass spectra data sets were used to search a decoy peptide database, created by reversing peptide sequences of all entries in the database used in this study. Aggregate false discovery rate (FDR) was calculated as: FDR (%) = 100 × (2 × Decoy IDs)/Total IDs. Decoy IDs is the number of “proteins” identified from the decoy database that pass the thresholds set for identifying proteins in the real database. Total IDs is the number of proteins identified from the real database using the same peptide data set. The data sets were manually filtered sequentially to exclude peptides leading to ≥2 protein identifications. From the filtered data sets, only those proteins identified across all three replicate experiments were retained for further analysis. A further filter applied to the data was to include only proteins whose response to SA treatment had a significant probability value (p ≤0.05) across all three replicates. This ensured that any changes arising from random errors within any individual replicate experiment were excluded. Finally, a comparison between a protein's response to SA and to ATP+SA was made by performing a Student's t test on the averages of SA/Control and “ATP+SA”/Control ratios. All proteins with a significant probability value (p ≤0.05) had their response to SA attenuated by inclusion of ATP. These constitute the final protein list of this study. Peptide sequences of all the proteins were analyzed using the SignalP 4.0 tool (28Petersen T.N. Brunak S. von Heijne G. Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions.Nat. Methods. 2011; 8: 785-786Crossref PubMed Scopus (7094) Google Scholar), which identifies the presence of an N-terminal signal peptide targeting the protein to the secretory pathway. AgriGo version 1.2 (29Du Z. Zhou X. Ling Y. Zhang Z. Su Z. agriGO: a GO analysis toolkit for the agricultural community.Nucleic Acids Res. 2010; 38: W64-W70Crossref PubMed Scopus (1951) Google Scholar) was used for Gene Ontology and enrichment analysis. A total of 33 SA- and ATP-responsive proteins were submitted for enrichment analysis against an Arabidopsis reference database (TAIR9) with 37,767. An FDR-adjusted p value <0.05 was used as a cut-off threshold for a significant enrichment. Analysis of protein samples using 2D-DiGE was performed as previously described (7Chivasa S. Tome D.F.A. Hamilton J.M. Slabas A.R. Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase β-subunit as a novel plant cell death regulator.Mol. Cell. Proteomics. 2011; 10M110.003905Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), with minor modifications. Four replicates of Control, SA, and ATP+SA protein samples were labeled with a two-dye system, in which each sample was labeled with Cye-5 and the pooled standard labeled with Cye-3. Using previous information regarding protein spot identity of Arabidopsis secreted proteins, CYCLASE1 protein spots were targeted for quantitative analysis. These same protein spots were excised from preparative gels and identified by tandem-MS as previously described (7Chivasa S. Tome D.F.A. Hamilton J.M. Slabas A.R. Proteomic analysis of extracellular ATP-regulated proteins identifies ATP synthase β-subunit as a novel plant cell death regulator.Mol. Cell. Proteomics. 2011; 10M110.003905Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We reasoned that an agonist and antagonist of FB1-induced cell death could be used to design an experimental system to identify plant pcd regulatory protein" @default.
• W2255979074 created "2016-06-24" @default.
• W2255979074 creator A5006320084 @default.
• W2255979074 creator A5015541284 @default.
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• W2255979074 creator A5025300568 @default.
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• W2255979074 date "2015-06-01" @default.
• W2255979074 modified "2023-09-30" @default.
• W2255979074 title "A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins*" @default.
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