The work deals with the testing and evaluation of the usability of Portal of the Public Administration. The work describes the main methods of usability testing and details specify the heuristic evaluation of the usability of Portal of the Public Administration. Using the proposed procedure is performed heuristic evaluation of the information system and in according to the results is described errors in usability of Portal and their repair.
Abstract Background Many flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion. Results Progression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle. Conclusions The current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.
Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. Tobacco mature pollen represents an extremely resistant structure filled with a desiccated cytoplasm that is surrounded by an extremely tough cell wall. This metabolically quiescent stage of male gametophyte has to reach stigma tissue in a viable state. After pollination, the rehydration and metabolic activation of a pollen grain starts. The pollen activation is represented by a time period when there is no pollen tube growth, and only metabolic processes within the original volume of cytoplasm occur together with cytoplasm hydration (1.Vogler F. Konrad S.S.A. Sprunck S. Knockin' on pollen's door: live cell imaging of early polarization events in germinating Arabidopsis pollen.Front. Plant Sci. 2015; 6: 246Crossref PubMed Scopus (18) Google Scholar). Within this period, the pollen aperture later used for pollen tube outgrowth is selected. After that, a rapid pollen tube tip growth starts in order to deliver the genetic information carried by two sperm cells to the ovaries. Desiccated mature pollen of many angiosperm species can be also rehydrated and activated in vitro (2.Mascarenhas J.P. Molecular mechanisms of pollen tube growth and differentiation.Plant Cell. 1993; 5: 1303-1314Crossref PubMed Scopus (363) Google Scholar). Here we aim to elucidate the regulation processes of pollen grain re-hydration and activation mediated by protein phosphorylation. Protein phosphorylation, representing one of the most frequent regulatory mechanisms, was shown to control a number of cellular processes, such as signal transduction, regulation of transcription and translation, regulation of cytoskeleton dynamics, cell cycle regulation, metabolism regulation, regulation of protein stability, and protein targeting (3.Mishra N.S. Tuteja R. Tuteja N. Signaling through MAP kinase networks in plants.Arch. Biochem. Biophys. 2006; 452: 55-68Crossref PubMed Scopus (264) Google Scholar, 4.Francis D. Halford N.G. The plant cell cycle.Physiol. Plant. 1995; 93: 365-374Crossref Scopus (36) Google Scholar, 5.van der Kelen K. Beyaert R. Inze D. de Veylder L. Translational control of eukaryotic gene expression.Crit. Rev. Biochem. Mol. Biol. 2009; 44: 143-168Crossref PubMed Scopus (100) Google Scholar). Similar to pollen activation, the rehydration of African xerophyte Craterostigma plantagineum was accompanied by changes in protein phosphorylation (6.Röhrig H. Colby T. Schmidt J. Harzen A. Facchinelli F. Bartels D. Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure.Proteomics. 2008; 8: 3548-3560Crossref PubMed Scopus (34) Google Scholar). Attachment of a phosphate group to the polypeptide chain shifts the pI of a protein to more acidic range (7.Darewicz M. Dziuba J. Minkiewicz P. Some properties of beta-casein modified via phosphatase.Acta Alimentaria. 2005; 34: 403-415Crossref Scopus (5) Google Scholar). Such pI shift usually causes changes of protein conformation within a single domain (8.Fletterick R.J. Sprang S.R. Glycogen-phosphorylase structures and function.Acc. Chem. Res. 1982; 15: 361-369Crossref Scopus (41) Google Scholar) or even influences domain-domain interactions (9.Kim J. Shen Y. Han Y.J. Park J.E. Kirchenbauer D. Soh M.S. Nagy F. Schafer E. Song P.S. Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction.Plant Cell. 2004; 16: 2629-2640Crossref PubMed Scopus (85) Google Scholar). In case of enzymes, phosphorylation sometimes inhibits activity by occupying the active site of the protein, as was documented for instance for isocitrate dehydrogenase (10.Garnak M. Reeves H.C. Phosphorylation of isocitrate dehydrogenase of Escherichia coli.Science. 1979; 203: 1111-1112Crossref PubMed Scopus (167) Google Scholar). In order to be able to identify phosphorylated proteins, it is inevitable to apply some of the various enrichment protocols (11.Fíla J. Honys D. Enrichment techniques employed in phosphoproteomics.Amino Acids. 2012; 43: 1025-1047Crossref PubMed Scopus (150) Google Scholar, 12.Dunn J.D. Reid G.E. Bruening M.L. Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry.Mass Spectrom. Rev. 2010; 29: 29-54PubMed Google Scholar) because of several reasons: (i) Phosphoproteins are mostly low abundant so they are overwhelmed by the excess of nonphosphorylated proteins. (ii) A given protein is expressed in many copies and contains many potential phosphorylation sites (Ser/Thr/Tyr residues), but individual phosphorylation sites are usually only partly phosphorylated— i.e. only a small share of the present protein molecules will be phosphorylated at a given position whereas the majority will be nonphosphorylated. (iii) The identification of phosphopeptides by mass spectrometry is still challenging from the technical point of view. The enrichment can be performed at two levels. The first possibility is to fish the intact phosphoproteins out of a protein mixture whereas the second approach relies on the enrichment of phosphorylated peptides of the protease-digested protein sample. A plethora of protocols are meanwhile available for both approaches, whereas for both advantages as well as disadvantages have been reported (11.Fíla J. Honys D. Enrichment techniques employed in phosphoproteomics.Amino Acids. 2012; 43: 1025-1047Crossref PubMed Scopus (150) Google Scholar). In order to broaden the phosphoproteome coverage, a tandem procedure applying first the former approach and then after protease cleavage also the latter one was suggested (13.Hoehenwarter W. Thomas M. Nukarinen E. Egelhofer V. Röhrig H. Weckwerth W. Conrath U. Beckers G.J.M. Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography.Mol. Cell. Proteomics. 2013; 12: 369-380Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14.Beckers G.J. Hoehenwarter W. Röhrig H. Conrath U. Weckwerth W. Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.Methods Mol. Biol. 2014; 1072: 621-632Crossref PubMed Scopus (11) Google Scholar). The first angiosperm pollen phosphoproteome published was that of Arabidopsis thaliana (15.Mayank P. Grossman J. Wuest S. Boisson-Dernier A. Roschitzki B. Nanni P. Nuehse T. Grossniklaus U. Characterization of the phosphoproteome of mature Arabidopsis pollen.Plant J. 2012; 72: 89-101Crossref PubMed Scopus (68) Google Scholar), which completed the pollen proteomic data because before that, three Arabidopsis pollen proteomic data sets achieved by the conventional in-gel approach (16.Holmes-Davis R. Tanaka C.K. Vensel W.H. Hurkman W.J. McCormick S. Proteome mapping of mature pollen of Arabidopsis thaliana.Proteomics. 2005; 5: 4864-4884Crossref PubMed Scopus (211) Google Scholar, 17.Noir S. Bräutigam A. Colby T. Schmidt J. Panstruga R. A reference map of the Arabidopsis thaliana mature pollen proteome.Biochem. Biophys. Res. Commun. 2005; 337: 1257-1266Crossref PubMed Scopus (125) Google Scholar, 18.Sheoran I.S. Sproule K.A. Olson D.J.H. Ross A.R.S. Sawhney V.K. Proteome profile and functional classification of proteins in Arabidopsis thaliana (Landsberg erecta) mature pollen.Sex. Plant Reprod. 2006; 19: 185-196Crossref Scopus (71) Google Scholar) and one high-throughput proteomic study (19.Grobei M.A. Qeli E. Brunner E. Rehrauer H. Zhang R. Roschitzki B. Basler K. Ahrens C.H. Grossniklaus U. Deterministic protein inference for shotgun proteomics data provides new insights into Arabidopsis pollen development and function.Genome Res. 2009; 19: 1786-1800Crossref PubMed Scopus (149) Google Scholar) were published. Mayank and colleagues identified many phosphopeptides, notable number of which played their roles in regulation of metabolism and protein function, metabolism, protein fate, binding other proteins, signal transduction, and cellular transport. Many kinases were identified in the data set, showing that these were indeed subject to phosphorylation, for instance AGC protein kinases, calcium-dependent protein kinases, and sucrose non-fermenting 1-related protein kinases (15.Mayank P. Grossman J. Wuest S. Boisson-Dernier A. Roschitzki B. Nanni P. Nuehse T. Grossniklaus U. Characterization of the phosphoproteome of mature Arabidopsis pollen.Plant J. 2012; 72: 89-101Crossref PubMed Scopus (68) Google Scholar). The tobacco pollen proteome was studied directly by a high-throughput approach but appeared only recently (20.Ischebeck T. Valledor L. Lyon D. Gingl S. Nagler M. Meijon M. Egelhofer V. Weckwerth W. Comprehensive cell-specific protein analysis in early and late pollen development from diploid microsporocytes to pollen tube growth.Mol. Cell. Proteomics. 2014; 13: 295-310Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this study, Ischebeck and colleagues compared the proteome of eight male gametophyte stages ranging from diploid microsporocytes to pollen tubes. Interestingly, the first tobacco pollen phosphoproteomic paper appeared earlier than the whole proteome was published (21.Fíla J. Matros A. Radau S. Zahedi R.P. Čapková V. Mock H.-P. Honys D. Revealing phosphoproteins playing role in tobacco pollen activated in vitro.Proteomics. 2012; 12: 3229-3250Crossref PubMed Scopus (24) Google Scholar). In order to identify phosphoproteins in tobacco mature pollen and pollen activated in vitro for 30 min, metal oxide/hydroxide affinity chromatography phosphoprotein enrichment employing an aluminum hydroxide matrix (Al(OH)3) was carried out (22.Wolschin F. Wienkoop S. Weckwerth W. Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC).Proteomics. 2005; 5: 4389-4397Crossref PubMed Scopus (274) Google Scholar). This approach led to the identification of only one phosphorylation site, so that additionally titanium dioxide (TiO2) 1The abbreviations used are:page: TiO2titanium dioxideAl(OH)3aluminium hydroxidebZIPbasic leucine zipperCAMK2Ca2+/calmodulin-dependent protein kinaseCDKcyclin-dependent protein kinaseCDPK—SnRKCa2+-dependent protein kinase–sucrose-nonfermenting-related kinaseCK2casein kinase 2CyscysteineDCN1defective in cullin neddylation protein 1DHB2,5–dihydroxybenzoic acidEPPEDTA/puromycine-resistant particleGOgene ontologyIMACimmobilized metal affinity chromatographyLEAlate embryogenesis abundantMAPKmitogen-activated protein kinaseMetmethionineMS/MStandem mass spectrometrynLCnano liquid chromatographyPB1octicosapeptide/PHOX/BEM1pPPI1peptidyl-prolyl cis-trans isomerase 1Rho GAPRho GTPase activation proteinRho GDI2Rho guanine nucleotide dissociation inhibitor 2RNF4RING FINGER PROTEIN 4SerserineSIMACsequential elution from IMACSMM-MESsucrose-mineral medium buffered with MESSNC1SUPRESSOR OF NPR1–1, CONSTITUTIVE 1ThrthreonineTyrtyrosineUBAubiquitin-associatedUBXubiquitin-likeUNC-89UNCOORDINATED-89WVD2WAVE-DAMPENED 2. enrichment was applied, identifying 51 more phosphorylation sites in the already-identified proteins from mature pollen. Among those proteins were for instance various translation initiation and elongation factors, metabolic proteins (for instance fructose bisphosphate-aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase), Rho guanine nucleotide dissociation inhibitor, and several ribosomal proteins. However, not many signaling proteins were identified in this study. The third male gametophyte phosphoproteome revealed to date was that of a gymnosperm Picea wilsonii. However, the proteome of this species was studied from the perspective of deficient growth media, and several phosphoproteins linked to Ca2+ and sucrose deficiency were identified (23.Chen Y. Liu P. Hoehenwarter W. Lin J. Proteomic and phosphoproteomic analysis of Picea wilsonii pollen development under nutrient limitation.J. Proteome Res. 2012; 11: 4180-4190Crossref PubMed Scopus (19) Google Scholar). titanium dioxide aluminium hydroxide basic leucine zipper Ca2+/calmodulin-dependent protein kinase cyclin-dependent protein kinase Ca2+-dependent protein kinase–sucrose-nonfermenting-related kinase casein kinase 2 cysteine defective in cullin neddylation protein 1 2,5–dihydroxybenzoic acid EDTA/puromycine-resistant particle gene ontology immobilized metal affinity chromatography late embryogenesis abundant mitogen-activated protein kinase methionine tandem mass spectrometry nano liquid chromatography octicosapeptide/PHOX/BEM1p peptidyl-prolyl cis-trans isomerase 1 Rho GTPase activation protein Rho guanine nucleotide dissociation inhibitor 2 RING FINGER PROTEIN 4 serine sequential elution from IMAC sucrose-mineral medium buffered with MES SUPRESSOR OF NPR1–1, CONSTITUTIVE 1 threonine tyrosine ubiquitin-associated ubiquitin-like UNCOORDINATED-89 WAVE-DAMPENED 2. The present study is a continuation of our male gametophyte phosphoproteomic studies. Herein, we employed phosphopeptide enrichment by metal oxide/hydroxide affinity chromatography with TiO2 matrix (24.Pinkse M.W.H. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J.R. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (825) Google Scholar) on three stages of male gametophyte, this time including two stages of activated pollen (5 min and 30 min) as well as mature pollen. Collectively, 471 phosphopeptides carrying 432 phosphorylation sites (phosphoRS probabilities >90%) have been identified in the three stages of male gametophyte. These phosphorylation sites belonged to 301 phosphoproteins that were classified into 13 functional categories; with transcription, protein synthesis, destination and storage, as well as signal transduction being the dominant functional groups. A phosphorylation motif search revealed 5 motifs with a central phosphoserine and one motif with a central phosphothreonine. Quantitative data led to the discovery of regulated phosphopeptides, which were grouped into seven categories based on their regulatory trends throughout the studied developmental stages. Tobacco plants (Nicotiana tabacum cv. Samsun) were grown in a greenhouse from April to September. Flower buds shortly before anthesis were collected between June and September. Anthers were removed from the buds and let dehisce at room temperature on a filtration paper overnight. Then, mature pollen was sieved by a stocking and stored at −20 °C (25.Petrů E. Hrabětová E. Tupý J. The technique of obtaining germinating pollen without microbial contamination.Biologia Plantarum. 1964; 6: 68-69Crossref Scopus (15) Google Scholar) until it was further used. The collected pollen represented bulk samples originating from three groups of 15 plants that were grown in separate parts of the greenhouse. These bulk samples were further referred to as the three biological replicates. Mature pollen was activated in vitro as a shaken suspension for 5 min, and 30 min, respectively, each stage in three biological replicates as mentioned above, at 27 °C in sucrose-mineral medium buffered with MES (SMM-MES; 175 mm sucrose, 1.6 mm boric acid, 3 mm Ca(NO3)2·4H2O, 0.8 mm MgSO4·H2O, 1 mm KNO3, 25 mm MES, pH 5.9) (26.Tupý J. Říhová L. Changes and growth effect of pH in pollen tube culture.J. Plant Physiol. 1984; 115: 1-10Crossref PubMed Scopus (43) Google Scholar). The activated pollen was then harvested by filtration on a vacuum pump-driven apparatus, and immediately frozen in liquid nitrogen. The three stages differ from each other as follows: mature pollen represents an oval-shaped structure with a desiccated cytoplasm. Upon re-hydration, 5-min activated pollen becomes round-shaped with a hydrated cytoplasm. Furthermore, one pollen aperture is usually chosen for pollen tube outgrowth after 30-min imbibition (see supplemental Fig. S1). The total proteins were extracted from all the above stages by TCA/acetone precipitation (27.Méchin V. Damerval C. Zivy M. Total protein extraction with TCA-acetone.in: Thiellement H. Zivy M. Damerval C. Méchin V. Methods in Mol. Biol. Springer, 2006: 1-8Crossref Google Scholar) with slight modifications (21.Fíla J. Matros A. Radau S. Zahedi R.P. Čapková V. Mock H.-P. Honys D. Revealing phosphoproteins playing role in tobacco pollen activated in vitro.Proteomics. 2012; 12: 3229-3250Crossref PubMed Scopus (24) Google Scholar) in three biological replicates as mentioned above (see Fig. 1 for workflow overview). In detail, mature or activated pollen was homogenized by a pestle in a mortar. The acquired fine powder was resuspended in 10 volumes of 10% w/v TCA in acetone supplemented with 1% w/v DTT. After 5 min sonication in an ultrasonic bath, the samples were briefly frozen in liquid nitrogen, incubated at −20 °C for 45 min, and centrifuged (23,000 × g, 15 min, 4 °C). After the removal of the supernatant, the samples were washed by 1.5 ml acetone with 1% w/v DTT, sonicated for 5 min, briefly frozen in liquid nitrogen, and kept at −20 °C for 30 min. After the centrifugation under the above conditions, the washing step was repeated. Finally, the pellet was dried and stored at −20 °C. The total protein extracts from all stages were resuspended in 0.2 m guanidinium chloride and 50 mm ammonium bicarbonate supplemented with PhosStop phosphatase inhibitor mixture (Roche, Penzberg, Germany), carbamidomethylated as described elsewhere (28.Kollipara L. Zahedi R.P. Protein carbamylation: In vivo modification or in vitro artefact?.Proteomics. 2013; 13: 941-944Crossref PubMed Scopus (98) Google Scholar) and subsequently trypsin-digested (trypsin-to-protein ratio 1:50; 37 °C, 12 h). There were three biological replicates for each studied stage. For each of the triplicates, 500 μg peptides were dissolved in loading buffer (80% v/v ACN, 6% v/v TFA, saturated with phthalic acid) and subjected to phosphopeptide enrichment by TiO2. Seven synthetic peptides were spiked to the peptide mixture in order to check the reproducibility of the replicates. The phosphopeptides bound to TiO2 beads were washed and eluted as described previously (21.Fíla J. Matros A. Radau S. Zahedi R.P. Čapková V. Mock H.-P. Honys D. Revealing phosphoproteins playing role in tobacco pollen activated in vitro.Proteomics. 2012; 12: 3229-3250Crossref PubMed Scopus (24) Google Scholar, 29.Beck F. Lewandrowski U. Wiltfang M. Feldmann I. Geiger J. Sickmann A. Zahedi R.P. The good, the bad, the ugly: Validating the mass spectrometric analysis of modified peptides.Proteomics. 2011; 11: 1099-1109Crossref PubMed Scopus (25) Google Scholar). The phosphopeptide-enriched samples were analyzed by nLC-MS/MS on an LTQ Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer coupled to an Ultimate 3000 nLC (Thermo Fisher Scientific). Peptides were pre-concentrated on a self-packed Synergi HydroRP trapping column (100 μm ID,4 μm particle size, 10 nm pore size, 2 cm length) and separated on a self-packed Synergi HydroRP main column (75 μm ID, 2.5 μm particle size, 10 nm pore size, 30 cm length) at 60 °C and a flow rate of 270 nl·min−1 using a binary gradient (A: 0.1% formic acid, B: 0.1% formic acid, 84% ACN) ranging from 3% to 45% B in 240 min. MS survey scans were acquired from 350–2000 m/z in the Orbitrap at a resolution of 60,000 using the polysiloxane m/z 445.120030 as lock mass. The ten most intense ions were subjected to collision-induced dissociation and MS/MS using normalized collision energy of 35% and an activation time of 30 ms and MS/MS were acquired in the LTQ. AGC values were set to 106 for MS and 104 for MS/MS scans. The acquired spectra were searched against the TIGR EST sequence database for Tobacco (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/; release version 10/04/2011, 48961 entries) using Proteome Discoverer 1.3 with Mascot. Quantification, false discovery assessment and phosphorylation site localization were performed using the following nodes: Precursor Ions Area Detector, Peptide Validator, and phoshoRS (30.Taus T. Koecher T. Pichler P. Paschke C. Schmidt A. Henrich C. Mechtler K. Universal and confident phosphorylation site localization using phosphoRS.J. Proteome Res. 2011; 10: 5354-5362Crossref PubMed Scopus (568) Google Scholar). Searches were conducted with the following settings: 10 ppm MS tolerance, 0.5 Da MS/MS tolerance, trypsin as a cleaving enzyme with max. two missed cleavage sites, carbamidomethylation (Cys) as fixed, and oxidation (Met) together with phosphorylation (Ser, Thr, Tyr) as variable modifications. Finally, the results were subjected to the filtering criteria of mass deviation ≤ 4 ppm and high confidence (corresponding to a false discovery rate <1% on the peptide-spectrum match level). The standard deviation of the peak areas of the synthetic peptides was below 25% so the results were considered reproducible. Peak areas were considered per peptide, i.e. different charge states were combined. Of all identified phosphopeptides, only the ones that showed a standard deviation <30% of the abundance between the biological replicates of the same stage, and that were identified in all of the replicates were listed in the result tables. Moreover, only phosphopeptides with an unambiguously assigned phosphorylation site with a probability higher than 90% (phoshoRS) were considered. All raw data and search results have been deposited in proteomeXchange (31.Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nature Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2070) Google Scholar) with the accession PXD003042. For each sample ∼1 μg of the trypsin digest was analyzed by nLC-MS/MS prior to TiO2 enrichment, using the same conditions as above. Data analysis was also conducted as above, however, omitting phosphorylation as variable modification. Only proteins meeting the following criteria were quantified: (1) at least 2 unique peptides quantified in at least 2 out of 3 biological replicates, (2) for all conditions standard deviations between biological replicates had to be <40%. Proteins that differed among any of two studied stages at least twofold in abundance were considered as regulated. The gene ontology (GO) and enzyme codes were originally acquired by Blast2GO ver 2.7.2 (https://www.blast2go.com); the identified tobacco ESTs translated in the longest reading frame were searched against the Arabidopsis proteome. For many of the sequences, the GO terms (divided into three groups: molecular function, biological process, and cellular compartment) together with the EC enzyme codes were assigned according to the homologous Arabidopsis sequences. However, some of the tobacco sequences lacked their Arabidopsis homologue in the proteome database and/or the gene ontology was not informative enough. So finally, the acquired GO terms were manually converted to protein categories and subcategories according to Bevan et al. (32.Bevan M. Bancroft I. Bent E. Love K. Goodman H. Dean C. Bergkamp R. Dirkse W. Van Staveren M. Stiekema W. Drost L. Ridley P. Hudson S.A. Patel K. Murphy G. Piffanelli P. Wedler H. Wedler E. Wambutt R. Weitzenegger T. Pohl T.M. Terryn N. Gielen J. Villarroel R. De Clerck R. Van Montagu M. Lecharny A. Auborg S. Gy I. Kreis M. Lao N. Kavanagh T. Hempel S. Kotter P. Entian K.D. Rieger M. Schaeffer M. Funk B. Mueller-Auer S. Silvey M. James R. Montfort A. Pons A. Puigdomenech P. Douka A. Voukelatou E. Milioni D. Hatzopoulos P. Piravandi E. Obermaier B. Hilbert H. Dusterhoft A. Moores T. Jones J.D.G. Eneva T. Palme K. Benes V. Rechman S. Ansorge W. Cooke R. Berger C. Delseny M. Voet M. Volckaert G. Mewes H.W. Klosterman S. Schueller C. Chalwatzis N. Project E.U.A.G. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.Nature. 1998; 391: 485-488Crossref PubMed Scopus (744) Google Scholar) to enable better categorization of the data. In case a protein had more functions, it was catalogued according to the prevailing function. All unambiguous phosphopeptides (supplemental Table S2) were analyzed for the significant phosphorylation motifs by Motif-X software (33.Chou M.F. Schwartz D. Biological sequence motif discovery using motif-x.Curr. Protoc. Bioinformatics. 2011; (Chapter 13, Unit 13.15–24)Crossref Scopus (298) Google Scholar, 34.Schwartz D. Gygi S.P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets.Nature Biotechnol. 2005; 23: 1391-1398Crossref PubMed Scopus (716) Google Scholar). Two searches were performed, one looking up phosphorylated serine and the other one searching for phosphorylated threonine (phosphotyrosine motifs were not searched because there was only one phosphorylated tyrosine in the phosphopeptide data set). The width of a phosphorylation motif was set to 13 (where the phosphoamino acid was placed into the central position), number of occurrences to 15, and significance score to 0.000001. As a background, data set of tobacco Uniprot sequences was uploaded. The regulated phosphopeptides were manually divided into seven categories according to their regulatory trends. The motif search was not performed on the regulated phosphopeptide data set because it contained only a limited number of phosphopeptides. The graphical representation of the peptide abundances in the various stages was performed by the VANTED software package (http://www.vanted.org, ref. 35.Rohn H. Junker A. Hartmann A. Grafahrend-Belau E. Treutler H. Klapperstueck M. Czauderna T. Klukas C. Schreiber F. VANTED v2: a framework for systems biology applications.BMC Syst. Biol. 2012; 6Crossref Scopus (126) Google Scholar). In this study, 471 phosphopeptides were identified with an unambiguously assigned position of the phosphorylation site (supplemental Tables S1 and S2). The vast majority of the identified phosphopeptides was singly phosphorylated (437), whereas only a minority was doubly (32.
Developmental variations in the amount and SDS-PAGE pattern of soluble proteins and of those. bound to heavy cell structures were examined during transition of the dividing microspore into mature pollen in tobacco cultivars Samsun and White Burley. Both protein fractions exhibited an initial rapid rise associated with young pollen grain filling with cytoplasm and a slight decrease during pollen maturation after starch deposition. These changes were more marked in the soluble fraction and the total protein content at its maximum was more than three times higher than at the stage of microspore mitosis. Most of about 70 distinct polypeptides detected were found in all stages. Stage specific variations concerned a progressive increase and changes in the proportion of existing proteins and in appearance of some minor peaks. Small differences between the cultivars were observed at the time of differentiation and in proportions of some polypeptides.
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