Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth
2013; Elsevier BV; Volume: 13; Issue: 1 Linguagem: Inglês
10.1074/mcp.m113.028100
ISSN1535-9484
AutoresTill Ischebeck, Luís Valledor, David Lyon, Stephanie Gingl, Matthias Nagler, Mónica Meijón, Volker Egelhofer, Wolfram Weckwerth,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoPollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development. Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development. Plants contain numerous specialized cell types, each of them expressing a specific set of proteins. In recent studies much effort has been put into isolating and analyzing proteins of these individual cell types (1Dai S. Chen S. Single-cell-type proteomics: toward a holistic understanding of plant function.Mol. Cell. Proteomics. 2012; 11: 1622-1630Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) rather than whole organs. New emerging methods have led to the in-depth analysis of different plant cell types, including guard cells (2Zhao Z. Zhang W. Stanley B.A. Assmann S.M. 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Skirpan A.L. Kao T.H. Gilroy S. Petunia phospholipase c1 is involved in pollen tube growth.Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (182) Google Scholar), this is not the case for earlier stages of pollen development. In angiosperms, mature pollen develops from microsporocytes in the anthers of the flower in a series of distinct stages (37Berger F. Twell D. Germline specification and function in plants.Annu. Rev. Plant Biol. 2011; 62: 461-484Crossref PubMed Scopus (141) Google Scholar). After the microsporocytes have completed meiosis, they form tetrads that release microspores with one central haploid nucleus. These microspores undergo polarization and asymmetric mitosis. The bigger vegetative cell internalizes the smaller cell, which later divides again and forms the two sperm cells. Finally, the pollen desiccates. When the pollen falls on the stigma, it rehydrates, and the vegetative cell forms a pollen tube that delivers the two sperm cells through the transmitting tract to the ovule (38Boavida L.C. Becker J.D. Feijo J.A. The making of gametes in higher plants.Int. J. Dev. Biol. 2005; 49: 595-614Crossref PubMed Scopus (84) Google Scholar). Even though pollen development studies using electron microscopy date back to the 1960s (39Echlin P. Godwin H. The ultrastructure and ontogeny of pollen in Helleborus foetidus L. II. Pollen grain development through the callose special wall stage.J. Cell Sci. 1968; 3: 175-186PubMed Google Scholar) and many mutants are described that are disrupted in this process (14Grobei M.A. Qeli E. Brunner E. Rehrauer H. Zhang R. Roschitzki B. Basler K. Ahrens C.H. Grossniklaus U. 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Cell-specific analysis of the tomato pollen proteome from pollen mother cell to mature pollen provides evidence for developmental priming.J. Proteome Res. 2013; 12: 4892-4903Crossref PubMed Scopus (67) Google Scholar). The transcriptome of Arabidopsis pollen has been analyzed from the microspore stage on (43Honys D. Twell D. Transcriptome analysis of haploid male gametophyte development in Arabidopsis.Genome Biol. 2004; 5: R85Crossref PubMed Google Scholar), but the earlier stages of microsporocytes, meiosis, and tetrads were not studied, most likely because of a limitation of available material. However, this study was able to show dramatic changes in the transcriptome during the development from microspores to the mature pollen. Similar studies have been performed with Brassica napus (44Whittle C.A. Malik M.R. Li R. Krochko J.E. Comparative transcript analyses of the ovule, microspore, and mature pollen in Brassica napus.Plant Mol. Biol. 2010; 72: 279-299Crossref PubMed Scopus (27) Google Scholar) and rice pollen (45Wei L.Q. Xu W.Y. Deng Z.Y. Su Z. Xue Y. Wang T. Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa.BMC Genomics. 2010; 11: 338Crossref PubMed Scopus (121) Google Scholar). A comparative analysis of the proteome from these stages, as presented in our study, can have special relevance, because in pollen the proteome can greatly differ from the transcriptome not only quantitatively, but also qualitatively, as has been shown for Arabidopsis pollen (14Grobei 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). It seems that often the mRNA is degraded while the protein persists or mRNA is stored in desiccated pollen to be transcribed after rehydration (14Grobei 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). Additionally, in our proteomic study, we extended the analysis to even earlier stages, including the stage of meiosis. We were able to compare, for the first time, the proteome of a total of eight stages: the diploid microsporocytes, cells undergoing meiosis, tetrads, microspores, polarized microspores (undergoing mitosis I), bipolar pollen, desiccated pollen, and finally pollen tubes. We found that the proteome underwent great changes during development, especially during the polarized microspore stage. Tobacco was grown under greenhouse conditions (12 h of light, 120 μmol m−2 s−1, 23 °C during the day, 20 °C at night, 60% humidity). Flowers of different sizes were collected, and the anthers of individual flowers were sampled in 200 μl of 10% mannitol. Anthers were gently squeezed open and vortexed, and the supernatant including the released pollen was transferred to a new tube. Pollen was spun down at 100 × g for 1 min and washed twice with 10% mannitol. A subfraction of the pollen of each individual flower was analyzed under a microscope to determine the developmental stage. Samples not representing a stage with at least 90% of their pollen were discarded. Pollen tubes were grown for 5 h in pollen tube medium (10% sucrose, 15 mm MES-KOH pH 5.9, 1 mm CaCl2, 1 mm KCl, 0.8 mm MgSO4, 1.6 mm H3BO3, 30 μm CuSO4) slightly modified according to Read et al. (46Read S.M. Clarke A.E. Bacic A. Stimulation of growth of cultured Nicotiana-Tabacum W-38 pollen tubes by poly(ethylene glycol) and Cu-(Ii) salts.Protoplasma. 1993; 177: 1-14Crossref Scopus (100) Google Scholar). Young leaves and roots were ground in liquid nitrogen, and proteins were extracted accordingly. Pollen samples were fixed in 10% mannitol and 4% formaldehyde overnight, collected via centrifugation, and resuspended in 1 μg/ml DAPI and 1% triton X-100 5 min prior to microscopy. Images were recorded with an upright point laser scanning confocal microscope (LSM780, Zeiss, Oberkochen, Germany) using a 405-nm diode laser for excitation and a band-pass filter ranging from 450–550 nm. Acquired images were processed using Fiji software. For each sample, pollen from between 5 and 30 flowers (depending on the stage) was pooled, freeze-dried, cooled in liquid nitrogen, and ground for 3 min in a shaking mill using three 2-mm steel balls per tube. The pollen fragments were resuspended in 200 μl of protein extraction buffer (62.5 mm Tris-HCl pH 6.5, 5% SDS (w/v), 10% glycerol (v/v), 10 mm DTT, 1.2% (v/v) plant protease inhibitor mixture (Sigma P9599)) and incubated for 5 min at room temperature. After this time, the samples were mixed again by pipetting, incubated for 3 min at 90 °C, and then centrifuged at 21,000 × g for 5 min at room temperature. Supernatants were carefully transferred to a new tube. After the addition of an equal volume of 1.4 m sucrose, proteins were extracted twice with Tris-EDTA buffer–equilibrated phenol. The combined phenolic phases were counter-extracted with 0.7 m sucrose and subsequently mixed with five volumes of 0.1 m ammonium-acetate in methanol to precipitate the proteins. After 16 h of incubation at −20 °C, samples were centrifuged for 5 min at 5000 × g at 5 °C. The pellet was washed twice with 0.1 m ammonium-acetate and once with acetone and then air-dried. Pellets were redissolved in 6 m urea, 5% SDS, and protein concentrations were estimated via bicinchoninic acid assay (47Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Measurement of protein using bicinchoninic acid.Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18586) Google Scholar). Proteins were analyzed via a new gel-LC-MS protocol (48Valledor L. Weckwerth W. An improved detergent-compatible gel-fractionation LC-LTQ-Orbitrap workflow for plant and microbial proteomics.Methods Mol. Biol. 2013; 1072: 347-358Crossref Scopus (42) Google Scholar). 40 μg of protein were loaded into a mini-protean cell and run for 1.5 cm. Gels were fixed and stained with methanol:acetic acid:water:Coomassie Brilliant Blue R-250 (40:10:50:0.001). Gels were destained in methanol:water (40:60), and then each lane was divided into two fractions. Gel pieces were destained, equilibrated, and digested with trypsin as previously described (49Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes.Nat. Protoc. 2007; 1: 2856-2860Crossref Scopus (3531) Google Scholar). Peptides were then desalted with the use of Bond-Elute C-18 stage tips (50Ishihama Y. Rappsilber J. Mann M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics.J. Proteome Res. 2006; 5: 988-994Crossref PubMed Scopus (224) Google Scholar) and concentrated in a SpeedVac. Prior to mass spectrometric measurement, protein digest pellets were dissolved in 4% (v/v) acetonitrile, 0.1% (v/v) formic acid. 10 μg of digested peptides were loaded per injection into a one-dimensional nano-flow LC-MS/MS system equipped with a pre-column (Eksigent, Redwood City, CA, USA). Peptides were eluted using a monolithic C18 column Chromolith RP-18r (Merck, Darmstadt, Germany) of 15-cm length and 0.1-mm internal diameter during an 80-min gradient from 5% to 50% (v/v) acetonitrile/0.1% (v/v) formic acid with a controlled flow rate of 500 nl/min. MS analysis was performed on an Orbitrap LTQ XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Specific tune settings for the MS were as follows: the spray voltage was set to 1.8 kV using a needle with a 30-μm inner diameter (PicoTip Emitter, New Objective, Woburn, MA), and the temperature of the heated transfer capillary was set at 180 °C. Fourier transform MS was operated as follows: full scan mode, centroid, resolution of 30,000, covering the range of 300–1800 m/z, and cyclomethicone used as a lock mass. Each full MS scan was followed by 10 dependent MS/MS scans performed in the ion trap, in which the 10 most abundant peptide molecular ions were dynamically selected with a dynamic exclusion window set to 90 s and an exclusion list set to 500. Dependent fragmentations were performed in collision-induced dissociation mode with a normalized collision energy of 35, an isolation width of 2.0, an activation Q of 0.250, and an activation time of 30 ms. Ions with an unassigned charge or a charge of +1 were excluded for fragmentation. The minimum signal threshold was set at 1000. Raw data were searched with the SEQUEST algorithm present in Proteome Discoverer version 1.3 (Thermo, Germany) as described elsewhere (51Valledor L. Recuenco-Munoz L. Egelhofer V. Wienkoop S. Weckwerth W. The different proteomes of Chlamydomonas reinhardtii.J. Proteomics. 2012; 75: 5883-5887Crossref PubMed Scopus (14) Google Scholar). In brief, identification confidence was set at a 5% false discovery rate, and the variable modifications were set as acetylation of the N terminus, oxidation of methionine, and carbamidomethyl cysteine formation, with mass tolerances of 10 ppm for the parent ion and 0.8 Da for the fragment ion. Up to two missed cleavage sites were permitted. Three different databases were employed (tobacco 7.0, a CDNA library from the gene index project with 120,122 entries; a tobacco protein database from UniProt 09.2011 with 4826 entries; and a genomic sequence database from the Tobacco Genome Initiative 11.2008 with 349,877 entries, resulting in 2,099,262 entries after six-frame translation). Databases were translated with an in-house tool, taking into consideration only the longest open reading frame of all reading frames. In the case of the genomic sequences, the longest open reading frames of all reading frames were considered. When the database from the gene index project was used, additional variable modifications were allowed: phosphorylation of threonine, serine, and tyrosine; methylation and dimethylation of lysine and arginine; and acetylation and trimethylation of lysine. Peptides were matched against these databases plus decoys, with a significant hit considered as one in which the peptide confidence was at least medium or high, and the xcorr score threshold was established at 2.5 for +2 ions and 3.5 for charge states of +3 or greater. The high thresholds were chosen to minimize false identifications based on the incomplete databases used. The identified proteins were quantitated via a label-free approach based on peptide count followed by a normalized spectral abundance factor (NSAF) 1The abbreviations used are:NSAFnormalized spectral abundance factorPCAprincipal components analysis. normalization strategy (52Paoletti A.C. Parmely T.J. Tomomori-Sato C. Sato S. Zhu D. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 18928-18933Crossref PubMed Scopus (408) Google Scholar), (NSAF)k=(PSM/L)k/∑i=1N(PSM/L)i in which the total number of spectra counts for the matching peptides from protein k (PSM) was divided by the protein length (L) and then divided by the sum of PSM/L for all N proteins. normalized spectral abundance factor principal components analysis. Multivariate statistical analyses such as principal components analysis (PCA) and k-means clustering were performed with the statistical toolbox COVAIN (53Sun X. Weckwerth W. COVAIN: a toolbox for uni- and multivariate statistics, time-series and correlation network analysis and inverse estimation of the differential Jacobian from metabolomics covariance data.Metabolomics. 2012; 8: 81-93Crossref Scopus (107) Google Scholar). The software and parameter settings can be accessed online. Missing values were estimated from the dataset, and data were log transformed before the PCA. For cluster analysis, the mean NSAF value of each developmental stage was calculated and normalized for each protein, setting the total amount throughout the stages to 1. All proteins in the three used databases were blasted for the closest Arabidopsis (TAIR10) homologue using an unpublished Python script in conjunction with stand-alone BLAST v2.2.26+ using the default matrix, and entries in the TAIR Arabidopsis MapMan mapping file (Ath_AGI_LOCUS_TAIR10_Aug2012) were replaced as previously described (54Staudinger C. Mehmeti V. Turetschek R. Lyon D. Egelhofer V. Wienkoop S. Possible role of nutritional priming for early salt and drought stress responses in Medicago truncatula.Front. Plant Sci. 2012; 3: 285Crossref PubMed Scopus (27) Google Scholar). This way, most tobacco protein accessions could be assigned to a functional bin and an Arabidopsis homologue. Tobacco and Arabidopsis microarray results were binned according to the MapMan mapping files Ntob_AGILENT44K_mapping and Ath_AGI_LOCUS_TAIR10_Aug2012, respectively. Tobacco bin numbers were slightly adjusted to fit the tobacco protein bins. Further blasting of the tobacco protein sequences versus the list of Arabidopsis proteins found in Arabidopsis pollen (14Grobei 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) and a list of pollen-affected Arabidopsis mutants (extended list from Ref. 14Grobei M.A. Qeli E
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