Proteomic and Phosphoproteomic Analysis of ... - ACS Publications

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Proteomic and Phosphoproteomic Analysis of Picea wilsonii Pollen Development under Nutrient Limitation Yanmei Chen,*,†,∥ Peng Liu,‡,∥ Wolfgang Hoehenwarter,§ and Jinxing Lin‡ †

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China ‡ Key Laboratory of Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China § Department Molecular Systems Biology, University of Vienna, Faculty of Life Sciences, Althanstrasse 14, A-1090, Vienna S Supporting Information *

ABSTRACT: The pollen tube is a tip-growing system that delivers sperm to the ovule and thus is essential for sexual plant reproduction. Sucrose and other microelements act as nutrients and signaling molecules through pathways that are not yet fully understood. Taking advantage of high-throughput liquid chromatography coupled to mass spectrometry (LC-MS), we performed a label-free shotgun proteomic analysis of pollen in response to nutrient limitation using mass accuracy precursor alignment. We compared 168 LC-MS analyses and more than 1 million precursor ions and could define the proteomic phenotypes of pollen under different conditions. In total, 166 proteins and 42 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, providing new insights into the multifaceted mechanism of nutrient function. The phosphorylation of proteins involved in cytoskeleton dynamics was found to be specifically responsive to Ca2+ and sucrose deficiency, suggesting that sucrose and extracellular Ca2+ influx are necessary for the maintenance of cytoskeleton polymerization. Sucrose limitation leads to widespread accumulation of proteins involved in carbohydrate metabolism and protein degradation. This highlights the wide range of metabolic and cellular processes that are modulated by sucrose but complicates dissection of the signaling pathways. KEYWORDS: pollen, proteomics, phosphoproteomics, calcium influx, cytoskeleton

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INTRODUCTION Pollen tubes grow at their tips through a relatively rapid and polarized cell growth process. They elongate within the female gametophyte to transfer sperm nuclei for fertilization. Pollination has been described as the movement of pollen from a plant's male sex organs to receptive female organs; in conifers, the process is complete only after the pollen tubes have entered the ovule and reached the nucellus.1 Therefore, they are an essential part of sexual reproduction in higher plants.2 Pollen tubes are ideally suited for the study of cell polarity establishment, cell differentiation, cell fate determination, and cell to cell recognition. The fast-growing pollen tube expands its membrane surface at the tip through vigorous and continuous secretion of newly synthesized proteins, cell membranes, and cell wall components. During its development, pollen accumulates large quantities of carbohydrates, which constitute a large part of its dry weight and provide the sugars needed for pollen germination and tube growth. Sucrose is used as the principle substrate for respiration and ATP production, which is required for tip growth, as confirmed by the high density of mitochondria with cristae in the subapical region.3 © 2012 American Chemical Society

Sucrose is a universal signal in organisms. At least three pathways are involved in sucrose signaling: G-protein-coupled receptors, hexokinase signaling, and SnRK kinase signaling.4 Following sugar deprivation, instantaneous responses such as cell growth, consumption of cellular carbohydrates, and degradation of lipids and proteins marked by post-translational modification and a general decline in glycolytic activitiy are observed.5 Other nutrients such as calcium are also central signaling molecules that mediate cellular function by interacting with various metabolic and signaling pathways. Inhibition of calcium pathways resulted in disrupted endo- and exocytosis, followed by perturbed pollen tube extension.6,7 Transcriptomics analysis revealed that more than 12000 genes are expressed in various stages of pollen development.8 However, the molecular mechanisms underlying post-translational regulation are generally less well understood both qualitatively and quantitatively. Furthermore, most studies of pollen germination and tube growth mechanisms are limited to model plants such as Arabidopsis thaliana or Nicotiana tabacum.9 Pollen tubes in Received: March 27, 2012 Published: June 18, 2012 4180

dx.doi.org/10.1021/pr300295m | J. Proteome Res. 2012, 11, 4180−4190

Journal of Proteome Research

Article

supernatant was centrifuged for 60 min at 25000g at 4 °C. The pellet was resuspended in 8 M urea and 100 mM NH4HCO3 for microsomal protein extraction. Cytosolic proteins were precipitated from the supernatant using methanol/chloroform precipitation.12 The protein concentration was determined by the Bradford method.13

gymnosperms differ greatly from those of angiosperms in their extended growth periods and extremely delayed gametogenesis, which represents a major evolutionary divergence in male gametophyte development in flowering plants.10,11 To improve our understanding of these mechanisms and evaluate their role in plant growth in response to nutrients, it would be helpful to identify targets on a large scale. Conventional two-dimensional (2D) gel-based proteomics allows the separation of hundreds of proteins; however, it suffers some drawbacks such as poor reproducibility and the under-representation of low abundant and hydrophobic proteins. In contrast, quantitative mass spectrometry can measure protein abundance and modification states on a large scale in a cell or organelle in a single experiment. It may therefore be the most powerful tool available for the analysis of the dynamics of differential protein phosphorylation. Our objective is to evaluate the effects of nutrients on the proteome and phosphoproteome of pollen during germination and tube growth in Picea wilsonii in an attempt to elucidate the mechanisms underlying the effects of nutrients on tip growth. For this purpose, we performed a label-free shotgun proteomics analysis of cultivated P. wilsonii pollen deprived of sucrose, calcium, and boron. To achieve deep coverage of the proteome, we used a combination of one-dimensional (1D) gel electrophoresis and shotgun proteomics to decipher protein/ phosphoprotein dynamics. We analyzed the proteins and phosphoproteins of pollen 24 h after nutrients deprivation. Each sample was measured using high mass accuracy LCOrbitrap-MS. To further increase proteome coverage, we enriched and analyzed microsomal proteins separately to the total protein extract with the intention of identifying components of the signal transduction network.

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One-Dimensional Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blot

Sample loading buffer (225 mM Tris, pH 6.9, 50% glycerol, 5% SDS, 0.05% bromophenol blue, and 250 mM DTT) was added to the protein extracts. Samples were votexed for 5 min and centrifuged for 15 min at 10000g at 4 °C. Thirty microgram proteins were loaded on a 12.5% acrylamide mini-gel. SDSPAGE electrophoresis was performed as described previously.14 Proteins were stained with Coomassie Brilliant Blue. After gel electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes (GE Healthcare Biosciences). Membranes were incubated with a specific antiserum against a rabbit anti-HSP70 or a plant antiactin antibody (SigmaAldrich) at a ratio of 1:1000 antibody to protein. In-Solution Trypsin Digestion

For in-solution digestion, protein pellets were resuspended with 8 M urea and 100 mM NH4HCO3. A 0.3 mg amount of total proteins was digested for 3 h with endoproteinase Lys-C (1/ 100 w/w) at room temperature. After 4-fold dilution with 10% ACN and 25 mM NH4HCO3, samples were digested overnight with trypsin (0.5/100 w/w) at 37 °C. In-Gel Trypsin Digestion

The gel lanes were cut into 40 slices and destained in 50 mM NH4HCO3 and 50% ACN (v/v). They were reduced with dithiothreitol, alkylated with iodoacetamide, and in-gel digested by adding trypsin to a concentration of 10 ng/μL overnight at 37 °C. Peptides were extracted from the gel pieces in three consecutive steps with acetonitrile (5, 50, and 90% in 1% formic acid, ddH2O), followed by vacuum concentration, and stored at −20 °C.

EXPERIMENTAL PROCEDURES

Plant Materials and Pollen Growth Conditions

Mature male cones were manually collected from P. wilsonii trees growing in the Botanical Garden of the Institute of Botany, Chinese Academy of Sciences. The pollen grains were air-dried and stored at −80 °C. For pollen germination in vitro, 100 mg of pollen grains was cultured in 100 mL of medium in an Erlenmeyer flask. All cultures were incubated at 25 °C on a 100 g shaker for 24 h. The standard medium for pollen culture contained 0.01% H3BO3, 0.03% Ca(NO3)2, and 12% sucrose. Nutrient-deprived cultures were fed with a defined mineral medium that was lacking either carbon (sucrose) or minerals [H3BO3 or Ca(NO3)2] with the other components in excess and at a constant residual concentration. Pollen samples were harvested directly from the flask after 24 h of cultivation. The cultures were placed on filters on sintered columns, the medium was removed by suction, and the pollen grains were washed with a double volume of distilled water, dried by suction for 30 s, and frozen in liquid nitrogen.

Enrichment of Phosphopeptides

Phosphopeptides were enriched with titanium dioxide at room temperature as described previously15 with some modifications. TiO2 tips were supplied by Glygen Inc. (Columbia, MD). Prior to loading samples, the beads were equilibrated with 80% acetonitrile and 0.1% TFA containing 20 mg/mL 2,5dihydroxybenzoic acid (DHB) as a selectivity enhancer. A 300 μg amount of digested soluble or microsomal proteins was incubated with 5 mg of beads for 30 min. After successively washing with 80% acetonitrile, 0.1% TFA, and 10% acetonitrile, 0.1% TFA, the bound peptides were eluted from the beads with 200 μL of 0.3 M NH4OH in 30% acetonitrile (pH > 10). Eluates were dried down almost to completion. Mass Spectrometry and Data Analysis

Protein Purification

The peptide mixture was separated by nanoscale C18 reverse phase liquid chromatography (Proxeon Biosystems, Odense, Denmark). The linear gradient increased from 5 to 30% acetonitrile in 0.5% acetic acid over 95 min. Peptides were eluted and electrosprayed into an LTQ-Orbitrap mass spectrometer (Thermo, Bremen, Germany) as described previously.15 Briefly, the LTQ-Orbitrap MS was performed in a positive ion mode using data-dependent acquisition to isolate and fragment the five most intensive ions. For phosphopeptide analysis, multistage activation was enabled with a neutral loss

About 100 mg of pollen tissue was homogenized in a blender with 5 mL of extraction buffer [50 mM HEPES-KOH (pH 7.5), 0.25 M sucrose, 10% (w/v) glycerol, 0.6% (w/v) PVP K-25, 5 mM EDTA, 1 mM PMSF, 5 mM DTT, 1 mM ascorbic acid, 50 mM NaF, 0.1% Proteinase Inhibitor Cocktail, 0.1% Phosphatase Inhibitor Cocktail 1, and 0.1% Phosphatase Inhibitor Cocktail 2]. The homogenate was filtered through a layer of nylon cloth (240 μm), and the filtrate was centrifuged for 10 min at 2800g at 4 °C. The pellet was discarded, and the 4181



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tube, which grows and reaches a nearby ovule.19 In standard medium, pollen tubes were healthy, and the growth rate was highest between 12 and 24 h of culture (Figure S2 in the Supporting Information). After 30 h of culture, tube growth was nearly arrested. However, pollen germination was completely inhibited in sucrose-deficient medium. A notable effect of sucrose limitation was that tube growth was severely limited even after 24 h of cultivation. In contrast, a lack of calcium and boron had no evident morphological effect on pollen germination. The mean growth rate of pollen tubes was 10 μm/h as compared with 17 μm/h in standard medium.

mass list of m/z 97.97, m/z 48.99, and m/z 32.66. Data processing was done with the strategy called mass accuracy precursor alignment (MAPA) developed recently.16 For peptide identification, raw data files were converted to mascot generic file (mgf) format with the default settings of DTASuperCharge v. 1.17 software with a tolerance of 10 ppm for precursor ion detection. The derived peak list was searched with the Mascot search engine (Matrix Science, MA) against an in-house green plants database (NCBInr) comprising 2120150 sequences and 82321271 residues, and common contaminants (such as trypsin and keratin) were included in the database. A mass tolerance of 10 ppm was set for MS, and 0.8 Da was set for MS/MS; carbamidomethylation was set as a fixed modification, and variable modifications for phosphoprotein mapping included oxidation (Met) and phosphorylation (Ser, Thr, and Tyr). Full tryptic specificity was required, and one missed cleavage was allowed. For protein identification, two peptides had an ion score of 30 or greater, which corresponds to 95% confidence for a peptide spectral match (PSM), of which one peptide was required to be a unique peptide. Mascot results and thermo raw files were imported into MSQuant (http://msquant.sourceforge.net) for peptide and phosphorylation site scoring. All spectra were manually validated; only peptides with more than six amino acid residues and extensive coverage of b- and/or y-ion series were retained further. Phosphopeptides that did not meet these criteria were accepted only if they were detected in two or more measurements or two or more forms (e.g., with or without methionine oxidation or a missed cleavage). In phosphopeptides with multiple potential phosphorylation sites, the probabilities for phosphorylation at each site were calculated from the post-translational modification scores (PTM score) as previously described.17

Sampling the Pollen Proteome by LC-MS/MS

Figure 1 is an overview of the procedure used to study the pollen proteome. We employed fractionation at the subcellular,

Figure 1. Schematic overview of quantitative proteomics approach used to study the effect of nutrient limitation on the pollen proteome. The cultivated P. wilsonii pollen grains were deprived of sucrose, calcium, and boron. The extracted proteins were analyzed by (1) 1D SDS-PAGE and in-gel digestion followed by enrichment of phosphorylated peptides and LC-MS/MS or direct measurement with LC-MS/MS foregoing phosphopeptide enrichment and (2) insolution digestion followed by phosphopeptide enrichment and LCMS/MS or direct measurement with LC-MS/MS forgoing phosphopeptide enrichment.

MAPA

Data alignment was done as described before.18 Briefly, raw data files were converted to mzXml format with the ReAdW program available online from the Institute for systems biology (WA). The data were imported into a matrix with the ProtMAX program developed in house that contained the measured ions m/z rounded to the second decimal in the rows, the identifier of each analysis in the columns, and the spectral count of each m/z in each respective analysis in the cells. The matrix was normalized to the mean total spectral count and transformed to log 10 values. Subsequently, the matrix was analyzed with the MetaGeneAlyse software. Independent component analysis (ICA) was used to determine the variables in the matrix (m/z) that most strongly affects the structure of the data. The resulting pattern is a lower dimensional visualization of the data and should reflect the experimental setup. The MS/MS spectra that most strongly determine the pattern are extracted and identified with database search and indicate which proteins are the most pronounced molecular features of the investigated phenotypes.

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protein, and peptide level to increase proteome coverage. First, we chose to fractionate and analyze microsomal proteins separately of total cytosolic proteins. Second, pollen proteins were separated with 1D SDS-PAGE, and third, peptides were separated and analyzed with LC-MS/MS methods. The extensive fractionation resulted in increased measurement time and sample consumption. Nevertheless, 1D SDS-PAGE and reversed phase chromatography are a fast and effective combination for protein and peptide separation. Proteins were extracted from control and nutrient-deprived pollen grains/tubes after 24 h of cultivation. Phosphopeptide enrichment and LC-MS/MS were performed for both the cytosolic and the microsomal fraction with the gel-free approach. For the gel-based approach, phosphopeptides were enriched with TiO2 from the in-gel digested proteins. To accurately quantify the effects of nutrients, three replicate experiments were performed independently. A total of more than 10000 MS/MS spectra were acquired, and more than 5000 peptides were identified using the mascot software, which calculates a probability PSM. Around 40% of the identified MS/ MS spectra represented unique peptide sequences. Because of the lack of a genome sequence of conifers, only 532 proteins

RESULTS

Pollen Tube Growth

It takes several weeks for the completion of conifer pollen tube growth in vivo. In in vitro cultivation, pollen germination was initiated after 10 h of incubation in standard culture medium and reached a maximum germination percentage after 24 h. At this point, biosynthesis is constant, and the developmental state of elongated pollen tubes corresponds to the in vivo pollen 4182



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were found to be identical to those already reported in the NCBInr database by cross-species matching. A putative function was assigned to these proteins based on sequence similarity with known proteins. Among the proteins identified, 352 (66%) were previously reported in conifers, and 180 (34%) were highly homologous to proteins found in other plants. However, a large number of the proteins that we identified could not be functionally annotated, so further work is needed to completely understand the molecular mechanisms underlying the effects of the different nutrients. The 1D gel LC-MS/ MS analysis revealed 83 proteins that were not accessible by gel free LC-MS/MS. Comparison of gel-free and gel-based proteomics approaches for analysis of nutrient responses in pollen revealed 80% overlap in the proteins and phosphoproteins identified with the respective approaches, illustrating that both are highly complementary. Proteome analysis without phosphopeptide enrichment resulted in the identification of three MS/MS spectra on average for each unique peptide sequence. This was caused by repeated sampling of the same peptide, sequencing of different charge states, sequencing of modified peptides, or sequencing of peptides with missed cleavage sites. We compared these results with previous conifer proteomics studies that used the technology available a few years ago. Less than 100 proteins were identified with more than one peptide employing 2D electrophoresis and more than 300 μg of protein.6,20,21 This demonstrates the high resolution of the conifer pollen tube proteome that we achieved with the combination of our fractionation and enrichment strategies and LC-MS/MS.

Figure 2. Sample pattern recognition in the ICA plot shows sample separation for nutrient limited samples.

(Figure 3); it shows that the expression of HSP 70 was increased in response to sucrose limitation, whereas it

Analysis of Nutrient Response Using MAPA

We recently developed a strategy termed MAPA with which we are capable of combining high sample throughput and advanced multivariate statistical procedures to distinguish proteomic phenotypes or similar proteins such as isoforms or posttranslational modified protein species.15,18 For comparative analysis, MAPA defines a set of proteomes by two parameters, the precursor ion mass to charge ratio (m/z) measured accurately with an error of less than 1 ppm, and the MS/MS spectral count, a proxy for peptide and protein abundance. We aligned and quantified all of the peptides recorded in a total of 168 proteomic analyses of fully fed and nutrient-deprived pollen cultures in one data matrix, in which we combined all of the measurements including the different fractionations and phospho- and nonphosphoproteomics of a nutrient condition. To mine the data, we employed ICA to compare protein/ phosphoprotein responses dependent on the respective nutrient supply. Figure 2 shows a plot of the reduced data in the optimal lower dimensional subspace spanned by the independent components. The sample pattern shows that the effects of the nutrients can be clearly distinguished from one another and the control on the two independent components, indicating nutrient-specific effects. In the ICA pattern, IC01 separates Ca2+ and boron-limited samples from the control; on the second dimension, IC02, sucrose, and boron-deprived samples are separated from the fully fed samples. The original variables can be reconstructed form the lower dimensional data plotted on the independent components with the minimal reconstruction error, making it clear which parts of the original data are prominent and determine the observed relationship. With this, we could detect the common and distinct influence of the nutrients on the pollen tube proteomes. We performed Western blot analysis to confirm the quantification method

Figure 3. Western blot showing the expression of HSP 70 is regulated by nutrient limitation. Western blot with antiactin was used as a control.

decreased in response to boron limitation, which is consistent with the results of MAPA. Proteome Responses to Nutrient Limitation

To investigate the response of P. wilsonii pollen to nutrient limitation, we focused on the 532 proteins identified and quantified in the label free experiment, of which 166 proteins and 42 phosphoproteins were found to be differentially regulated in this study (see Table 1 and Table S1 in the Supporting Information). As many as 82% of the proteins were quantified using two or more peptides, 51% with five or more. Ninety-three proteins were regulated in response to sucrose limitation alone, whereas 59 proteins were specifically regulated under calcium and boron limitation (Figure 4a). This set of 166 proteins was further evaluated to determine if proteins that belong to certain functional categories were specifically regulated in response to carbon or microelement limitation. When comparing the effects of sucrose to microelement limitation, we found that the category “metabolism” was overrepresented in response to sucrose limitation. This reflects major metabolic rearrangement in pollen in adaptation to the altered sucrose supply. In contrast to the significant response to sucrose, fewer proteins in this functional category were regulated by Ca2+ or boron. Interestingly, the metabolic changes under sucrose limitation mainly involved carbon metabolism-related proteins. Particularly, proteins localized in mitochondria such as F1-ATPase α-subunit, malate dehydrogenase, mitochondrial HSP23, mitochondrial HSP70, mito4183


gi no.

4184

gi|330253636 gi|332659298 gi|115646743 gi|332197826 gi|332004072 gi|332196505 gi|330251348

gi|88196757 gi|31711810 gi|332660290 gi|50897170 gi|332189116 gi|332641499 gi|115646736 gi|222423891 gi|222423984 gi|332661655 gi|332643496 gi|332004164 gi|332009775 gi|332009326 gi|239948910

gi|332643413 gi|332005927 gi|332004475 gi|222424863 gi|75318061 gi|227204241 gi|332006838 gi|332645422 gi|343455578 gi|30023716 gi|222423887

gi|330254250

gi|332642494 gi|332197341 gi|332189513 gi|75186527

protein name

26S proteasome regulatory subunit UBX domain-containing protein wrky family transcription factor 1 chromatin protein family transcription elongation factor transcription regulator, calmodulin binding unknown protein

protein kinase protein kinase protein kinase leucine-rich repeat transmembrane protein kinase (LRRkinase) leucine-rich repeat transmembrane protein kinase (LRRkinase) transmembrane kinase (TMKL1) calcium-dependent protein kinase PBS1 kinase (AVRPPHB SUSCEPTIBLE 1) IQD31 (IQ-domain 31), calmodulin binding calcium-binding EF hand family protein phospholipase C lipase class 3 family protein syntaxin 122(SNAP receptor) dynamin vesicle associated membrane protein epsin N-terminal homology (ENTH) domain-containing protein/clathrin assembly protein-related golgi snare 12 (GOS12), SNARE binding ARF GTPase activator calcium-transporting ATPase (ACA10) sugar transporter 60S acidic ribosomal protein P1 (RPP1A) 60S acidic ribosomal protein P0 UDP-glucose 6-dehydrogenase methyltransferase phosphoenolpyruvate carboxylase microtubule-associated protein formin, actin binding protein kinesin motor protein-related myosin heavy chain-related heat shock protein 81-3 glucan synthase-like 7

sequence with modifications

A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana Hordeum vulgare A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana

DNQpTPTQSVVSAPTSTLQNLK FAApSSLSEDDDDDDDDDPDYVEEEEEPLVSHRPR LVPHTVASQSEVDVApSPVSEK ASGSPPVPVMHpSPPRPVTVK FNQPGDLEPPSLIADEDpSPVQK GFRQDVESTEDpSEDEDILK LENSVQQGpSSPREAGSGAPSLLETGK

FTQGGYVDpTGSPTVGSGR TPAFLSSSLpSK DVEAGTSpSFTEYEDSPFDIASTK SSGEIpSPEREPLIK KDEPAEEpSDGDLGFGLFD KEEpSDEEDYEGGFGLFDEE FDWDHPLHLQPMpSPTTVK YVEEWVGPGpSPMNpSPR MApSIDVHLR SLpSNLFLQDK STFISIpSPpS(ox)MSPK SDAALLNLEEGSpSPIPNPSTAAEDSR SEpSGNRLSETDVGALYSQLK TIEKEIpSDDEEEEEK IHSpSVIpTLVELLLK

SSIEpSEDDLEEGDEEDEIGEK TEpSAIFR LNPVDEpSNHGQK SGGMLETQNVGPEEIpSDDEIELPEGK DNDVPVSYpSGSGGPTK NKpSEAKDDLDGNDDDDDDDDEDK SNpSGEFVLNDNVVPER TSVADGSpSPPHSHNIEMSK ATSPQPDGPSSTGGpSLK VVYVAPPRPPpSPVREGSEEGpSSPR pSFGDVNEIGAR

thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana thaliana

A. A. A. A. A. A. A. A. A. A. A.

VSNSHLTEESDVLpSPR HPWLSYPYEPIpSA KNVETNTPEHVpSQTETSAK LIEEVSHSSGSPNPVpSD GGIpSDEFSR

thaliana thaliana thaliana thaliana

A. thaliana

A. A. A. A.

plant species

Table 1. All Phosphopeptides Identified from P. wilsonii Pollens in Response to the Lack of Three Nutrientsa

1pST 1pST 1pST 1pST 1pST 1pST 1pST

1pST 1pST 1pST 1pST 1pST 1pST 1pST 2pST 1pST 1pST 1pST, 1(ox)M 1pST 1pST 1pST 2pST

1pST 1pST 1pST 1pST 1pST 1pST 1pST 1pST 1pST 2pST 1pST

1pST

1pST 1pST 1pST 1pST

no. of modifications

1155.05 1362.55 1130.05 1100.52 1238.57 1146.48 1340.13

933.40 609.29 1288.53 811.39 1010.91 1152.42 973.12 982.89 561.26 622.79 779.31 1380.11 1117.52 951.89 862.94

1217.46 452.20 709.30 1427.12 815.86 1106.51 928.41 1038.94 782.35 903.75 815.86

524.21

925.42 827.37 1090.49 917.40

precursor ion

52 82 82 40 130 84 76

40 48 166 80 77 215 40 87 56 115 74 109 73 89 58

98 56 50 67 87 60 114 75 96 50 64

60

114 119 47 40

PTM score

2 3 2 2 2 2 2

2 2 2 2 2 2 3 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2 2 2 2

2

2 2 2 2

charge state

K R K R K R K

R K K K K K R K K K R R K K K

K R K R R K R K R R R

R

K K K R

left

E R A D A V A

Q V Q A R L E K E

S K N E

A S A K

E C K S K N

S

A

L

right

flanking AA

Journal of Proteome Research Article


3 112 910.43 1pST AHGPAVGLPTEDDMGNpSEVGHNALGAGR unknown protein gi|116786902

In those cases in which the site of phosphorylation could be determined, it is noted by pS for phosphoserine, pT for phosphothreonine, or pY for phosphotyrosine.

K

I

Article

Figure 4. Venn diagram showing the overlap of identified proteins/ phosphoproteins between independent experimental sets. (a) Effects of various nutrient deficiencies on changes in protein abundance of P. wilsonii pollen. (b) Effects of various nutrient deficiencies on protein phosphorylations of P. wilsonii pollen. The cultivated pollen grains were deprived of sucrose, calcium, and boron. Proteins were extracted from the cytoplasm and microsome, the digested peptides were subjected to LC-Orbitrap-MS, and data were analyzed by MAPA. In total, 166 proteins and 42 phosphoproteins were found to be regulated by nutrients. The diagram shows the number of proteins that were regulated specifically in response to the various treatments as well as proteins that were regulated by deprivation of two or three nutrients.

chondrial elongation factor Tu, and NADH-ubiquinone oxidoreductase were upregulated under sucrose limitation. NADH-ubiquinone oxidoreductase was also increased in calcium-deprived pollen, which may reflect an altered NAD+ status. Three Calvin cycle enzymes, succinate dehydrogenase, succinyl-CoA ligase, and malate dehydrogenase, were increased based on spectral count and extracted ion chromatogram quantification following sucrose deprivation; their abundance was unchanged in the microelement-limited pollen. In total, we identified 107 proteins that were responsive to sucrose deficiency, 26 of which were down-regulated at least 3fold. Sucrose deprivation mostly repressed the expression of peptidases, proteins involved in nucleotide metabolism, the translational machinery, and the biosynthesis of amino acids. Proteins related to the degradation of amino acids increased in abundance in response to limited sucrose. Several proteins involved in the synthesis of sucrose were also up-regulated under these conditions (6-phosphogluconate dehydrogenase, UDP-D-glucuronate decarboxylase, ADP-glucose pyrophosphorylase, and starch synthase). Conversely, proteins whose function is fructose phosphorylation or dephosphorylation were down-regulated in sucrose-deprived pollen tubes (fructokinase and fructose-1,6-bisphosphatase). Several proteins that function in proteasome and ubiqiutination showed differential regulation

a

R

right left

R K K 2 2 2 48 60 40 1190.95 702.1 2084.78 1pST 1pST 1pST APEEDEEDpSGDEDDDRPPKR SVpSSGNLSSMDMVEHK VEEKEEpSDEDMGFSLFD

A. thaliana A. thaliana Picea sitchensis P. sitchensis unknown protein unknown protein unknown protein gi|30102918 gi|332660007 gi|116782363

gi no.

Table 1. continued

protein name

plant species

sequence with modifications

no. of modifications

precursor ion

PTM score

charge state

flanking AA


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in response to nutrient deficiency. 20S proteasome α-subunits C1, G1, and E1 were increased after sucrose limitation, but ubiquitin-related proteins (ubiquitin 5, SCF ubiquitin ligase, and SUMO2) were decreased in response to calcium limitation. Especially proteins with functions in cell wall structure, lipid modification, stress responses, and a large number of proteins with unknown functions showed the strongest changes in response to sucrose deficiency. Tubulin abundance was decreased in response to Ca2+ and boron. Two RNA-binding proteins (glycine-rich RNA-binding protein and nuclear-localized RNA binding protein) increased in abundance under boron deprivation, and one of these (nuclear-localized RNA binding protein) also increased under calcium deficiency. The increase of histone H2B protein and lysyl-tRNA synthetase suggests an elevated transcriptional and/ or translational activity in pollen tubes in response to boron limitation. While several of these proteins increased in response to both limited calcium and boron, a ribosomal protein S15 was increased in abundance in response to calcium deficiency but decreased under boron limitation. A translation elongation factor-1 and a protein homologous to transcription factor BTF3 were decreased in response to calcium, while rRNA processing protein and poly (A)-binding protein were regulated in response to sucrose deficiency. Two 14-3-3 proteins were decreased in response to both calcium and boron limitation. Kinases and other proteins involved in signaling were strongly regulated under mineral limitation. In contrast, proteins with functions in carbohydrate metabolism mostly did not show response to microelement limitation. As expected, two calciumrelated proteins (calmodulin and calreticulin) were regulated under calcium limitation, while a calcium-sensing receptor was increased in response to calcium and sucrose deficiency. Calreticulin is the most potent Ca2+-binding protein; downregulation of its abundance leads to a decrease of Ca2+ in the ER and Golgi-coated vesicles.22 Therefore, we can conclude that extracellular calcium deficiency depletes the intracellular Ca2+ stores and redirects the Ca2+ gradient in the pollen tube tip. Profilin, a pollen allergen, plays a pivotal role in the control of actin polymerization.23 Three members of this protein family were regulated differently by calcium and boron deficiency. The abundance of gi|548597 and gi|30841324 increased in response to calcium limitation, while after boron deficiency, gi| 332660220 increased in abundance, and gi|548597 decreased. Profilin is distributed throughout the pollen tube, but the actinsequestering activity of some profilins is sensitive to Ca2+; thus, the changed Ca2+ concentration can profoundly affect profilin activity both spatially and temporally. A protein homologous to the Arabidopsis actin depolymerizing factor (ADF) was downregulated under sucrose deficiency. ADF binds actin filaments and regulates the proper balance of actin polymerization and depolymerization needed for an optimum pollen tube growth process; therefore, the decreased amount of ADF in pollen may disrupt actin organization and induce depolarized growth.

indicating nutrient-specific phosphorylation (see Figure 4b). Proteins involved in cell signaling form the largest fraction of the identified phosphoproteins. A considerable fraction of these phosphoproteins were involved in kinase and calcium signaling. The next largest set was membrane/protein trafficking, followed by cell wall remodeling, transcription, and translation. The function and interaction of different proteins in pollen are better understood through this comprehensive phosphoproteome analysis than by simply profiling protein abundance. Six protein kinases were regulated by nutrients. In total, three RLKs were indentified in this study, and the phosphorylation level of two LRR kinases (gi|75186527 and gi|330254250) was increased 3-fold by calcium and decreased by boron deprivation. In addition, another RLK gi|332004475 showed increased phosphorylation in its N terminus following boron deprivation, while phosphorylation remained constant in the sucrose and calcium-deprived pollen. This suggests that the activity of the RLK is dependent on boron. The phosphorylation level of a transmembrane protein kinase was increased by calcium deficiency. A cytosolic protein kinase gi|332189513 showed increased phosphorylation when pollen were deprived of sucrose, whereas a significant decrease was detected after calcium limitation. Another two protein kinases, gi|332642494 and gi|332197341 displayed decreased phosphorylation in response to calcium and sucrose deficiency. Phosphorylation in the kinase domain of a calcium-dependent protein kinase (CDPK) was strongly decreased in response to Ca2+ limitation and increased by 2-fold following sucrose and boron deprivation. This indicates that Ca2+ signaling differs from sucrose and boron responses. A transmembrane protein kinase was exclusively increased by calcium limitation, indicating a nutrient-specific response. Six vesicle trafficking related proteins were found to be differentially phosphorylated after nutrient limitation. Syntaxin and epsin N-terminal homology (ENTH) domain-containing protein/clathrin assembly protein-related were regulated by sucrose and boron. Dynamin was exclusively regulated by calcium, while phosphorylation of Golgi snare protein was found to be down-regulated by boron alone. Dynamin is a GTP-binding protein and involved in various aspects of endomembrane and intracellular organelle dynamics, such as endocytosis, Golgi network trafficking, and mitochondrial fusion.24 In pollen, dynamin is required for plasma membrane maintenance during microspore maturation. Moreover, phosphorylation of dynamin enchances GTPase activity;25 therefore, we conclude that calcium deficiency could regulate GTPase activity in growing tubes. A phosphorylation site of another GTP-binding protein, ARF GTPase activator, was found to be up-regulated by the limitation of calcium and boron. In contrast, phosphorylation of vesicle-associated membrane protein (VAMP) was shown to be decreased after calcium and sucrose limitation. These proteins constitute the major vesicle-associated component. They are specifically localized throughout the secretory pathways including the Golgi apparatus and plasma membrane and play an important role in vesicle docking. Thus, the differentially regulated phosphorylation levels suggest that different nutrients have different effects in the regulation of membrane trafficking pathways in growing pollen tubes. Our analysis revealed four differentially phosphorylated proteins involved in cytoskeletal dynamics. All four are actin or microtubule-related proteins, including formin, myosin, kinesin, and microtubule-associated protein. Phosphorylation of

Phosphoproteome Response to Nutrient Limitation

To investigate the effect of various nutrients on protein phosphorylation in pollen, we analyzed phosphoproteins under different nutrient conditions by gel-free and gel- based approaches. The results show that a similar overall pattern of phosphorylation is observed between calcium and boronlimited pollen. However, there are substantial differences between sucrose and calcium or boron-limited samples, 4186



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pollen tubes remain speculative. Previously, there was no comprehensive survey of the proteome or phosphoproteome following nutrient deficiency in a polarized system. The regulation of nutrient response in pollen tube signaling is of utmost interest in understanding the metabolic processes involved in plant fertility and reproduction. In this study, we have performed a global analysis of the effects of nutrients on protein abundance and phosphorylation and identified known associations with nutrient pathways as well as proteins that contain strongly induced phosphorylation sites. The conifer proteome is far from completely annotated, and a large number of proteins still have no function assigned to them. In that respect, studies of tip growth and the analysis of phosphorylation under different conditions may provide a valuable contribution to the functional categorization of poorly annotated proteins. For example, two “unknown” proteins (gi| 116787106 and gi|148906365) were identified in this study that showed strong responses similar to those of sugar transporters. Similarly, the uncharacterized protein kinase gi|42562040 could be involved in calcium signaling. The overall picture that emerges is that sucrose limitation strongly impacted protein expression in pollen. Following sucrose deprivation, proteins assigned to glycolysis, the tricarboxylic acid (TCA) cycle, mitochondrial electron transport and ATP synthesis, and cell wall precursor synthesis were increased. Concurrently, there was a highly significant induction of proteins assigned to DNA/ RNA synthesis or processing and protein degradation. Furthermore, phosphorylation of 26S proteasome regulatory subunit was increased in response to sucrose and calcium deficiencies, suggesting that a proteasome-mediated degradation mechanism was induced due to phosphorylation changes in pollen tubes. Also, the phosphorylation of sucrose transporter was decreased. This indicates that carbon depletion in pollen leads to an inhibition of nutrient assimilation, biosynthesis, and cell wall remodeling, thereby inhibiting pollen germination and tube growth. This resembles the response in other species.30,31 Regulated phosphoproteins were mostly signaling molecules and vesicle transporters, indicating that nutrient deficiency largely affected the signaling cascades and membrane/protein trafficking in the polarized cells. Proteins homologous to Arabidopsis RLK were differentially phosphorylated in response to nutrient starvation. Several receptor like kinases were shown to be critical for anther and pollen development.32 The extracellular domain of pollen RLK interacts with distinct pollen and stigma factors prior to and after germination.33 Boron starvation-induced N-terminal phosphorylation of RLK suggests possible interactions in the active state. In addition, our results provide a link between nutrient-induced phosphorylation and the regulation of GTPase activity, such as ARF GTPase activator and dynamin. Pollen RLKs are known to associate with and phosphorylate GTP-binding proteins, thus altering their nucleotide exchange activity, inducing changes in downstream signaling.34 Overexpression of the phosphorylated or dephosphorylated form of RLK dissociated with GTPbinding proteins and induced pollen tube depolarization.29 Our findings suggest an alternative means of regulation, namely, that nutrient-dependent multisite phosphorylation of GTP-binding proteins is autoregulative, leading to a different affinity to its effectors and a failure to induce pollen tube tip growth. The formation of SNARE protein complexes is known to be regulated by phosphorylation.35 SNARE protein complexes mediate the recognition and docking of vesicles and cellular

myosin heavy chain and formin was up-regulated in the growing tubes after calcium deficiency. Previous studies have revealed that formin is an actin-binding protein, stimulates actin assembly mainly around the subapical membrane in pollen tubes, and controls tip growth by facilitating assembly of subapical actin structures and apical vesicular trafficking.2 Therefore, they are essential for polar pollen tube growth. These results suggest that actin cables and dynamics in the growing tubes may depend on phosphorylation. The phosphorylation of microtubule-associated protein and kinesin motor protein was decreased following sucrose limitation. Because microtubule-associated protein and kinesin motor protein function in organelle transport, the sucrose limitationinduced down-regulation of phosphorylation levels could affect the speed of organelle movement in pollen, thus leading to the inhibition of pollen germination and tube growth. Two transport proteins were found to be regulated by the nutrient deficiency (sucrose transporter and Ca2+-ATPase). It is well-known that phosphorylation of transporters influences the activities of ion channels initiating a downstream response. A phosphorylation site was identified for the sucrose transporter in the N-terminal part of the protein. The abundance of this phosphopeptide decreased 3-fold in sucrose-limited pollen as compared to that of control samples. The sucrose transporter activity is regulated by phosphorylation;26 decreased phosphorylation suggests that long time carbon starvation led to an adaptive response and sucrose channel closing. Similarly, phosphorylation of an N-terminal site of the Ca2+ -ATPase decreased in response to calcium and boron deficiency. Previous studies reported that this region regulates pump activities through a calmodulin binding domain.27 Furthermore, as calcium is an important second messenger, decreased phosphorylation of the Ca2+-ATPase could decrease the intercellular Ca2+ concentration and modulate various signals and responses. This is consistent with the decreased phosphorylation of calcium-binding EF hand family protein following calcium deficiency. In addition, the phosphorylation of camodulin-binding protein, IQ-domain 31, was significantly regulated by the lack of three different nutrients. The phosphorylation of two lipase-involved proteins was also affected by nutrient deprivation. Lipase class 3 family protein was regulated by sucrose and Ca 2+ limitation, while phospholipase C was regulated exclusively by the lack of Ca2+. Phospholipase C is known to play a role in tip growth not only by controlling InsP3-gated Ca2+ fluxes but also by altering the spectrum of PI lipids in the tube apex.28 The regulated phospholipase C could impact downstream second messengers such as IP3 and diacylglycerol.

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DISCUSSION Pollen tubes elongate through rapid and polarized cell growth and transport sperms to the female gametophytes for fertilization. Polarized tip growth results from a dynamic actin cytoskeleton and highly active membrane trafficking system that provide the secretory activities needed for growth. Furthermore, a polarized cytoplasm with vesicles and tipfocused Ca2+ concentration gradients are essential for the process of polar cell growth. Nutrients such as sucrose provide carbon for new plasma membrane and cell wall components and remodel the cell wall composition. A delicate balance between the exocytosis of cell wall components, the cell wall assembly, and the membrane trafficking is important for pollen tube growth.29 The precise mechanisms behind this process in 4187



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Figure 5. Functional classifications of differentially regulated proteins. Pie charts of functional categories of all responsive proteins identified from the total number of tryptic peptides (166 proteins).

Figure 6. Model showing the potential nutrient-mediated phosphorylation signaling events that lead to pollen tip growth. Nutrient deficiency is perceived by receptors, leading to downstream activation of protein kinases. The activated protein kinases regulate the phosphorylation of transporters, GTPases, cytoskeleton-related proteins, ubiquitins, and transcription factors. The regulated phosphorylation levels of transporters such as Ca2+-ATPase are known to control transporter activities, which induce a decrease in cytosolic Ca2+ concentration, and this in turn regulates the activities of protein kinases. As a consequence, the cytoskeleton is remodeled, and vesicle trafficking is activated, resulting in cell wall reconstruction. Together, these pathways lead to the disruption of tip growth.

phosphorylation of the kinase domain of a potential Ca2+ sensor, CDPK, was shown to be decreased by Ca2+ limitation, suggesting regulated kinase activity. These observations are consistent with the well-known regulation of CDPK in Ca2+ signaling. Perturbation of membrane-localized CDPK was reported to result in increased intracellular Ca2+ and abolition of the tip-focused [Ca2+] gradient.37 The regulated phosphorylation suggested that CDPK may participate in Ca 2+ homeostasis by phosphorylation of Ca2+-ATPase. As a consequence, after Ca2+ starvation, pollen could balance the supply and utilization of calcium with specific Ca2+ signaling pathways. Cytoskeleton-binding proteins play important roles in cytoskeleton organization to determine plant directional cell growth. Phosphorylation of cytoskeleton binding protein is known to stabilize microfilaments or microtubules and

membranes. Taken together, our data demonstrate that differential phosphorylation patterns for different SNARE isoforms could reduce or enhance their interactions and trigger selective modulation of membrane trafficking pathways. An N-terminal phosphorylation site of Ca2+-ATPase was downregulated in response to Ca2+ deficiency. N-terminal phosphorylation of Ca2+-ATPase is known to be crucial for ATPase activity and overrides regulation via calmodulinbinding protein. Elongated pollen tubes exhibit a tip-focused cytosolic Ca2+ ([Ca2+]c) gradient that is largely maintained by extracellular Ca2+ influx. Mutation of Ca2+-ATPase is male deficient and shows reduced tube growth rates.36 We can conclude that the reduced phosphorylation would reduce Ca2+ influx in pollen tubes after long time Ca2+ starvation, consistent with the finding of down-regulated calreticulin. Interestingly, 4188



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stimulate their assembly.38,39 Cytoskeleton-related proteins identified in this study are diverse and respond selectively to different signals. The specificities in phosphorylation responses imply that both microfilaments and microtubules function schematically with different programs in specific cell regions or organelles transported during pollen tube growth. In summary, the nontargeted label-free proteomics analysis that we carried out has expanded our knowledge of pollen development by identifying and quantifying a number of proteins and protein phosphorylation sites in response to nutrient limitation in P. wilsonii. It was revealed that lack of nutrients regulated the phosphorylation level of transporters; the regulated phosphorylation of transporters was suggested to control transporter activities, which induces a decrease in cytosolic Ca2+ concentration through the regulation of both the influx of Ca2+ from the extracellular space and the release of Ca2+ from intracellular stores, and this in turn regulates the activities of protein kinases, affecting the phosphorylation level of downstream targets. As a consequence, the cytoskeleton is remodeled, and vesicle trafficking is activated, resulting in cell wall reconstruction. Taken together, these pathways lead to the disruption of tip growth, as summarized in Figure 6. Our investigation provides a general rationale for the role of nutrients in processes critical to pollen function and development. Furthermore, we took a first step in including a dynamic component in pollen phosphoproteomics, especially in gymnosperm species, which indicates new candidates possibly involved in the key process of nutrient transport and regulation. More importantly, we found evidence for a new complex network of polarized tip growth.

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ASSOCIATED CONTENT

Changes in relative phosphorylation of various proteins in response to the lack of nutrients (Figure S1). Micrographs of P. wilsonii pollen under different conditions (Figure S2): (A) control pollen tubes under standard medium, (B) pollen tubes deprived of H3BO4, (C) pollen tubes deprived of calcium, and (D) pollen tubes deprived of sucrose. Complete list of proteins identified to be differentially regulated in pollen of P. wilsonii under nutrient limitation (Table S1), and statistical analysis of regulated phosphoproteins (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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*Tel: +86-10-62733433. Fax: +86-10-62731128. E-mail: [email protected]. Author Contributions ∥

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S Supporting Information *

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Article

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Tong Chen (Institute of Botany, Chinese Academy of Sciences, China) for male cones harvesting. This work was supported by the program for Chinese Universities Scientific Fund (2012XJ005). 4189



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