Proteomic and Phosphoproteomic Analysis of Picea wilsonii

Proteomic and Phosphoproteomic Analysis of Picea wilsonii PollenDevelopment under Nutrient LimitationYanmei Chen,*,†,∥ Peng Liu,‡,∥ Wolfgang Hoehenwarter,§ and Jinxing Lin‡

†State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing100193, 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 deliverssperm to the ovule and thus is essential for sexual plant reproduction.Sucrose and other microelements act as nutrients and signaling moleculesthrough pathways that are not yet fully understood. Taking advantage ofhigh-throughput liquid chromatography coupled to mass spectrometry(LC-MS), we performed a label-free shotgun proteomic analysis of pollenin response to nutrient limitation using mass accuracy precursoralignment. We compared 168 LC-MS analyses and more than 1 millionprecursor ions and could define the proteomic phenotypes of pollenunder different conditions. In total, 166 proteins and 42 phosphoproteinswere identified as differentially regulated. These proteins are involved in avariety of signaling pathways, providing new insights into themultifaceted mechanism of nutrient function. The phosphorylation ofproteins involved in cytoskeleton dynamics was found to be specificallyresponsive to Ca2+ and sucrose deficiency, suggesting that sucrose and extracellular Ca2+ influx are necessary for the maintenanceof cytoskeleton polymerization. Sucrose limitation leads to widespread accumulation of proteins involved in carbohydratemetabolism and protein degradation. This highlights the wide range of metabolic and cellular processes that are modulated bysucrose but complicates dissection of the signaling pathways.

KEYWORDS: pollen, proteomics, phosphoproteomics, calcium influx, cytoskeleton

■ INTRODUCTIONPollen tubes grow at their tips through a relatively rapid andpolarized cell growth process. They elongate within the femalegametophyte to transfer sperm nuclei for fertilization.Pollination has been described as the movement of pollenfrom a plant's male sex organs to receptive female organs; inconifers, the process is complete only after the pollen tubeshave entered the ovule and reached the nucellus.1 Therefore,they are an essential part of sexual reproduction in higherplants.2 Pollen tubes are ideally suited for the study of cellpolarity establishment, cell differentiation, cell fate determi-nation, and cell to cell recognition. The fast-growing pollentube expands its membrane surface at the tip through vigorousand continuous secretion of newly synthesized proteins, cellmembranes, and cell wall components. During its development,pollen accumulates large quantities of carbohydrates, whichconstitute a large part of its dry weight and provide the sugarsneeded for pollen germination and tube growth. Sucrose is usedas the principle substrate for respiration and ATP production,which is required for tip growth, as confirmed by the highdensity of mitochondria with cristae in the subapical region.3

Sucrose is a universal signal in organisms. At least threepathways are involved in sucrose signaling: G-protein-coupledreceptors, hexokinase signaling, and SnRK kinase signaling.4

Following sugar deprivation, instantaneous responses such ascell growth, consumption of cellular carbohydrates, anddegradation of lipids and proteins marked by post-translationalmodification and a general decline in glycolytic activitiy areobserved.5 Other nutrients such as calcium are also centralsignaling molecules that mediate cellular function by interactingwith various metabolic and signaling pathways. Inhibition ofcalcium pathways resulted in disrupted endo- and exocytosis,followed by perturbed pollen tube extension.6,7 Transcriptom-ics analysis revealed that more than 12000 genes are expressedin various stages of pollen development.8 However, themolecular mechanisms underlying post-translational regulationare generally less well understood both qualitatively andquantitatively. Furthermore, most studies of pollen germinationand tube growth mechanisms are limited to model plants suchas Arabidopsis thaliana or Nicotiana tabacum.9 Pollen tubes in

Received: March 27, 2012Published: June 18, 2012

Article

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

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gymnosperms differ greatly from those of angiosperms in theirextended growth periods and extremely delayed gametogenesis,which represents a major evolutionary divergence in malegametophyte development in flowering plants.10,11 To improveour understanding of these mechanisms and evaluate their rolein plant growth in response to nutrients, it would be helpful toidentify targets on a large scale.Conventional two-dimensional (2D) gel-based proteomics

allows the separation of hundreds of proteins; however, itsuffers some drawbacks such as poor reproducibility and theunder-representation of low abundant and hydrophobicproteins. In contrast, quantitative mass spectrometry canmeasure protein abundance and modification states on a largescale in a cell or organelle in a single experiment. It maytherefore be the most powerful tool available for the analysis ofthe dynamics of differential protein phosphorylation. Ourobjective is to evaluate the effects of nutrients on the proteomeand phosphoproteome of pollen during germination and tubegrowth in Picea wilsonii in an attempt to elucidate themechanisms underlying the effects of nutrients on tip growth.For this purpose, we performed a label-free shotgun proteomicsanalysis 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 electro-phoresis and shotgun proteomics to decipher protein/phosphoprotein dynamics. We analyzed the proteins andphosphoproteins of pollen 24 h after nutrients deprivation.Each sample was measured using high mass accuracy LC-Orbitrap-MS. To further increase proteome coverage, weenriched and analyzed microsomal proteins separately to thetotal protein extract with the intention of identifyingcomponents of the signal transduction network.

■ EXPERIMENTAL PROCEDURESPlant Materials and Pollen Growth Conditions

Mature male cones were manually collected from P. wilsoniitrees growing in the Botanical Garden of the Institute ofBotany, Chinese Academy of Sciences. The pollen grains wereair-dried and stored at −80 °C. For pollen germination in vitro,100 mg of pollen grains was cultured in 100 mL of medium inan Erlenmeyer flask. All cultures were incubated at 25 °C on a100 g shaker for 24 h. The standard medium for pollen culturecontained 0.01% H3BO3, 0.03% Ca(NO3)2, and 12% sucrose.Nutrient-deprived cultures were fed with a defined mineralmedium that was lacking either carbon (sucrose) or minerals[H3BO3 or Ca(NO3)2] with the other components in excessand at a constant residual concentration. Pollen samples wereharvested directly from the flask after 24 h of cultivation. Thecultures were placed on filters on sintered columns, themedium was removed by suction, and the pollen grains werewashed with a double volume of distilled water, dried bysuction for 30 s, and frozen in liquid nitrogen.

Protein Purification

About 100 mg of pollen tissue was homogenized in a blenderwith 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, 5mM EDTA, 1 mM PMSF, 5 mM DTT, 1 mM ascorbic acid, 50mM NaF, 0.1% Proteinase Inhibitor Cocktail, 0.1% Phospha-tase Inhibitor Cocktail 1, and 0.1% Phosphatase InhibitorCocktail 2]. The homogenate was filtered through a layer ofnylon cloth (240 μm), and the filtrate was centrifuged for 10min at 2800g at 4 °C. The pellet was discarded, and the

supernatant was centrifuged for 60 min at 25000g at 4 °C. Thepellet was resuspended in 8 M urea and 100 mM NH4HCO3for microsomal protein extraction. Cytosolic proteins wereprecipitated from the supernatant using methanol/chloroformprecipitation.12 The protein concentration was determined bythe Bradford method.13

One-Dimensional Sodium Dodecyl Sulfate−PolyacrylamideGel 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 addedto the protein extracts. Samples were votexed for 5 min andcentrifuged for 15 min at 10000g at 4 °C. Thirty microgramproteins were loaded on a 12.5% acrylamide mini-gel. SDS-PAGE electrophoresis was performed as described previously.14

Proteins were stained with Coomassie Brilliant Blue. After gelelectrophoresis, proteins were electrotransferred to polyvinyli-dene difluoride membranes (GE Healthcare Biosciences).Membranes were incubated with a specific antiserum againsta rabbit anti-HSP70 or a plant antiactin antibody (Sigma-Aldrich) at a ratio of 1:1000 antibody to protein.

In-Solution Trypsin Digestion

For in-solution digestion, protein pellets were resuspended with8 M urea and 100 mM NH4HCO3. A 0.3 mg amount of totalproteins 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 overnightwith 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 mMNH4HCO3 and 50% ACN (v/v). They were reduced withdithiothreitol, alkylated with iodoacetamide, and in-gel digestedby adding trypsin to a concentration of 10 ng/μL overnight at37 °C. Peptides were extracted from the gel pieces in threeconsecutive steps with acetonitrile (5, 50, and 90% in 1%formic acid, ddH2O), followed by vacuum concentration, andstored at −20 °C.Enrichment of Phosphopeptides

Phosphopeptides were enriched with titanium dioxide at roomtemperature as described previously15 with some modifications.TiO2 tips were supplied by Glygen Inc. (Columbia, MD). Priorto loading samples, the beads were equilibrated with 80%acetonitrile and 0.1% TFA containing 20 mg/mL 2,5-dihydroxybenzoic acid (DHB) as a selectivity enhancer. A300 μg amount of digested soluble or microsomal proteins wasincubated with 5 mg of beads for 30 min. After successivelywashing with 80% acetonitrile, 0.1% TFA, and 10% acetonitrile,0.1% TFA, the bound peptides were eluted from the beads with200 μL of 0.3 M NH4OH in 30% acetonitrile (pH > 10).Eluates were dried down almost to completion.

Mass Spectrometry and Data Analysis

The peptide mixture was separated by nanoscale C18 reversephase liquid chromatography (Proxeon Biosystems, Odense,Denmark). The linear gradient increased from 5 to 30%acetonitrile in 0.5% acetic acid over 95 min. Peptides wereeluted and electrosprayed into an LTQ-Orbitrap massspectrometer (Thermo, Bremen, Germany) as describedpreviously.15 Briefly, the LTQ-Orbitrap MS was performed ina positive ion mode using data-dependent acquisition to isolateand fragment the five most intensive ions. For phosphopeptideanalysis, multistage activation was enabled with a neutral loss

Journal of Proteome Research Article

dx.doi.org/10.1021/pr300295m | J. Proteome Res. 2012, 11, 4180−41904181

mass list of m/z 97.97, m/z 48.99, and m/z 32.66. Dataprocessing was done with the strategy called mass accuracyprecursor alignment (MAPA) developed recently.16 Forpeptide identification, raw data files were converted to mascotgeneric file (mgf) format with the default settings ofDTASuperCharge v. 1.17 software with a tolerance of 10ppm for precursor ion detection. The derived peak list wassearched with the Mascot search engine (Matrix Science, MA)against an in-house green plants database (NCBInr) comprising2120150 sequences and 82321271 residues, and commoncontaminants (such as trypsin and keratin) were included in thedatabase. A mass tolerance of 10 ppm was set for MS, and 0.8Da was set for MS/MS; carbamidomethylation was set as afixed modification, and variable modifications for phosphopro-tein mapping included oxidation (Met) and phosphorylation(Ser, Thr, and Tyr). Full tryptic specificity was required, andone missed cleavage was allowed. For protein identification,two peptides had an ion score of 30 or greater, whichcorresponds to 95% confidence for a peptide spectral match(PSM), of which one peptide was required to be a uniquepeptide. Mascot results and thermo raw files were importedinto MSQuant (http://msquant.sourceforge.net) for peptideand phosphorylation site scoring. All spectra were manuallyvalidated; only peptides with more than six amino acid residuesand extensive coverage of b- and/or y-ion series were retainedfurther. Phosphopeptides that did not meet these criteria wereaccepted only if they were detected in two or moremeasurements or two or more forms (e.g., with or withoutmethionine oxidation or a missed cleavage). In phosphopep-tides with multiple potential phosphorylation sites, theprobabilities for phosphorylation at each site were calculatedfrom the post-translational modification scores (PTM score) aspreviously described.17

MAPA

Data alignment was done as described before.18 Briefly, rawdata files were converted to mzXml format with the ReAdWprogram available online from the Institute for systems biology(WA). The data were imported into a matrix with theProtMAX program developed in house that contained themeasured ions m/z rounded to the second decimal in the rows,the identifier of each analysis in the columns, and the spectralcount of each m/z in each respective analysis in the cells. Thematrix was normalized to the mean total spectral count andtransformed to log 10 values. Subsequently, the matrix wasanalyzed with the MetaGeneAlyse software. Independentcomponent analysis (ICA) was used to determine the variablesin the matrix (m/z) that most strongly affects the structure ofthe data. The resulting pattern is a lower dimensionalvisualization of the data and should reflect the experimentalsetup. The MS/MS spectra that most strongly determine thepattern are extracted and identified with database search andindicate which proteins are the most pronounced molecularfeatures of the investigated phenotypes.

■ RESULTSPollen Tube Growth

It takes several weeks for the completion of conifer pollen tubegrowth in vivo. In in vitro cultivation, pollen germination wasinitiated after 10 h of incubation in standard culture mediumand reached a maximum germination percentage after 24 h. Atthis point, biosynthesis is constant, and the developmental stateof elongated pollen tubes corresponds to the in vivo pollen

tube, which grows and reaches a nearby ovule.19 In standardmedium, pollen tubes were healthy, and the growth rate washighest between 12 and 24 h of culture (Figure S2 in theSupporting Information). After 30 h of culture, tube growthwas nearly arrested. However, pollen germination wascompletely inhibited in sucrose-deficient medium. A notableeffect of sucrose limitation was that tube growth was severelylimited even after 24 h of cultivation. In contrast, a lack ofcalcium and boron had no evident morphological effect onpollen germination. The mean growth rate of pollen tubes was10 μm/h as compared with 17 μm/h in standard medium.

Sampling the Pollen Proteome by LC-MS/MS

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

protein, and peptide level to increase proteome coverage. First,we chose to fractionate and analyze microsomal proteinsseparately of total cytosolic proteins. Second, pollen proteinswere separated with 1D SDS-PAGE, and third, peptides wereseparated and analyzed with LC-MS/MS methods. Theextensive fractionation resulted in increased measurementtime and sample consumption. Nevertheless, 1D SDS-PAGEand reversed phase chromatography are a fast and effectivecombination for protein and peptide separation.Proteins were extracted from control and nutrient-deprived

pollen grains/tubes after 24 h of cultivation. Phosphopeptideenrichment and LC-MS/MS were performed for both thecytosolic and the microsomal fraction with the gel-freeapproach. For the gel-based approach, phosphopeptides wereenriched with TiO2 from the in-gel digested proteins. Toaccurately quantify the effects of nutrients, three replicateexperiments were performed independently. A total of morethan 10000 MS/MS spectra were acquired, and more than 5000peptides were identified using the mascot software, whichcalculates a probability PSM. Around 40% of the identified MS/MS spectra represented unique peptide sequences. Because ofthe lack of a genome sequence of conifers, only 532 proteins

Figure 1. Schematic overview of quantitative proteomics approachused 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) 1DSDS-PAGE and in-gel digestion followed by enrichment ofphosphorylated peptides and LC-MS/MS or direct measurementwith LC-MS/MS foregoing phosphopeptide enrichment and (2) in-solution digestion followed by phosphopeptide enrichment and LC-MS/MS or direct measurement with LC-MS/MS forgoing phospho-peptide enrichment.



http://msquant.sourceforge.net

were found to be identical to those already reported in theNCBInr database by cross-species matching. A putativefunction was assigned to these proteins based on sequencesimilarity 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 identifiedcould not be functionally annotated, so further work is neededto completely understand the molecular mechanisms under-lying the effects of the different nutrients. The 1D gel LC-MS/MS analysis revealed 83 proteins that were not accessible by gelfree LC-MS/MS. Comparison of gel-free and gel-basedproteomics approaches for analysis of nutrient responses inpollen revealed 80% overlap in the proteins and phosphopro-teins identified with the respective approaches, illustrating thatboth are highly complementary.Proteome analysis without phosphopeptide enrichment

resulted in the identification of three MS/MS spectra onaverage for each unique peptide sequence. This was caused byrepeated sampling of the same peptide, sequencing of differentcharge states, sequencing of modified peptides, or sequencingof peptides with missed cleavage sites. We compared theseresults with previous conifer proteomics studies that used thetechnology available a few years ago. Less than 100 proteinswere identified with more than one peptide employing 2Delectrophoresis and more than 300 μg of protein.6,20,21 Thisdemonstrates the high resolution of the conifer pollen tubeproteome that we achieved with the combination of ourfractionation and enrichment strategies and LC-MS/MS.

Analysis of Nutrient Response Using MAPA

We recently developed a strategy termed MAPA with which weare capable of combining high sample throughput and advancedmultivariate statistical procedures to distinguish proteomicphenotypes or similar proteins such as isoforms or post-translational modified protein species.15,18 For comparativeanalysis, MAPA defines a set of proteomes by two parameters,the precursor ion mass to charge ratio (m/z) measuredaccurately with an error of less than 1 ppm, and the MS/MSspectral count, a proxy for peptide and protein abundance. Wealigned and quantified all of the peptides recorded in a total of168 proteomic analyses of fully fed and nutrient-deprivedpollen cultures in one data matrix, in which we combined all ofthe measurements including the different fractionations andphospho- and nonphosphoproteomics of a nutrient condition.To mine the data, we employed ICA to compare protein/phosphoprotein responses dependent on the respectivenutrient supply. Figure 2 shows a plot of the reduced data inthe optimal lower dimensional subspace spanned by theindependent components. The sample pattern shows that theeffects of the nutrients can be clearly distinguished from oneanother and the control on the two independent components,indicating nutrient-specific effects. In the ICA pattern, IC01separates Ca2+ and boron-limited samples from the control; onthe second dimension, IC02, sucrose, and boron-deprivedsamples are separated from the fully fed samples. The originalvariables can be reconstructed form the lower dimensional dataplotted on the independent components with the minimalreconstruction error, making it clear which parts of the originaldata are prominent and determine the observed relationship.With this, we could detect the common and distinct influenceof the nutrients on the pollen tube proteomes. We performedWestern blot analysis to confirm the quantification method

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

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

To investigate the response of P. wilsonii pollen to nutrientlimitation, we focused on the 532 proteins identified andquantified in the label free experiment, of which 166 proteinsand 42 phosphoproteins were found to be differentiallyregulated in this study (see Table 1 and Table S1 in theSupporting Information). As many as 82% of the proteins werequantified using two or more peptides, 51% with five or more.Ninety-three proteins were regulated in response to sucroselimitation alone, whereas 59 proteins were specifically regulatedunder calcium and boron limitation (Figure 4a). This set of 166proteins was further evaluated to determine if proteins thatbelong to certain functional categories were specificallyregulated in response to carbon or microelement limitation.When comparing the effects of sucrose to microelementlimitation, we found that the category “metabolism” wasoverrepresented in response to sucrose limitation. This reflectsmajor metabolic rearrangement in pollen in adaptation to thealtered sucrose supply. In contrast to the significant response tosucrose, fewer proteins in this functional category wereregulated by Ca2+ or boron. Interestingly, the metabolicchanges under sucrose limitation mainly involved carbonmetabolism-related proteins. Particularly, proteins localized inmitochondria such as F1-ATPase α-subunit, malate dehydro-genase, mitochondrial HSP23, mitochondrial HSP70, mito-

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

Figure 3. Western blot showing the expression of HSP 70 is regulatedby nutrient limitation. Western blot with antiactin was used as acontrol.



Table

1.AllPho

spho

peptides

Identified

from

P.w

ilsoniiPollens

inRespo

nseto

theLack

ofThree

Nutrientsa

flankingAA

gino.

proteinname

plant

species

sequence

with

modificatio

nsno.o

fmodificatio

nsprecursor

ion

PTM

score

charge

state

left

right

gi|332642494

proteinkinase

A.thaliana

VSN

SHLT

EESD

VLp

SPR

1pST

925.42

114

2K

Lgi|332197341

proteinkinase

A.thaliana

HPW

LSYPY

EPIpSA

1pST

827.37

119

2K

gi|332189513

proteinkinase

A.thaliana

KNVET

NTPE

HVpSQTET

SAK

1pST

1090.49

472

KA

gi|75186527

leucine-richrepeat

transm

embraneproteinkinase

(LRR-

kinase)

A.thaliana

LIEE

VSH

SSGSP

NPV

pSD

1pST

917.40

402

R

gi|330254250

leucine-richrepeat

transm

embraneproteinkinase

(LRR-

kinase)

A.thaliana

GGIpSD

EFSR

1pST

524.21

602

RS

gi|332643413

transm

embranekinase

(TMKL1

)A.thaliana

SSIEpSED

DLE

EGDEE

DEIGEK

1pST

1217.46

982

KE

gi|332005927

calcium-dependent

proteinkinase

A.thaliana

TEp

SAIFR

1pST

452.20

562

RC

gi|332004475

PBS1

kinase

(AVRPP

HBSU

SCEP

TIBLE

1)A.thaliana

LNPV

DEp

SNHGQK

1pST

709.30

502

KK

gi|222424863

IQD31

(IQ-dom

ain31),calmodulin

binding

A.thaliana

SGGMLE

TQNVGPE

EIpSDDEIEL

PEGK

1pST

1427.12

672

RS

gi|75318061

calcium-binding

EFhand

family

protein

A.thaliana

DNDVPV

SYpSGSG

GPT

K1pST

815.86

872

RK

gi|227204241

phospholipaseC

A.thaliana

NKpSEA

KDDLD

GNDDDDDDDDED

K1pST

1106.51

602

KN

gi|332006838

lipaseclass3family

protein

A.thaliana

SNpSGEF

VLN

DNVVPE

R1pST

928.41

114

2R

gi|332645422

syntaxin

122(SN

APreceptor)

A.thaliana

TSV

ADGSpSP

PHSH

NIEMSK

1pST

1038.94

752

KA

gi|343455578

dynamin

A.thaliana

ATSP

QPD

GPS

STGGpSLK

1pST

782.35

962

RS

gi|30023716

vesicleassociated

mem

braneprotein

A.thaliana

VVYVAPP

RPP

pSPV

REG

SEEG

pSSP

R2pST

903.75

502

RA

gi|222423887

epsinN-terminalhomology(ENTH)domain-containing

protein/clathrin

assemblyprotein-related

A.thaliana

pSFG

DVNEIGAR

1pST

815.86

642

RK

gi|88196757

golgisnare12

(GOS12),S

NAREbinding

A.thaliana

FTQGGYVDpT

GSP

TVGSG

R1pST

933.40

402

RS

gi|31711810

ARFGTPase

activator

A.thaliana

TPA

FLSSSL

pSK

1pST

609.29

482

KK

gi|332660290

calcium-transportingATPase

(ACA10)

A.thaliana

DVEA

GTSpSF

TEY

EDSP

FDIAST

K1pST

1288.53

166

2K

Ngi|50897170

sugartransporter

A.thaliana

SSGEIpSPE

REP

LIK

1pST

811.39

802

KE

gi|332189116

60Sacidicrib

osom

alproteinP1

(RPP

1A)

A.thaliana

KDEP

AEE

pSDGDLG

FGLF

D1pST

1010.91

772

Kgi|332641499

60Sacidicrib

osom

alproteinP0

A.thaliana

KEE

pSDEE

DYEG

GFG

LFDEE

1pST

1152.42

215

2K

gi|115646736

UDP-glucose6-dehydrogenase

A.thaliana

FDWDHPL

HLQ

PMpSPT

TVK

1pST

973.12

403

RQ

gi|222423891

methyltransferase

A.thaliana

YVEE

WVGPG

pSPM

NpSPR

2pST

982.89

872

KV

gi|222423984

phosphoenolpyruvatecarboxylase

A.thaliana

MApSID

VHLR

1pST

561.26

562

KQ

gi|332661655

microtubule-associatedprotein

A.thaliana

SLpSNLF

LQDK

1pST

622.79

115

2K

Agi|332643496

form

in,actin

bindingprotein

A.thaliana

STFISIpSPp

S(ox)M

SPK

1pST

,1(ox)M

779.31

742

RR

gi|332004164

kinesinmotor

protein-related

A.thaliana

SDAALL

NLE

EGSpSP

IPNPS

TAAED

SR1pST

1380.11

109

2R

Lgi|332009775

myosinheavychain-related

A.thaliana

SEpSGNRLS

ETDVGALY

SQLK

1pST

1117.52

732

KE

gi|332009326

heat

shockprotein81-3

A.thaliana

TIEKEIpSDDEE

EEEK

1pST

951.89

892

KK

gi|239948910

glucan

synthase-like

7Hordeum

vulgare

IHSpSV

IpTLV

ELLL

K2pST

862.94

582

KE

gi|330253636

26Sproteasomeregulatory

subunit

A.thaliana

DNQpT

PTQSV

VSA

PTST

LQNLK

1pST

1155.05

522

KE

gi|332659298

UBXdomain-containing

protein

A.thaliana

FAApSSL

SEDDDDDDDDDPD

YVEE

EEEP

LVSH

RPR

1pST

1362.55

823

RR

gi|115646743

wrkyfamily

transcrip

tionfactor

1A.thaliana

LVPH

TVASQ

SEVDVApSPV

SEK

1pST

1130.05

822

KA

gi|332197826

chromatin

proteinfamily

A.thaliana

ASG

SPPV

PVMHpSPP

RPV

TVK

1pST

1100.52

402

RD

gi|332004072

transcrip

tionelongatio

nfactor

A.thaliana

FNQPG

DLE

PPSL

IADED

pSPV

QK

1pST

1238.57

130

2K

Agi|332196505

transcrip

tionregulator,calmodulin

binding

A.thaliana

GFR

QDVES

TED

pSED

EDILK

1pST

1146.48

842

RV

gi|330251348

unknow

nprotein

A.thaliana

LENSV

QQGpSSP

REA

GSG

APS

LLET

GK

1pST

1340.13

762

KA



chondrial elongation factor Tu, and NADH-ubiquinoneoxidoreductase were upregulated under sucrose limitation.NADH-ubiquinone oxidoreductase was also increased incalcium-deprived pollen, which may reflect an altered NAD+

status. Three Calvin cycle enzymes, succinate dehydrogenase,succinyl-CoA ligase, and malate dehydrogenase, were increasedbased on spectral count and extracted ion chromatogramquantification following sucrose deprivation; their abundancewas 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 3-fold. Sucrose deprivation mostly repressed the expression ofpeptidases, proteins involved in nucleotide metabolism, thetranslational machinery, and the biosynthesis of amino acids.Proteins related to the degradation of amino acids increased inabundance in response to limited sucrose. Several proteinsinvolved in the synthesis of sucrose were also up-regulatedunder these conditions (6-phosphogluconate dehydrogenase,UDP-D-glucuronate decarboxylase, ADP-glucose pyrophos-phorylase, and starch synthase). Conversely, proteins whosefunction is fructose phosphorylation or dephosphorylation weredown-regulated in sucrose-deprived pollen tubes (fructokinaseand fructose-1,6-bisphosphatase). Several proteins that functionin proteasome and ubiqiutination showed differential regulationT

able

1.continued

flankingAA

gino.

proteinname

plant

species

sequence

with

modificatio

nsno.o

fmodificatio

nsprecursor

ion

PTM

scor-

echarge

state

left

right

gi|30102918

unknow

nprotein

A.thaliana

APE

EDEE

DpSGDED

DDRPP

KR

1pST

1190.95

482

Rgi|332660007

unknow

nprotein

A.thaliana

SVpSSG

NLS

SMDMVEH

K1pST

702.1

602

KR

gi|116782363

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



in response to nutrient deficiency. 20S proteasome α-subunitsC1, G1, and E1 were increased after sucrose limitation, butubiquitin-related proteins (ubiquitin 5, SCF ubiquitin ligase,and SUMO2) were decreased in response to calcium limitation.Especially proteins with functions in cell wall structure, lipidmodification, stress responses, and a large number of proteinswith unknown functions showed the strongest changes inresponse to sucrose deficiency.Tubulin abundance was decreased in response to Ca2+ and

boron. Two RNA-binding proteins (glycine-rich RNA-bindingprotein and nuclear-localized RNA binding protein) increasedin abundance under boron deprivation, and one of these(nuclear-localized RNA binding protein) also increased undercalcium deficiency. The increase of histone H2B protein andlysyl-tRNA synthetase suggests an elevated transcriptional and/or translational activity in pollen tubes in response to boronlimitation. While several of these proteins increased in responseto both limited calcium and boron, a ribosomal protein S15 wasincreased in abundance in response to calcium deficiency butdecreased under boron limitation. A translation elongationfactor-1 and a protein homologous to transcription factor BTF3were decreased in response to calcium, while rRNA processingprotein and poly (A)-binding protein were regulated inresponse to sucrose deficiency. Two 14-3-3 proteins weredecreased in response to both calcium and boron limitation.Kinases and other proteins involved in signaling were stronglyregulated under mineral limitation. In contrast, proteins withfunctions in carbohydrate metabolism mostly did not showresponse to microelement limitation. As expected, two calcium-related proteins (calmodulin and calreticulin) were regulatedunder calcium limitation, while a calcium-sensing receptor wasincreased in response to calcium and sucrose deficiency.Calreticulin is the most potent Ca2+-binding protein; down-regulation of its abundance leads to a decrease of Ca2+ in theER and Golgi-coated vesicles.22 Therefore, we can concludethat extracellular calcium deficiency depletes the intracellularCa2+ stores and redirects the Ca2+ gradient in the pollen tubetip.Profilin, a pollen allergen, plays a pivotal role in the control

of actin polymerization.23 Three members of this protein familywere regulated differently by calcium and boron deficiency. Theabundance of gi|548597 and gi|30841324 increased in responseto calcium limitation, while after boron deficiency, gi|332660220 increased in abundance, and gi|548597 decreased.Profilin is distributed throughout the pollen tube, but the actin-sequestering activity of some profilins is sensitive to Ca2+; thus,the changed Ca2+ concentration can profoundly affect profilinactivity both spatially and temporally. A protein homologous tothe Arabidopsis actin depolymerizing factor (ADF) was down-regulated under sucrose deficiency. ADF binds actin filamentsand regulates the proper balance of actin polymerization anddepolymerization needed for an optimum pollen tube growthprocess; therefore, the decreased amount of ADF in pollen maydisrupt actin organization and induce depolarized growth.

Phosphoproteome Response to Nutrient Limitation

To investigate the effect of various nutrients on proteinphosphorylation in pollen, we analyzed phosphoproteins underdifferent nutrient conditions by gel-free and gel- basedapproaches. The results show that a similar overall pattern ofphosphorylation is observed between calcium and boron-limited pollen. However, there are substantial differencesbetween sucrose and calcium or boron-limited samples,

indicating nutrient-specific phosphorylation (see Figure 4b).Proteins involved in cell signaling form the largest fraction ofthe identified phosphoproteins. A considerable fraction of thesephosphoproteins 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 arebetter understood through this comprehensive phosphopro-teome 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 phosphorylationlevel of two LRR kinases (gi|75186527 and gi|330254250) wasincreased 3-fold by calcium and decreased by borondeprivation. In addition, another RLK gi|332004475 showedincreased phosphorylation in its N terminus following borondeprivation, while phosphorylation remained constant in thesucrose and calcium-deprived pollen. This suggests that theactivity of the RLK is dependent on boron. The phosphor-ylation level of a transmembrane protein kinase was increasedby calcium deficiency. A cytosolic protein kinase gi|332189513showed increased phosphorylation when pollen were deprivedof sucrose, whereas a significant decrease was detected aftercalcium limitation. Another two protein kinases, gi|332642494and gi|332197341 displayed decreased phosphorylation inresponse to calcium and sucrose deficiency. Phosphorylationin the kinase domain of a calcium-dependent protein kinase(CDPK) was strongly decreased in response to Ca2+ limitationand increased by 2-fold following sucrose and borondeprivation. This indicates that Ca2+ signaling differs fromsucrose and boron responses. A transmembrane protein kinasewas exclusively increased by calcium limitation, indicating anutrient-specific response.Six vesicle trafficking related proteins were found to be

differentially phosphorylated after nutrient limitation. Syntaxinand epsin N-terminal homology (ENTH) domain-containingprotein/clathrin assembly protein-related were regulated bysucrose and boron. Dynamin was exclusively regulated bycalcium, while phosphorylation of Golgi snare protein wasfound to be down-regulated by boron alone. Dynamin is aGTP-binding protein and involved in various aspects ofendomembrane and intracellular organelle dynamics, such asendocytosis, Golgi network trafficking, and mitochondrialfusion.24 In pollen, dynamin is required for plasma membranemaintenance during microspore maturation. Moreover, phos-phorylation of dynamin enchances GTPase activity;25 therefore,we conclude that calcium deficiency could regulate GTPaseactivity in growing tubes. A phosphorylation site of anotherGTP-binding protein, ARF GTPase activator, was found to beup-regulated by the limitation of calcium and boron. Incontrast, phosphorylation of vesicle-associated membraneprotein (VAMP) was shown to be decreased after calciumand sucrose limitation. These proteins constitute the majorvesicle-associated component. They are specifically localizedthroughout the secretory pathways including the Golgiapparatus and plasma membrane and play an important rolein vesicle docking. Thus, the differentially regulated phosphor-ylation levels suggest that different nutrients have differenteffects in the regulation of membrane trafficking pathways ingrowing pollen tubes.Our analysis revealed four differentially phosphorylated

proteins involved in cytoskeletal dynamics. All four are actinor microtubule-related proteins, including formin, myosin,kinesin, and microtubule-associated protein. Phosphorylation of



myosin heavy chain and formin was up-regulated in thegrowing tubes after calcium deficiency. Previous studies haverevealed that formin is an actin-binding protein, stimulates actinassembly mainly around the subapical membrane in pollentubes, and controls tip growth by facilitating assembly ofsubapical 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 thegrowing tubes may depend on phosphorylation. Thephosphorylation of microtubule-associated protein and kinesinmotor protein was decreased following sucrose limitation.Because microtubule-associated protein and kinesin motorprotein function in organelle transport, the sucrose limitation-induced down-regulation of phosphorylation levels could affectthe speed of organelle movement in pollen, thus leading to theinhibition of pollen germination and tube growth.Two transport proteins were found to be regulated by the

nutrient deficiency (sucrose transporter and Ca2+-ATPase). It iswell-known that phosphorylation of transporters influences theactivities of ion channels initiating a downstream response. Aphosphorylation site was identified for the sucrose transporterin the N-terminal part of the protein. The abundance of thisphosphopeptide decreased 3-fold in sucrose-limited pollen ascompared to that of control samples. The sucrose transporteractivity is regulated by phosphorylation;26 decreased phosphor-ylation suggests that long time carbon starvation led to anadaptive response and sucrose channel closing. Similarly,phosphorylation of an N-terminal site of the Ca2+ -ATPasedecreased in response to calcium and boron deficiency.Previous studies reported that this region regulates pumpactivities through a calmodulin binding domain.27 Furthermore,as calcium is an important second messenger, decreasedphosphorylation of the Ca2+-ATPase could decrease theintercellular Ca2+ concentration and modulate various signalsand responses. This is consistent with the decreasedphosphorylation of calcium-binding EF hand family proteinfollowing calcium deficiency. In addition, the phosphorylationof camodulin-binding protein, IQ-domain 31, was significantlyregulated by the lack of three different nutrients.The phosphorylation of two lipase-involved proteins was also

affected by nutrient deprivation. Lipase class 3 family proteinwas regulated by sucrose and Ca2+ limitation, whilephospholipase C was regulated exclusively by the lack ofCa2+. Phospholipase C is known to play a role in tip growth notonly by controlling InsP3-gated Ca2+ fluxes but also by alteringthe spectrum of PI lipids in the tube apex.28 The regulatedphospholipase C could impact downstream second messengerssuch as IP3 and diacylglycerol.

■ DISCUSSIONPollen tubes elongate through rapid and polarized cell growthand transport sperms to the female gametophytes forfertilization. Polarized tip growth results from a dynamic actincytoskeleton and highly active membrane trafficking systemthat provide the secretory activities needed for growth.Furthermore, a polarized cytoplasm with vesicles and tip-focused Ca2+ concentration gradients are essential for theprocess of polar cell growth. Nutrients such as sucrose providecarbon for new plasma membrane and cell wall componentsand remodel the cell wall composition. A delicate balancebetween the exocytosis of cell wall components, the cell wallassembly, and the membrane trafficking is important for pollentube growth.29 The precise mechanisms behind this process in

pollen tubes remain speculative. Previously, there was nocomprehensive survey of the proteome or phosphoproteomefollowing nutrient deficiency in a polarized system. Theregulation of nutrient response in pollen tube signaling is ofutmost interest in understanding the metabolic processesinvolved in plant fertility and reproduction. In this study, wehave performed a global analysis of the effects of nutrients onprotein abundance and phosphorylation and identified knownassociations with nutrient pathways as well as proteins thatcontain strongly induced phosphorylation sites.The conifer proteome is far from completely annotated, and

a large number of proteins still have no function assigned tothem. In that respect, studies of tip growth and the analysis ofphosphorylation under different conditions may provide avaluable contribution to the functional categorization of poorlyannotated proteins. For example, two “unknown” proteins (gi|116787106 and gi|148906365) were identified in this study thatshowed strong responses similar to those of sugar transporters.Similarly, the uncharacterized protein kinase gi|42562040 couldbe involved in calcium signaling. The overall picture thatemerges is that sucrose limitation strongly impacted proteinexpression in pollen. Following sucrose deprivation, proteinsassigned to glycolysis, the tricarboxylic acid (TCA) cycle,mitochondrial electron transport and ATP synthesis, and cellwall precursor synthesis were increased. Concurrently, therewas a highly significant induction of proteins assigned to DNA/RNA synthesis or processing and protein degradation.Furthermore, phosphorylation of 26S proteasome regulatorysubunit was increased in response to sucrose and calciumdeficiencies, suggesting that a proteasome-mediated degrada-tion mechanism was induced due to phosphorylation changesin pollen tubes. Also, the phosphorylation of sucrose trans-porter was decreased. This indicates that carbon depletion inpollen leads to an inhibition of nutrient assimilation, biosyn-thesis, and cell wall remodeling, thereby inhibiting pollengermination and tube growth. This resembles the response inother species.30,31

Regulated phosphoproteins were mostly signaling moleculesand vesicle transporters, indicating that nutrient deficiencylargely affected the signaling cascades and membrane/proteintrafficking in the polarized cells. Proteins homologous toArabidopsis RLK were differentially phosphorylated in responseto nutrient starvation. Several receptor like kinases were shownto be critical for anther and pollen development.32 Theextracellular domain of pollen RLK interacts with distinctpollen and stigma factors prior to and after germination.33

Boron starvation-induced N-terminal phosphorylation of RLKsuggests possible interactions in the active state. In addition,our results provide a link between nutrient-induced phosphor-ylation and the regulation of GTPase activity, such as ARFGTPase activator and dynamin. Pollen RLKs are known toassociate with and phosphorylate GTP-binding proteins, thusaltering their nucleotide exchange activity, inducing changes indownstream signaling.34 Overexpression of the phosphorylatedor dephosphorylated form of RLK dissociated with GTP-binding proteins and induced pollen tube depolarization.29 Ourfindings suggest an alternative means of regulation, namely, thatnutrient-dependent multisite phosphorylation of GTP-bindingproteins is autoregulative, leading to a different affinity to itseffectors and a failure to induce pollen tube tip growth. Theformation of SNARE protein complexes is known to beregulated by phosphorylation.35 SNARE protein complexesmediate the recognition and docking of vesicles and cellular



membranes. Taken together, our data demonstrate thatdifferential phosphorylation patterns for different SNAREisoforms could reduce or enhance their interactions and triggerselective modulation of membrane trafficking pathways.An N-terminal phosphorylation site of Ca2+-ATPase was

downregulated in response to Ca2+ deficiency. N-terminalphosphorylation of Ca2+-ATPase is known to be crucial forATPase activity and overrides regulation via calmodulin-binding protein. Elongated pollen tubes exhibit a tip-focusedcytosolic Ca2+ ([Ca2+]c) gradient that is largely maintained byextracellular Ca2+ influx. Mutation of Ca2+-ATPase is maledeficient and shows reduced tube growth rates.36 We canconclude that the reduced phosphorylation would reduce Ca2+

influx in pollen tubes after long time Ca2+ starvation, consistentwith the finding of down-regulated calreticulin. Interestingly,

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 areconsistent with the well-known regulation of CDPK in Ca2+

signaling. Perturbation of membrane-localized CDPK wasreported to result in increased intracellular Ca2+ and abolitionof the tip-focused [Ca2+] gradient.37 The regulated phosphor-ylation suggested that CDPK may participate in Ca2+

homeostasis by phosphorylation of Ca2+-ATPase. As aconsequence, after Ca2+ starvation, pollen could balance thesupply and utilization of calcium with specific Ca2+ signalingpathways. Cytoskeleton-binding proteins play important rolesin cytoskeleton organization to determine plant directional cellgrowth. Phosphorylation of cytoskeleton binding protein isknown to stabilize microfilaments or microtubules and

Figure 5. Functional classifications of differentially regulated proteins. Pie charts of functional categories of all responsive proteins identified from thetotal 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 isperceived by receptors, leading to downstream activation of protein kinases. The activated protein kinases regulate the phosphorylation oftransporters, GTPases, cytoskeleton-related proteins, ubiquitins, and transcription factors. The regulated phosphorylation levels of transporters suchas Ca2+-ATPase are known to control transporter activities, which induce a decrease in cytosolic Ca2+ concentration, and this in turn regulates theactivities 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.



stimulate their assembly.38,39 Cytoskeleton-related proteinsidentified in this study are diverse and respond selectively todifferent signals. The specificities in phosphorylation responsesimply that both microfilaments and microtubules functionschematically with different programs in specific cell regions ororganelles transported during pollen tube growth.In summary, the nontargeted label-free proteomics analysis

that we carried out has expanded our knowledge of pollendevelopment by identifying and quantifying a number ofproteins and protein phosphorylation sites in response tonutrient limitation in P. wilsonii. It was revealed that lack ofnutrients regulated the phosphorylation level of transporters;the regulated phosphorylation of transporters was suggested tocontrol transporter activities, which induces a decrease incytosolic Ca2+ concentration through the regulation of both theinflux of Ca2+ from the extracellular space and the release ofCa2+ from intracellular stores, and this in turn regulates theactivities of protein kinases, affecting the phosphorylation levelof downstream targets. As a consequence, the cytoskeleton isremodeled, and vesicle trafficking is activated, resulting in cellwall reconstruction. Taken together, these pathways lead to thedisruption of tip growth, as summarized in Figure 6. Ourinvestigation provides a general rationale for the role ofnutrients in processes critical to pollen function and develop-ment. Furthermore, we took a first step in including a dynamiccomponent in pollen phosphoproteomics, especially ingymnosperm species, which indicates new candidates possiblyinvolved in the key process of nutrient transport and regulation.More importantly, we found evidence for a new complexnetwork of polarized tip growth.

■ ASSOCIATED CONTENT

*S Supporting Information

Changes in relative phosphorylation of various proteins inresponse 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 tubesdeprived of H3BO4, (C) pollen tubes deprived of calcium, and(D) pollen tubes deprived of sucrose. Complete list of proteinsidentified to be differentially regulated in pollen of P. wilsoniiunder nutrient limitation (Table S1), and statistical analysis ofregulated phosphoproteins (Table S2). This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author

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

Author Contributions∥These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank Dr. Tong Chen (Institute of Botany, ChineseAcademy of Sciences, China) for male cones harvesting. Thiswork was supported by the program for Chinese UniversitiesScientific Fund (2012XJ005).

■ REFERENCES(1) Jin, B.; Zhang, L.; Lu, Y.; Wang, D.; Jiang, X. X.; Zhang, M.;Wang, L. The mechanism of pollination drop withdrawal in Ginkgobiloba L. BMC Plant Biol. 2012, 12, 59.(2) Hepler, P. K.; Vidali, L.; Cheung, A. Y. Polarized cell growth inhigher plants. Annu. Rev. Cell Dev. Biol. 2001, 17, 159−187.(3) Lovy-Wheeler, A.; Cardenas, L.; Kunkel, J. G.; Hepler, P. K.Differential organelle movement on the actin cytoskeleton in lilypollen tubes. Cell Motil. Cytoskeleton 2007, 64 (3), 217−232.(4) Rolland, F.; Baena-Gonzalez, E.; Sheen, J. Sugar sensing andsignaling in plants: Conserved and novel mechanisms. Annu. Rev. PlantBiol. 2006, 57, 675−709.(5) Koch, K. Sucrose metabolism: Regulatory mechanisms andpivotal roles in sugar sensing and plant development. Curr. Opin. PlantBiol. 2004, 7 (3), 235−246.(6) Chen, T.; Wu, X. Q.; Chen, Y. M.; Li, X. J.; Huang, M.; Zheng,M. Z.; Baluska, F.; Samaj, J.; Lin, J. X. Combined proteomic andcytological analysis of Ca2+-calmodulin regulation in Picea meyeripollen tube growth. Plant Physiol. 2009, 149 (2), 1111−1126.(7) Wu, X. Q.; Chen, T.; Zheng, M. Z.; Chen, Y. M.; Teng, N. J.;Samaj, J.; Baluska, F.; Lin, J. X. Integrative proteomic and cytologicalanalysis of the effects of extracellular Ca2+ influx on Pinus bungeanapollen tube development. J. Proteome Res. 2008, 7 (10), 4299−4312.(8) Feijo, J. A.; Costa, S. S.; Prado, A. M.; Becker, J. D.; Certal, A. C.Signalling by tips. Curr. Opin. Plant Biol. 2004, 7 (5), 589−598.(9) Zhao, Y.; Yan, A.; Feijo, J. A.; Furutani, M.; Takenawa, T.;Hwang, I.; Fu, Y.; Yang, Z. Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis andtobacco. Plant Cell 2011, 22 (12), 4031−4044.(10) Justus, C. D.; Anderhag, P.; Goins, J. L.; Lazzaro, M. D.Microtubules and microfilaments coordinate to direct a fountainstreaming pattern in elongating conifer pollen tube tips. Planta 2004,219 (1), 103−109.(11) Chen, Y. M.; Chen, T.; Shen, S. H.; Zheng, M. Z.; Guo, Y. M.;Lin, J. X.; Baluska, F.; Samaj, J. Differential display proteomic analysisof Picea meyeri pollen germination and pollen-tube growth afterinhibition of actin polymerization by latrunculin B. Plant J. 2006, 47(2), 174−195.(12) Wessel, D.; Flugge, U. I. A method for the quantitative recoveryof protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984, 138 (1), 141−143.(13) Bradford, M. M. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 1976, 72, 248−254.(14) de Godoy, L. M.; Olsen, J. V.; de Souza, G. A.; Li, G.;Mortensen, P.; Mann, M. Status of complete proteome analysis bymass spectrometry: SILAC labeled yeast as a model system. GenomeBiol. 2006, 7 (6), R50.(15) Chen, Y.; Hoehenwarter, W.; Weckwerth, W. Comparativeanalysis of phytohormone-responsive phosphoproteins in Arabidopsisthaliana using TiO2-phosphopeptide enrichment and mass accuracyprecursor alignment. Plant J. 2010, 63 (1), 1−17.(16) Hoehenwarter, W.; Larhlimi, A.; Hummel, J.; Egelhofer, V.;Selbig, J.; van Dongen, J. T.; Wienkoop, S.; Weckwerth, W. MAPAdistinguishes genotype-specific variability of highly similar regulatoryprotein isoforms in Potato Tuber. J. Proteome Res. 2011, 10 (7), 2979−2991.(17) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.;Mortensen, P.; Mann, M. Global, in vivo, and site-specificphosphorylation dynamics in signaling networks. Cell 2006, 127 (3),635−648.(18) Hoehenwarter, W.; van Dongen, J. T.; Wienkoop, S.; Steinfath,M.; Hummel, J.; Erban, A.; Sulpice, R.; Regierer, B.; Kopka, J.;Geigenberger, P.; Weckwerth, W. A rapid approach for phenotype-screening and database independent detection of cSNP/proteinpolymorphism using mass accuracy precursor alignment. Proteomics2008, 8 (20), 4214−4225.(19) Lazzaro, M. D.; Cardenas, L.; Bhatt, A. P.; Justus, C. D.; Phillips,M. S.; Holdaway-Clarke, T. L.; Hepler, P. K. Calcium gradients in



http://pubs.acs.org

mailto:[email protected]

conifer pollen tubes; dynamic properties differ from those seen inangiosperms. J. Exp. Bot. 2005, 56 (420), 2619−2628.(20) Lippert, D.; Zhuang, J.; Ralph, S.; Ellis, D. E.; Gilbert, M.;Olafson, R.; Ritland, K.; Ellis, B.; Douglas, C. J.; Bohlmann, J.Proteome analysis of early somatic embryogenesis in Picea glauca.Proteomics 2005, 5 (2), 461−473.(21) Fernando, D. D. Characterization of pollen tube development inPinus strobus (Eastern white pine) through proteomic analysis ofdifferentially expressed proteins. Proteomics 2005, 5 (18), 4917−4926.(22) Nardi, M. C.; Feron, R.; Navazio, L.; Mariani, P.; Pierson, E.;Wolters-Arts, M.; Knuiman, B.; Mariani, C.; Derksen, J. Expressionand localization of calreticulin in tobacco anthers and pollen tubes.Planta 2006, 223 (6), 1263−1271.(23) Valenta, R.; Duchene, M.; Pettenburger, K.; Sillaber, C.; Valent,P.; Bettelheim, P.; Breitenbach, M.; Rumpold, H.; Kraft, D.; Scheiner,O. Identification of profilin as a novel pollen allergen; IgEautoreactivity in sensitized individuals. Science 1991, 253 (5019),557−560.(24) Konopka, C. A.; Backues, S. K.; Bednarek, S. Y. Dynamics ofArabidopsis dynamin-related protein 1C and a clathrin light chain atthe plasma membrane. Plant Cell 2008, 20 (5), 1363−1380.(25) Hinshaw, J. E. Dynamin and its role in membrane fission. Annu.Rev. Cell Dev. Biol. 2000, 16, 483−519.(26) Ransom-Hodgkins, W. D.; Vaughn, M. W.; Bush, D. R. Proteinphosphorylation plays a key role in sucrose-mediated transcriptionalregulation of a phloem-specific proton-sucrose symporter. Planta2003, 217 (3), 483−489.(27) Baekgaard, L.; Luoni, L.; De Michelis, M. I.; Palmgren, M. G.The plant plasma membrane Ca2+ pump ACA8 contains overlappingas well as physically separated autoinhibitory and calmodulin-bindingdomains. J. Biol. Chem. 2006, 281 (2), 1058−1065.(28) Dowd, P. E.; Coursol, S.; Skirpan, A. L.; Kao, T. H.; Gilroy, S.Petunia phospholipase c1 is involved in pollen tube growth. Plant Cell2006, 18 (6), 1438−1453.(29) Cheung, A. Y.; Wu, H. M. Structural and signaling networks forthe polar cell growth machinery in pollen tubes. Annu. Rev. Plant Biol.2008, 59, 547−572.(30) Osuna, D.; Usadel, B.; Morcuende, R.; Gibon, Y.; Blasing, O. E.;Hohne, M.; Gunter, M.; Kamlage, B.; Trethewey, R.; Scheible, W. R.;Stitt, M. Temporal responses of transcripts, enzyme activities andmetabolites after adding sucrose to carbon-deprived Arabidopsisseedlings. Plant J. 2007, 49 (3), 463−491.(31) Devaux, C.; Baldet, P.; Joubes, J.; Dieuaide-Noubhani, M.; Just,D.; Chevalier, C.; Raymond, P. Physiological, biochemical andmolecular analysis of sugar-starvation responses in tomato roots. J.Exp. Bot. 2003, 54 (385), 1143−1151.(32) Wan, J.; Patel, A.; Mathieu, M.; Kim, S. Y.; Xu, D.; Stacey, G. Alectin receptor-like kinase is required for pollen development inArabidopsis. Plant Mol. Biol. 2008, 67 (5), 469−482.(33) Tang, W.; Ezcurra, I.; Muschietti, J.; McCormick, S. A cysteine-rich extracellular protein, LAT52, interacts with the extracellulardomain of the pollen receptor kinase LePRK2. Plant Cell 2002, 14 (9),2277−2287.(34) Wengier, D.; Valsecchi, I.; Cabanas, M. L.; Tang, W. H.;McCormick, S.; Muschietti, J. The receptor kinases LePRK1 andLePRK2 associate in pollen and when expressed in yeast, but dissociatein the presence of style extract. Proc. Natl. Acad. Sci. U.S.A. 2003, 100(11), 6860−6865.(35) Risinger, C.; Bennett, M. K. Differential phosphorylation ofsyntaxin and synaptosome-associated protein of 25 kDa (SNAP-25)isoforms. J. Neurochem. 1999, 72 (2), 614−624.(36) Schiott, M.; Romanowsky, S. M.; Baekgaard, L.; Jakobsen, M.K.; Palmgren, M. G.; Harper, J. F. A plant plasma membrane Ca2+

pump is required for normal pollen tube growth and fertilization. Proc.Natl. Acad. Sci. U.S.A. 2004, 101 (25), 9502−9507.(37) Yoon, G. M.; Dowd, P. E.; Gilroy, S.; McCubbin, A. G. Calcium-dependent protein kinase isoforms in Petunia have distinct functions inpollen tube growth, including regulating polarity. Plant Cell 2006, 18(4), 867−878.

(38) Fauquant, C.; Redeker, V.; Landrieu, I.; Wieruzseski, J. M.;Verdegem, D.; Laprevote, O.; Lippens, G.; Gigant, B.; Knossow, M.Systematic identification of tubulin interacting fragments of themicrotubule-associated protein TAU leads to a highly efficientpromoter of microtubule assembly. J. Biol. Chem. 2011, 286, 33358−33368.(39) Liu, B. Q.; Jin, L.; Zhu, L.; Li, J.; Huang, S.; Yuan, M.Phosphorylation of microtubule-associated protein SB401 fromSolanum berthaultii regulates its effect on microtubules. J. Integr.Plant Biol. 2009, 51 (3), 235−242.



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Proteomic and Phosphoproteomic Analysis of Picea wilsonii