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Extracellular Ca2+ Influx on Pinus bungeana Pollen Tube Development
Xiaoqin Wu, Tong Chen, Maozhong Zheng, Yanmei Chen, Nianjun Teng, Jozef S#amaj, Frantis#ek Balus#ka, and Jinxing Lin J. Proteome Res., 2008, 7 (10), 4299-4312 • DOI: 10.1021/pr800241u • Publication Date (Web): 21 August 2008
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1155 Sixteenth Street N.W., Washington, DC 20036 Integrative Proteomic and Cytological Analysis of the Effects of
Extracellular Ca2+ Influx on Pinus bungeana Pollen Tube
Xiaoqin Wu,†,‡,# Tong Chen,†,§,# Maozhong Zheng,†,§ Yanmei Chen,† Nianjun Teng,†,§
ˇamaj, ,⊥ Frantisˇek Balusˇka, ,¶l and Jinxing Lin*,†
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, Chinese Academy of Sciences, 100093 Beijing, China, Systematic and Evolutionary Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China, Graduate School of Chinese Academy of Sciences, Beijing 100049, China, Department of Plant Cell Biology, Institute of Cellular and Molecular Botany, Rheinische Friedrich-Wilhelms-University Bonn, D-53115 Bonn, Germany, Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, SK-95007 Nitra, Slovak Republic, and Institute of Botany, Slovak Academy of Sciences, SK-84223 Bratislava, Slovak Republic Received April 1, 2008
Ca2+ is an essential ion in the control of pollen germination and tube growth. However, the control ofpollen tube development by Ca2+ signaling and its interactions with cytoskeletal components, energy-providing pathways, and cell-expansion machinery remain elusive. Here, we used nifedipine (Nif) tostudy Ca2+ functions in differential protein expression and other cellular processes in Pinus bungeanapollen tube growth. Proteomics analysis indicated that 50 proteins showed differential expression withvarying doses of Nif. Thirty-four of these were homologous to previously reported proteins and wereclassified into different functional categories closely related to tip-growth machinery. Blocking the L-typeCa2+ channel with Nif in the pollen tube membrane induced several early alterations within a shorttime, including a reduction of extracellular Ca2+ influx and a subsequently dramatic decrease in cytosolicfree Ca2+ concentration ([Ca2+] ), concomitant with ultrastructural abnormalities and changes in the abundance of proteins involved in energy production and signaling. Secondary alterations includedactin filament depolymerization, disrupted patterns of endocytosis/exocytosis, and cell wall remodeling,along with changes in the proteins involved in these processes. These results suggested that extracellularCa2+ influx was necessary for the maintenance of the typical tip-focused [Ca2+] gradient in the P. bungeana pollen tube, and that reduced adenosine triphosphate production (ATP), depolymerizationof the cytoskeleton, and abnormal endocytosis/exocytosis, together with enhanced rigidity of cell walls,were responsible for the growth arrest observed in pollen tubes treated with Nif.
Keywords: calcium • nifedipine • Pinus bungeana • proteomics • pollen tube
conifers differs from that in angiosperms in several parameters,such as growth rate, cytoskeleton organization, and endo/ Pollen tubes represent a robust model system for investigat- exocytosis.6-9 Therefore, it is unlikely that the mechanism of ing tip growth. Basic information regarding angiosperm pollen polarized growth is the same in angiosperm and gymnosperm tube biology has long been known.1,2 It is only recently, species. Previous research has created a foundation for a better however, that major new insights regarding intracellular signal understanding of the biochemical processes of gymnosperm cascades, the cytoskeleton, and endo/exocytosis in angiospermpollen tubes have come to light.3-5 Pollen tube growth in pollen tube development.10-12 Nevertheless, gymnosperm pol-len tube growth is still far from fully understood, particularly * To whom correspondence should be addressed. Prof. Jinxing Lin, Key with regard to the role of cytosolic Ca2+ dynamics which differ Laboratory of Photosynthesis and Molecular Physiology, Institute of Botany, from those in angiosperms.12 Chinese Academy of Sciences, Beijing 100093, China. Tel: 0086-10-62836211.
Fax: 0086-10-62590833. E-mail: [email protected].
The inhibitor nifedipine (Nif), or 1, 4-dihydro-2, 6-dimethyl- † Institute of Botany, Chinese Academy of Sciences.
4-(2-nitrophenyl)-3, 5 pyridine-dicarboxylic acid dimethyl ester, ‡ South China Botanical Garden, Chinese Academy of Sciences.
§ has been used successfully to treat various types of angina and Graduate School of Chinese Academy of Sciences.
# These authors contributed equally to this work.
hypertension.13 There is increasing evidence of a striking effect Rheinische Friedrich-Wilhelms-University Bonn.
of Nif on extracellular Ca2+ influx by blocking voltage-gated ⊥ Institute of Plant Genetics and Biotechnology, Slovak Academy of L-type Ca2+ channels. This makes the inhibitor useful for ¶l Institute of Botany, Slovak Academy of Sciences.
elucidating the biological processes connected with Ca2+ influx 10.1021/pr800241u CCC: $40.75  2008 American Chemical Society Journal of Proteome Research 2008, 7, 4299–4312 4299
Published on Web 08/21/2008 research articles and [Ca2+] gradient.14-16 Using a laser scanning confocal Microinjection of Calcium Green-1 Dextran. Pollen grains
microscope (LSCM) and patch-clamp, Shang et al. reported that were fixed to a coverslip forming the bottom of a microscope a hyperpolarization-activated Ca2+-permeable channel on pol- slide chamber with a thin layer of media supplemented with len protoplast membrane could be suppressed by Nif and 1% agarose (type VII; Sigma). Microinjection was performed [Ca2+] subsequently decreased.17 Moreover, studies on lily on an Axiovert 200 M inverted microscope (Eppendorf Trans- pollen tubes established that Nif treatment altered the tip- ferMan NK2, Germany). A total of 2.5 mM Calcium Green-1 focused [Ca2+] gradient markedly accompanied by irregular dextran (CG-1D, 10 000 MW, Molecular Probes, Inc., Eugene, tube growth, resulting in growth arrest.18,19 The improved Ca2+ OR) in 5 mM HEPES buffer, pH 7.0, was pressure-injected into detection techniques including relevant fluorescence imaging the pollen tube. The pipet tip reached no more than 3 pm into and ion-selective vibrating electrode enabled the detection of the cytoplasm of pollen tubes, and agents were gently loaded extracellular Ca2+ influx and the [Ca2+] gradient in growing into the cytoplasm. Ca2+ dynamics of the injected pollen tubes pollen tubes.2,20 These results demonstrated that a tip-focused was recorded using a LSM 510 META LSCM (Zeiss Co., [Ca2+] gradient regulated the direction of pollen tube growth, Germany) in a time-course mode.
endo/exocytosis in the pollen tube apex, and the organization Protein Extraction and Two Dimensional (2D)-PAGE
of the actin cytoskeleton.19,21-23 However, these studies were Analysis. Pollen tubes at different developmental stages in the
largely focused on angiosperm pollen tubes. Much less is control medium and media with 100 mM Nif and 200 mM Nif known about the precise roles of the tip-focused [Ca2+] in were collected from media, ground in liquid nitrogen and gymnosperm pollen tube growth. Moreover, the mechanisms suspended in 10% (w/v) TCA in acetone with 0.07% (v/v) that maintain the [Ca2+] gradient remain unclear.

-mercaptoethanol at -20 °C for 2 h with interval stirring. The Proteomics is an emerging molecular tool for identifying samples was centrifuged at 17 000g for 15 min at 4 °C, then proteins involved in specific biological responses.10,24 Recent the pellets were washed twice with cold acetone containing studies have indicated that the pollen-specific gene-expression 0.07% (v/v)
-mercaptoethanol and once with 80% acetone, pattern, which is required for normal pollen development, can followed by centrifugation for 15 min at 17 000g. The resulting be altered in response to treatment with inhibitors.11,25 Pro- pellets were lyophilized and then resuspended in lysis buffer, teomics would permit the exploration of pollen tube growth containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.2% (v/v) pH in additional detail. However, no study to date has systemati- 3.5-10.0 Pharmalyte, 1% DTT, 1 mM PMSF. The protein cally linked extracellular Ca2+ influx to the cytoskeleton and extracts were stirred for 5 min at 4 °C, water bathed for 30 min cell wall components in gymnosperms at the proteomic level.
at 30 °C, and then centrifuged at 17 000g for 30 min at 20 °C.
Such studies may be important for the integration of data at Protein concentration was determined by the Bradford method biochemical, physiological, and cellular levels.
with a spectrophotometer (DU 640 Spectrophotometer, Beck- The objective of this investigation was to explore the effects of extracellular Ca2+ influx on pollen tube development in Immobilized pH gradient strips (24 cm, 3-10 L; 11 cm, 4-7 Pinus bungeana by studying the proteomic expression profiles, L) were rehydrated overnight with rehydrated buffer (8 M urea, the cell wall components, and the ultrastructure of pollen tubes.
2% CHAPS, 0.5% IPG buffer, 20 mM DTT, 0.002% BRP) at 20 °C.
The results are compared to published data for angiosperms, Sample load was about 600 µg (24 cm) and 300 µg (11 cm) of and their relevance for tip growth is discussed.
protein, respectively. IFF was conducted at 20 °C in EttanIPGphor system (GE Healthcare Life Sciences). 2D-electro- Materials and Methods
phoresis was performed according to the description in Am-ersham Biosciences' handbook. SDS-PAGE in the second Plant Materials and Culture Conditions. Pollen grains of
dimension was run on Ettan DALTsix (GE Healthcare Life P. bungeana were cultured in the standard media containing Sciences). For each sample analyzed, three replicas of 2-D gels 0.01% H BO , 0.01% CaCl and 15% sucrose. For inhibitors were done to confirm reproducibility. Gels were stained with experiments, Nif was dissolved in the container to 0.01 M stock Coomassie brilliant blue G-250 (CBB). Image analysis was in 0.1% DMSO and then diluted to the appropriate concentra- carried out with the ImageMaster 2-D Platinum version 5.0 tions in the standard media. Pollen cultures in flasks were software (GE Healthcare Life Sciences). The optimized param- incubated on a shaker (100 rpm) at 25 °C in the dark.
eters were set as saliency 2.0, partial threshold 5, and minimum Pollen Tube Growth Determination and Morphological
area 50. All gels from each treatment were matched to each Measurements. The average pollen germination rate and pollen
other and to the other treatments, and spots were assigned tube growth rate were calculated according to Hao et al.11 The arbitrary identifiers. The quantitative comparison of the spots digital images of germinated tubes and the morphology of was based on total spot volume normalization. When the pollen tubes were captured under a Zeiss Q500 IW light percentage of the protein volume from Nif-treated pollen tubes microscope (Zeiss Co., Germany) with a Spot Π camera was up- or down-regulated by 1.5-fold compared to the control (Diagnostic Instruments, Inc.).
in any stage of the culture time, that is, 36 + 1, 36, 60 and 84 h, Measurement of Extracellular Ca2+ Influx. Pollens were
it was regarded as differentially expressed protein. The identi- incubated in the standard media for 72 h and then treated with fied protein spots were manually rechecked.
100, 250 and 500 µM Nif, respectively. Net Ca2+ flux was Mass Spectrometry and Database Search. The digestion of
measured at Xu-Yue Sci. & Tech. Co. Ltd. (www.xuyue.net) proteins was carried out exactly as described previously.10 The using the noninvasive, scanning ion-selective electrode tech- interested spots were manually excised from the gels and cut nique (SIET) as described previously.26 The obtained data by in about 1 mm2 pieces. Gel slices were destained with 50 mM the ion selective probe technique were analyzed by Excel NH HCO in 50% (v/v) methanol for 1 h at 40 °C. The step spreadsheet to convert data from the background -mV estima- was repeated until the color disappeared. Gel particles were tion of concentration and microvolt difference estimation of then mixed with 10 mM DTT in 100 mM NH HCO for 1 h at the local gradient into specific ion influx (pmol cm-2 s-1).
60 °C to reduce the proteins. The gels were dried in a freeze- Journal of Proteome Research • Vol. 7, No. 10, 2008
Calcium Involved in Pollen Tube Growth
research articles vacuum centrifuge for 30 min prior to incubating with 40 mM been cultured for 36 h and then collected after treatment for iodoacetamide in 100 mM NH HCO for 30 min at ambient only 1 h (designed as 36 + 1 h time point here). The fixation of temperature in the dark to alkylate the proteins. The gel pieces pollen tube for electron microscopy was performed after Hao were washed several times with water and completely dried in et al.11 Sections were cut using a Leica ULTRACUT-R ultrami- a vacuum centrifuge. Enzymatic digestion was carried out by crotome (Leica, Germany), stained with 2% uranyl acetate (w/ adding gel pieces into the digestion buffer containing 100 mM v) in 70% methanol (v/v) and 0.5% lead citrate, and then examined with a JEM-1230 electron microscope (JEOL Ltd., µL trypsin. The reaction mixture was kept at 37 °C for 16 h. Digested peptides were extracted by three changes of 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile.
Fourier Transform Infrared (FTIR) Microspectroscopy.
The collected solutions were concentrated to 10 µL and then Pollen tubes were collected after 84 h of incubation, washed desalted with ZipTipC18 (Millipore, Bedford, MA). Peptides with deionized water three times, and then frozen in vapor over were eluted from the column with 2 µL of 0.1% TFA in 50% liquid nitrogen immediately. After that, the samples were dried acetonitrile, loaded into borosilicate nanoflow tip (Micromass, in a layer on a barium fluoride window. Spectra were obtained U.K.), and then analyzed by electrospray ionization quadrupole from the tip regions of pollen tubes with a MAGNA 750 FTIR time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS, spectrometer (Nicolet Corporation, Japan). Spectra were ob- Micromass, Altrincham, U.K.). The instrument was externally tained at a resolution of 8 cm-1, with 128 coadded interfero- calibrated using the fragmentation spectrum of the doubly grams, and normalized to obtain relative absorbance.
charged 1571.68 Da (785.84 m/z) ion of fibrinopeptide B beforeloading the digested peptide samples. Protein identification was performed by searching against Mascot servers (http://www.
Extracellular Ca2+ Flux in P. bungeana Pollen Tubes.
matrixscience.com). The applied spray voltage was 800 V, with Pharmacological treatment demonstrated the inhibition of a sample cone working on 30 V. Dependent on the mass and germination and tube growth by Nif in a dose-dependent charge state of the peptides, the collision energy was varied manner (Supplementary Figure 1). Furthermore, Nif altered from 14 to 40 V. Peptide precursor ions were acquired over normal pollen tube morphology (Supplementary Figure 2).
the m/z range 400-1900 Da in TOF-MS mode. Multiple charged LaCl , another Ca2+ commonly used channel-modulating drug, (2+, 3+) ions rising above predefined threshold intensity were also showed dose-dependent inhibitory effects on the pollen automatically selected for MS/MS analysis, and product ion tube development (Supplementary Figure 3). Using vibrating spectra collected from m/z 50-2000. Tandem MS data were electrode technique, we measured Ca2+ influx at the extreme processed using the MaxEnt 3.0 (Micromass) to create peak apex of growing pollen tubes. Ca2+ influx prevailed in the list file. Database searches were carried out for monoisotopic control tube apex and oscillated over time (n ) 5; Figure 1).
peptide masses using the following parameters: A taxonomy The mean maximal Ca2+ influx at the peak of the oscillations confined to Viridiplantae. Trypsin was specified as the pro- was 104.4 pmol cm-2 s-1 ((3.81, n ) 5). Although the treatment teolytic enzyme, and one missed cleavages was allowed. Fixed with 250 and 500 µM Nif markedly decreased the magnitude modifications are not selected and variable modifications of Ca2+ influx, it did not completely block Ca2+ influx at the including carbamidomethylation of cysteine and oxidation of extreme apex. The mean maximal influxes at the extreme apex methionine are selected for searching. A peptide tolerance of after different concentrations of Nif treatment were 54.5 pmol (1.2 Da for the precursor ions and a MS/MS tolerance of (0.6 cm-2 s-1 ((5.58, n ) 5), 46.3 pmol cm-2 s-1 ((5.58, n ) 5) Da for the fragment ions were set. Peptide charge of +2 and and 33.83 pmol cm-2 s-1 ((9.65, n ) 5), respectively, indicating +3 and monoisotopic mass was chosen, data format was set that the net cytosolic Ca2+ concentration derived from extra- as Micromass (.PKL), and the instrument type was set to ESI- cellular Ca2+ bulk was substantially reduced in a dose-depend- QUAD-TOF. Mascot uses a probability based on "Mowse Score" to evaluate data from MS/MS, which is reported as -10 × Confocal Imaging of [Ca2+]
Change Detected with
Log10 (p) where p is the probability that the observed match Calcium Green-1 Dextran. The control pollen tubes showed a
between the experimental data and the database sequence is strong intracellular fluorescence at the tips of the pollen tubes, a random event. This means that the best match is the one whereas the other regions emitted only faint fluorescence with the highest score. Mowse scores greater than 47 were (Figure 2). Thus, a steep gradient of [Ca2+] was seen from the considered significant (p < 0.05). The apparent pI and molec- tip to the base. This typical tip-focused [Ca2+] appeared ular weight on the original gels as well as the species investi- relatively constant during the first 60 s. After application of Nif gated were also referred during protein identification.
at 75 s, the fluorescence appeared to be significantly weaker F-Actin Staining, FM4-64 Dye Loading and Immunolabel-
over the time in both the extreme apex and in the shank of ing of Pectins. Immunofluorescence labeling of F-actin and
the tube, particularly at 180 and 195 s. Finally, after ap- pectins was performed according to the method described in proximately 195 s, the [Ca2+] gradient was completely dis- detail by Chen et al.27 FM4-64 loading was achieved according sipated and only weak fluorescence was observed in the to Wang et al.9 The samples were observed under LSCM with extreme tip. [Ca2+] gradient dissipation was not observed in excitation at 488 nm for pectins observation and 514 nm for the [Ca2+] dynamics of control pollen tube as long as 195 s actin and FM4-64, respectively. Optical sections were acquired (Supplementary Figure 4).
at 1 or 0.5 µM intervals in the Z-axis. Three dimensional Proteomic Profiles of Germinated Pollen Following Nif
reconstructions were performed with LSM 510 software (Zeiss Treatment. Total proteins were extracted from pollen tubes
Co., Germany).
after 36, 60, and 84 h treatment with two different concentra- Transmission Electron Microscopy. The control, 250 µM Nif
tions of Nif (100 and 250 µM) to investigate the temporal treated pollen tubes cultured for 36 and 84 h were collected characteristics of protein variation. In addition, the proteins from the media. To determine the immediate effects of the in the pollen tubes were also extracted as early as 1 h after inhibitor, pollen tubes were treated with Nif after they have inhibitor application to distinguish the immediate effects of Journal of Proteome Research • Vol. 7, No. 10, 2008 4301

research articles Figure 1. Dose-dependent inhibitory effects of exogenous Nif on extracellular Ca2+ influx at the pollen tube tips. A typical extracellular
Ca2+ influx can be detected in the very tip of control pollen tube, and 100 µM Nif treatment decreased the Ca2+ influx, while 250 and
500 µM Nif treatment greatly reduced this Ca2+ influx.
Because of the lack of complete genome information forconifers and the low levels of some proteins, only 34 proteinsof the 50 analyzed were found to match sequences of proteinsreported previously. Information about candidate proteins withhigh scores of confidence levels was listed in SupplementaryTable 1 and Supporting Information. Of these 34 proteins, 22were found to be highly homologous to proteins in conifersand 12 were found in other plants. Multiple spots appearingin a single gel could often be attributed to the same protein,such as spots 1 and 2, because of post-translational or chemicalmodification. Among the identified proteins, 15 spots showedincreased expression after Nif treatment (Figure 3B), while theother 19 decreased (Figure 3B). Two proteins (spots 13 and 22)were not identified in routine searches against the NationalCenter for Biotechnology Information databases. However, asearch against the Expressed Sequence Tag (EST) databaseresulted in positive matches to known EST sequences. ESTviridiplantae database searching for some proteins with onlyone matched peptide were performed as well (SupportingInformation). This technique has been proven reliable forproteomics studies in organisms lacking sufficient genomeinformation.28 The 34 identified proteins were classified into nine functional categories (Supplementary Figure 6). Because of the limitationsof 2D gel-based separation and CBB staining sensitivity, theproteins were resolved in relatively higher abundance (32%,i.e., proteins related to carbohydrate and energy metabolism).
The biological functions of some proteins (spots 12, 14 and 33),however, were not clearly linked to changes in the [Ca2+]c Figure 2. Time course analysis of [Ca2+] changes upon Nif
gradient in pollen tube growth. In addition, two proteins (spots treatment. Control pollen tubes exhibited a typical tip-focused 15 and 34) identified in this study were difficult to classify based gradient, whereas Nif application (500 µM) rapidly on the available information.
decreased this [Ca2+] gradient within a couple of seconds. Finally Time-Dependent Variations in Protein Expression Pat-
the [Ca2+] gradient was completely dissipated and only weak terns with Varying Doses of Nif. To determine the proteins
fluorescence can be observed in the extreme tip after ap- related to the primary and secondary responses to Nif, we proximately 195 s. Bar )20 µM.
analyzed different patterns of spot variation (Figure 4, Supple-mentary Figure 5 and 7). On time course analysis, 20 spots Nif on the pollen tube. Two-dimensional electrophoresis was changed their intensity in a time-dependent manner. Some performed, and approximately 700 protein spots were detected proteins (spots 3, 4, 6, and 21-24) showed little quantitative (Figure 3). Most of these proteins occurred at pI 4.0-7.5 with change in expression at 36 + 1 and 36 h after inhibitor application, molecular weights ranging from 14 to 97 kDa. Thus, a narrow- while the changes were more pronounced later and maximized range immobilized pH gradient (IPG; pH 4-7) was further used at 60 and 84 h. In contrast, other spots were up-regulated (spots to prevent the spot overlap and crowding (Supplementary 5 and 7-9) or down-regulated (spots 16, 17, 19, 25, 26 and 29) Figure 5). In response to Nif treatment, 50 differentially during the early stage of pollen tube growth with marked expressed proteins were identified by ESI-Q-TOF MS/MS.
quantitative changes. Narrow range IPG 2D maps showed similar Journal of Proteome Research • Vol. 7, No. 10, 2008

Calcium Involved in Pollen Tube Growth
research articles tion of actin filaments (Figure 5). Under LSCM, the actinfilaments in the control pollen tubes were organized into acontiguous network of bundles throughout the tube. Theseactin bundles were mainly parallel to the growth axis andexcluded from the growing tip (Figure 5A,B). The organizationof the actin cytoskeleton was noticeably disrupted by Nif in adose-dependent manner. With a lower concentration of Nif inthe media, the actin cytoskeleton in pollen tubes was slightlyfragmented (Figure 5C). Severe disruption and fragmentationappeared in the presence of 100 and 250 µM Nif (Figure 5D,E).
When the Nif concentration was increased to 500 µM, the actincytoskeleton was completely broken into short actin filaments,which was accompanied by inhibition of pollen germination(Figure 5F).
Time Course of FM4-64 Internalization. FM4-64 uptake into
the control pollen tubes showed distinct time-dependentinternalization (Figure 6A-F). Fluorescence at the plasmamembrane could be discerned as early as 1 min following dyeapplication. Initial dye internalization could be seen as a slightstaining of the apical cytoplasm (Figure 6B,C). This wasfollowed by further internalization of the dye within 20 µM fromthe apex (Figure 6D). After 9-13 min, the typical FM4-64staining pattern was apparent, that is, bright staining of theentire apical region in contrast to the weak staining of otherregions, which corresponded to the so-called clear zone (Figure6E,F). In Nif-treated pollen tubes, the dye internalization alsobegan in the apical region (Figure 6G-L). However, in com-parison to the control pollen tubes, FM4-64 internalization wasweaker at the same time points. Furthermore, a clump-likepattern of fluorescence was distributed throughout the pollentubes later on (Figure 6K).
Ultrastructural Changes in the Pollen Tubes in Re-
sponse to Nif Treatment. Transmission electron microscopy
(TEM) indicated an electron-translucent zone in the very tip
of the control pollen tubes with a subapical region containing
larger organelles and other cytoplasmic inclusions. We did not
find specific accumulation of organelles in the extreme tip
Figure 3. 2-D PAGE pattern of P. bungeana pollen tube proteins
regions (Figure 7A,D,E). The cell walls of the control pollen cultured for 84 h. The proteins were separated by first-dimen- tubes were about 0.2 µM thick at 36 h and 0.5 µM thick at 84 h, sional pH 3-10 linear IPG strips and 12% vertical slab gels in respectively (Figure 7A,D,E). Mitochondria showed intact mem- the second dimension. Some selected regions are enlarged and branes and possessed numerous well-developed cristae (Figure shown in (A). Pollen tube proteins without Nif treatment (B).
8A). Golgi stacks contained 4-6 flattened cisternae with a Fifteen up-regulated proteins (1-15) and 19 down-regulated distinct cis-trans polarity (Figure 8B). The ER was mostly flat proteins (16-34) from 250 µM Nif-treated pollen tubes.
with a large amount of ribosomes densely attached to the alterations in expression of the proteins over time, further surfaces of the membranes (Figure 8C).
confirming the accuracy of the proteomics data (Supplementary In comparison to the control pollen tube wall, Nif treatment Figure 5). We also examined the effects of Nif concentration on for 1 and 36 h did not produce obvious alteration in the the protein expression in P. bungeana pollen subjected to 100 and thickness of the tube wall (Figure 7B,C). However, some 250 µM Nif for 84 h. Variation in expression level of most proteins organelles emerged in the extreme apex of the tubes (Figure was dose-dependent, with slight changes observed with exposure 7B). Furthermore, short-term Nif treatment (1 h) affected to 100 µM Nif (Supplementary Figure 7). These results demon- the organellar ultrastructure. Mitochondria showed slight strated that signaling proteins (i.e., receptor protein kinase and enlargement and marked swelling of cristae (Figure 8D,G). Golgi adenosine kinase), some metabolic and energy producing proteins cisternae were disintegrated and ruptured into vesicular struc- as primary responses, along with proteins involved in amino acid tures (Figures 8E,H). The ER membranes became irregular, and protein synthesis and in folding and RNA binding as second- although the ribosomes were still present on the surface of ER ary responses were up-regulated. In contrast, some other proteins membrane (Figure 8F,I). Thus, ultrastructural abnormalities in involved in metabolism, energy production, and signaling as mitochondria and Golgi stacks occurred at early time points, primary responses as well as cytoskeletal proteins and proteins therefore, representing a primary organelle-specific response involved in cell wall expansion as secondary responses were induced by Nif treatment.
Long-term treatment induced pronounced ultrastructural Nif Inhibited Pollen Tube Development and Disrupted
changes (Figure 8J-L). An increasing number of vacuoles the Actin Cytoskeleton. Inhibition of Ca2+ channels by Nif had
accumulated at the tips of Nif-treated pollen tubes, and a profound effects on pollen tube growth, leading to disorganiza- significant decrease in the thickness of pollen tube walls (0.2 Journal of Proteome Research • Vol. 7, No. 10, 2008 4303

research articles Figure 4. Continued
Journal of Proteome Research • Vol. 7, No. 10, 2008

Calcium Involved in Pollen Tube Growth
research articles Figure 4. Variations in protein spots during pollen tube growth. Proteins were extracted from the control and Nif-treated pollen tubes
cultured for 36, 60 and 84 h. To demonstrate the very short effects of Nif treatment, proteins were also extracted from the pollen tubes
cultured in the standard media for 36 h and subsequently in the media with Nif for 1 h. Both up-regulated (A) and down-regulated
proteins (B) showed time-dependent changes of the differentially expressed proteins. Numbered spots corresponded to Supplementary
Table 1. The histograms showed the relative abundance ratio of protein (% Volume). "+" represented the change in abundance more
than 1.5-fold and less than 2.0-fold after Nif treatement. "++" represented the change in abundance more than 2.0-fold after Nif
treatement.
Journal of Proteome Research • Vol. 7, No. 10, 2008 4305

research articles showing growth suppression by 250 µM Nif (Figure 9D).
Nondestructive FTIR spectroscopy confirmed immunolocal-ization result. The saturated ester peak at 1735 cm-1, and thecarbohydrate peaks between 1200 and 900 cm-1 were de-creased, whereas the carboxylic acids bands at 1604 and 1419cm-1 increased in the differential spectrum (Figure 10).
Nif Induced Rapid Reduction of Extracelluar Ca2+ Influx
and [Ca2+] . Ca2+ is necessary for pollen tube elongation.2
Although there is no doubt that the disruption of cytosolic[Ca2+] leads to cessation of pollen tube extension, the identity of the Ca2+ channels responsible for the extracellular Ca2+influx in pollen tubes is still unclear. Franklin-Tong et al. foundCa2+ influx at both the apex and shank of pollen tubes couldbe inhibited or blocked by Ca2+ channel blockers, includingGd3+ and La3+.20 Our result of noninvasive Ca2+ flux detectioncorresponded well with the previous conclusion. However, themean maximal Ca2+ influx at the peak of the P. bungeanapollen tubes were significantly higher than that in the an-giosperm pollen tubes. Deducing from the thicker cell walls(0.5 µM in P. bungeana in contrast to 0.2 µM in Lily), wespeculated that higher Ca2+ influx was necessary for gymno- Figure 5. Nif treatment resulted in reorganization and disruption
sperm pollen tube wall construction since incorporation of of the actin cytoskeleton in P. bungeana germinating pollen and Ca2+ into the tube wall was necessary for pollen tube growth.29,30 pollen tubes. (A and B) The control pollen cultured for 60 and From the results of noninvasive Ca2+ flux detection and 84 h showing long pollen tubes and normal organization of actin Calcium Green-1 dextran microinjection, we further found that filaments and bundles. (C) Pollen treated with 10 µM Nif for 84 h, the application of Nif led to a rapid reduction of extracellular showing slightly twisted and wavy actin cables. (D) Pollen treated Ca2+ influx, resulting in the dissipation of tip-focused [Ca2+] .
with 100 µM Nif for 84 h. Inhibition of growth is accompanied These observations indicated that the [Ca2+] gradient derives by reorganization of actin cytoskeleton in the form of short and directly from influx of extracellular Ca2+ across the plasma wavy actin filaments. (E) Pollen treated with 250 µM Nif for 84 h.
Actin cytoskeleton is completely broken down to very short membrane. Similar result was indeed reported in the previous filaments and phalloidin-stained aggregates. (F) Pollen treated investigations on angiosperm pollen tubes.31 As alteration in with 500 µM Nif for 84 h. Inhibition of pollen germination was [Ca2+] represented a rapid response to environmental stimuli accompanied by the severe fragmentation of actin filaments in and subsequently trigger downstream cytological changes, we pollen grains. Bar ) 120 µM.
concluded that Nif treatment induced a rapid decrease inextracellular Ca2+ influx as the first primary response and then µM) was observed (Figure 7F,G). Mitochondria showed a gave rise to the reduce in the [Ca2+] .
reduced number of cristae and were disrupted into vacuole- Nif Induced Early Alterations in Organellar Ultra-
like structures to various extents (Figure 8J). The Golgi ap- structure. A significant alteration in organellar ultrastructure
paratus tended to be fragmented, especially the trans-Golgi occurred in most pollen tubes as early as 1 h after application network (TGN), which ruptured into vesicle-like structures of Nif; the changes included swelling and loss of cristae in (Figure 8J-K). The ER was swollen, dilated, and fragmented, mitochondria, swelling and loss of Golgi stacks, and an irregular which led to the formation of irregularly shaped vesicular ER structure. These events represented primary responses, structures. In addition, the ribosomes appeared detached from which was further reinforced by TEM observations of pollen the ER membrane (Figure 8L). These ultrastructural abnormali- tubes after short-term treatment with La3+ (Supplementary ties in the endomembrane system may result in the breakdown Figure 8), indicating the ultrastructural changes were common of secretory pathway activities, and of the protein synthesis and responses to changes in Ca2+ dynamics. These substantial modification system as secondary responses to Nif treatment.
alterations in organellar ultrastructure were similar to those Moreover, the ultrastructural abnormalities caused by La3+ observed in the SI system of the Papaver rhoeas pollen tubes were also similar to that of nifedipine treatment (Supplemental with respect to swelling of the mitochondria and/or rupture of their cristae, suggesting that metabolic activity may be Changes in the Distribution/Configuration of Tube Wall
severely affected at this early time point.32 Nevertheless, further Components. The JIM5 epitope was present along the cell walls
disruption of Golgi stacks and ER together with severe vacu- and at the germinating site in pollen tubes cultured in standard olation of the mitochondria was observed after long-term media but was excluded from the tip. In the Nif-treated pollen treatment of Nif as secondary response to Nif.
tubes; however, the JIM5 fluorescence was distributed all over Proteins Involved in Energy Production. A highly active
the cell walls including the apex of pollen tube, and showed a metabolism is a prerequisite for pollen germination and tip relatively homogeneous staining pattern (Figure 9A,B). The growth of the pollen tube.33,34 In our proteomic analysis, esterified pectin labeled with JIM7 was found in the walls of metabolic enzymes represented the most abundant category the tip region of control pollen tubes, in contrast to the of identified proteins. Six of them were up-regulated proteins distribution pattern of acidic pectin (Figure 9C). Esterified including enoyl-ACP reductase, alcohol dehydrogenase (ADH), pectin was found in the walls of the entire tube in those probable mannitol dehydrogenase, and putative glutamine Journal of Proteome Research • Vol. 7, No. 10, 2008

Calcium Involved in Pollen Tube Growth
research articles Figure 6. FM4-64-uptake time course in growing P. bungeana pollen tubes cultured for 84 h in standard media (A-F) and media containing
250 µM Nif (G-L). (A-E) and (G-K) Confocal fluorescence images of the control and Nif-treated pollen tubes at different times; (F and
L), the corresponding bright field image. In the tip region of the control pollen tube, an extremely high rate of endocytosis and membrane
traffic occurred. FM4-64 internalization process suggested Nif treatment did not inhibit the internalization of FM4-64 dye through
endocytosis, but the dispersed FM4-64 distributing pattern indicated Nif inhibited transportation of secretory vesicles to the tube tip.
Bar ) 25 µM.
Figure 7. TEM observation of P. bungeana pollen tubes cultured in the standard media in the absence or presence of 250 µM Nif. (A)
The control tube (36 h) showing the apical clear zone and distribution of different organelles. Bar ) 1 µM. (B) The Nif-treated (1 h)
pollen tube wall showing no significant alteration in thickness. Bar ) 2 µM. (C) The Nif-treated (36 h) pollen tube wall showing no
significant alteration in thickness. Bar ) 1 µM. (D) The control pollen tube (84 h) showing the apical clear zone and distribution of
different organelles. Bar ) 4 µM. (E) Detail of cell wall structure in tip of control pollen tube in (D). The thickness of the cell wall was
about 0.5 µM. Bar ) 0.5 µM. (F) The Nif-treated (84 h) pollen tube showed accumulation of vacuoles in the swollen tip. Bar ) 8 µM. (G)
Details of cell wall structure in the tip of Nif-treated pollen tube in (F). The cell wall became pronouncedly thinner (0.2 µM) as compared
to the control. Bar ) 0.5 µM.
synthetase. Another five were down-regulated proteins, includ- or intermediate synthesis in TCA cycle, were significantly down- ing the ATP synthase beta subunit, putative nicotinamide regulated in Nif-treated pollen tubes, indicating potential adenine dinucleotide (NAD) malate dehydrogenase, aconitate reduction in both the source and intermediary products hydratase, and enolase. In particular, the expression of enzymes involved in energy production of TCA cycle. ADH is another involved in the tricarboxylic acid (TCA) cycle, such as NAD- up-regulated protein which is abundant in tobacco pollen, but malate dehydrogenase and aconitate hydratase together with its functions in pollen remains unclear.35 We propose that the ATP synthases, decreased as early as 1 h after Nif treatment.
increased expression of ADH in Nif-treated pollen tubes The up-regulation of glutamine synthetase and enoyl-ACP facilitated ethanolic fermentation to generate additional ATP, reductase, both of which are key regulators for the component which may partly compensate for the reduction of ATP in Journal of Proteome Research • Vol. 7, No. 10, 2008 4307
research articles Figure 8. Organellar ultrastructure of P. bungeana pollen tubes cultured in the standard media in the absence or presence of 250 µM
Nif. Mitochondria (A, bar ) 0.5 µM), Golgi apparatus (B, bar ) 0.4 µM), and endoplasmic reticulum (C, bar ) 0.5 µM) in the control tube
(84 h), showing normal ultrastructure; mitochondria in Nif-treated tube, showing membrane swelling and less cristae (D, 1 h; G, 36 h;
bar ) 0.2 µM); the fragmentation of Golgi outer cisternae in Nif-treated tube (E, 1 h; H, 36 h; bar ) 0.2 µM); endoplasmic reticulum in
Nif-treated tube, showing irregular membrane structure. (F, 1 h; I, 36 h; bar ) 0.2 µM); mitochondria (J, bar ) 0.5 µM), Golgi apparatus
(K, bar ) 0.2 µM), and endoplasmic reticulum (L, bar ) 0.5 µM) in Nif-treated control tube (84 h), showing accelerated swelling and
disrupted ultrastructure. Control (A and C) and 250 µM Nif-treated (B and D) pollen tubes cultured for 84 h were labeled with JIM 5 and
JIM7, respectively, and visualized by LSCM.
glycolysis due to the production of NAD+ during conversion High rates of synthesis and delivery of new plasma mem- of acetaldehyde to ethanol by ADH, corresponding well with brane precursors to the apex of the pollen tube are necessary recent results in Pinus strobus using a proteomic approach.36 for tip growth, which is an ATP-consuming process requiring Furthermore, the level of NAD-dependent mannitol dehydro- specific carbon precursor.33 We found that the variation in most genase, which catalyzes mannitol to mannose, unexpectedly of the proteins mentioned above reduced the metabolism increased after treatment with Nif. We speculate that restraint intermediate in TCA and ultimately decreased the synthesis of of the TCA cycle may trigger alternative energy-producing ATP, which was also supported by evidence from TEM for bypasses to meet the enormous demand for energy and enable mitochondria degradation at the early stage. This is consistent FM endocytic uptake into pollen tubes.9,37 with evidence showing that a decline in cellular ATP associated The second group of proteins included five that were down- with glucose depletion and oxidative phosphorylation results regulated. Enzymes involved in oxidative respiration included in disorganization of actin cytoskeleton.38,39 On the basis of two mitochondrial ATP synthases, which would directly affect our results, it is reasonable to speculate that the primary effects the generation of ATP. Enzymes in the TCA cycle, including of Nif on inhibition of ATP generation arrest of pollen tube aconitate hydratase and NAD-malate dehydrogenase, are im- growth. Furthermore, low [Ca2+] also triggers metabolism portant for the regulation of carbon flow rate in the TCA cycle.
bypasses to compensate for the reduction in energy resulting Enolase is a glycolytic enzyme that catalyzes the conversion of from Nif treatment, indicating precise self-regulation in pollen 2-phosphoglycerate to phosphoenol pyruvate.
tube growth.
Journal of Proteome Research • Vol. 7, No. 10, 2008
Calcium Involved in Pollen Tube Growth
research articles Figure 9. Effects of Nif treatment on the distribution of acidic and esterified pectin in P. bungeana pollen tube walls. Bar ) 20 µM.
present time course analysis revealed that Nif treatmentinduced a rapidly increase in expression of protein receptor-like kinases (RLKs) and adenosine kinase (ADK) after 1 and 36 htreatment of Nif. Although the precise links between these RLKsand Ca2+ signaling remains unknown, the depression inreceptor kinase could be considered a direct consequence ofthe blockage of L-type Ca2+ channel by Nif. Adenosine kinasecatalyzes the salvage synthesis of adenine monophosphate fromadenosine and ATP.41 Previous investigation reported thatadenosine kinase deficiency induced developmental abnor-malities and reduced transmethylation.41 By immunolocaliza-tion and biochemical techniques, Pereira et al. also revealedthat deficiency of adenosine kinase activity led to the higherabundance and the altered distribution of low methyl-esterifiedpectin in cell walls of Arabidopsis thaliana.42 Interestingly, inthe present study, we also found the enhanced acid pectin inthe apex pollen tube wall after Nif treatment, indicating thatthere is a direct correlation among Ca2+ influx, ADK expressionand pectin methyl-esterification in pollen tubes.
VCP displaying ATPase activity may modulate protein-protein Figure 10. FTIR spectra from the tip regions of normal pollen
interactions in membrane transport processes of clathrin- tubes, pollen tubes treated with 250 µM Nif, and the FTIR coated pits and vesicles, which would be critical for endocyto- differential spectrum generated by digital subtraction of the sis.43,44 We found that decreased expression of VCP in pollen control spectra from the spectra of Nif-treated tube. The spectra tubes of P. bungeana disturbed the endocytotic process, which revealed that there was less saturated ester pectin and carbo- disrupted the balance of endo/exocytosis and led to the hydrate, but more acid pectin present in the Nif-treated tubes.
accumulation of vesicles and vacuolation at the tips of pollen Signaling Proteins. Of the differentially expressed spots
tubes. Moreover, the disruption of TGN in Nif-treated pollen investigated, however, only five proteins identified were in- tubes, as observed under TEM, further confirmed that the volved in signal transduction: two receptor protein kinases, a endocytic pathway was disturbed, which disrupted the balance valosin-containing protein (VCP) homologue, adenosine kinase of endo/exocytosis and led to the accumulation of vesicles and and hydroxyproline-rich glycoprotein family protein. This was vacuolation at the tips of pollen tubes. Of particular impor- mainly because of the lack of complete pine genome sequence tance, these results suggest that apical endocytosis in P. data, and the temporal and spatial features in the expression bungeana pollen tubes is regulated by the [Ca2+] gradient as of proteins involved in signaling transduction. High levels of reported in angiosperm pollen tubes.45 However, there existed protein kinase activity have been detected in the growing pollen differences in endocytic pattern and growth rate between tubes of maize.40 In P. strobus, protein kinase is expressed only angiosperm and gymnosperm pollen tubes.9,46,47 In our study, in growing pollen tubes rather than in pollen grains.36 The the FM4-64 staining pattern in P. bungeana did not demon- Journal of Proteome Research • Vol. 7, No. 10, 2008 4309
research articles strate the previously described vesicle-rich, V-shaped region nase (UDPGDH), showed significant changes in their expres- as reported in angiosperm species and the cell walls in P. sion with long-term treatment of Nif, that is, 60 and 84 h. RGP bungeana pollen tube tip were much thicker than in an- was reported previously to be localized to the Golgi and to be giosperm.9 On the basis of these differences, we may conclude involved in cell wall polysaccharide biosynthesis through that the slow growth rate of P. bungeana pollen tubes was reversible glycosylation.56 The significant decrease of RGP over largely attributed to the smaller region of secretory vesicles at time indirectly supported the evident decrease in the amount the tip and the higher requirements of materials for tube wall of cell wall polysaccharide in the FTIR spectra. UDPGDH is a key enzyme in the biosynthesis of uronic acids, primarily Hydroxyproline-rich glycoproteins are important compo- D-galacturonic acid residues in the backbone of acidic pectic nents of plant cell wall and play various functions, particularly polysaccharides. In Escherichia coli, the overexpression of serving in cell-cell interactions and communication in plant UDPGDH resulted in a decrease in level of glucuronic acid in reproduction.48 It has been reported that the pistil and pollen the backbone of acidic pectic polysaccharides.57 Therefore, we tube extracellular matrix are full of these highly glycosylated propose that reduced expression of UDPGDH in the Nif-treated proteins, most of which are likely to be contributed by pollen tube would increase the acidic pectin, which forms egg- arabinogalactan proteins (AGPs) and extension-like proteins, carton patterns with Ca2+ through its carboxyl group, further regulating the pistil-pollen recognition and pollen tube growth leading to an increase in rigidity and a decrease in viscoelas- to realize the successful fertilization.48,49 In our study, the ticity of the cell wall.32,58,59 Our immunolabeling and FTIR expression of hydroxyproline-rich glycoproteins increased over analysis further revealed an enhanced acidic pectin synthesis the culture time. After short-term exposure to Nif (1 and 36 h), and a decrease in esterified pectin in the apex wall of Nif- this protein decreased in expression significantly. Therefore, treated tubes, thus, supporting our proteomic analysis. Taken it can be rationally deduced that the inhibition of pollen tube together, these observations indicated that the Nif-induced growth induced by Nif was partially attributed to the biosyn- decrease in expression of proteins involved in cell wall expan- thesis of cell wall components as well as wall properties.
sion and the altered composition of the cell walls led to changes in the chemical and physical properties of the cell walls, and Organization. The directional transport of vesicles containing
consequently to growth inhibition.
cell wall precursors is necessary for the rapid growth of pollen In summary, the present study mainly focused on the tubes and is dependent on the dynamics of cytoskeletal proteomic and cytological variations upon the rapid decrease components.50,51 In addition, the pollen tube cytoskeleton is in the extracellular Ca2+ influx induced by Nif treatment. There involved in controlling cytoplasmic reorganization during tube were three major findings of the present study: (1) extracellular elongation.7 Besides, the reduced ATP production by Nif Ca2+ influx was indispensable for maintenance of the typical treatment would affect the ATP-dependent cytoskeleton de/ tip-focused [Ca2+] gradient in P. bungeana pollen tubes; (2) polymerization and further disturb actin-dependent delivery the rapid decrease of extracellular Ca2+ influx, subsequent of secretory and endocytic vesicles; two down-regulated protein reduction in [Ca2+] , and dissipation of the tip-focused [Ca2+] spots matched the cytoskeletal proteins and would directly gradient, in addition to early alterations in mitochondrial disarrange cytoskeletal dynamics upon Nif treatment. Alpha- ultrastructure and changes in the abundance of signaling tubulin is a structural unit of microtubules, along with beta- proteins, and proteins involved in energy production, appeared tubulin. An earlier study revealed that the stability of the within a short time as primary responses; (3) the primary microtubule cytoskeleton relies on Ca2+ concentration in a response subsequently triggered serial downstream alterations, calmodulin-dependent manner, which is supported by the including actin depolymerization, unbalanced endo/exocytosis, presence of short microtubules, or the complete absence of and cell wall remodeling along with the differential regulation microtubules, in the apical zone.6,52 Thion et al. demonstrated of proteins with roles in the cytoskeleton, cell wall modeling that the plasma membrane voltage-dependent Ca2+-permeable and other functional categories, which further led to the growth channels of carrot cells could be activated by the disruption of arrest of P. bungeana pollen tubes. The combined proteomic microtubules.53 Therefore, we postulate that reciprocal regula- and cytological study provided new insights into the multifac- tion of microtubules and Ca2+ may occur in pollen tube growth eted mechanistic framework for the functions of Ca2+ in the and the decreased expression of alpha-tubulin would definitely polarized tip growth of pollen tubes.
affect the assembly of microtubules, which would account for Abbreviations: CW, cell wall; ER, endoplasmic reticulum; M,
the swollen tips of pollen tubes.
mitochondrion; G, Golgi apparatus; S, starch granule; V, The myosin-like protein, an actin-associated protein that vacuole; L, lipid droplet.
participates in the transport of secretory vesicles by ATP- Acknowledgment. This work was funded by the
dependent sliding along actin microfilaments, was also found National Key Basic Research Program (2007CB108703) from to be down-regulated. The decrease in its expression was MOST, the grants from NSFC (30570100 and 30700040) accompanied by depolymerization of actin microfilaments, together with grants from Deutsche Forschungsgemeinschaft similar to the results reported in Nif-treated cultured rat to J.Sˇ. (DFG, SA 1564/2-1), from EU Research Training lactotrope cells.54 Previous studies indicated that high [Ca2+]c Network TIPNET (project HPRN-CT-2002-00265) obtained in the extreme apex of the tube would invoke fragmentation from Brussels, Belgium;, from Deutsches Zentrum fu of filamentous actin.55 However, low [Ca2+] induced by Nif and Raumfahrt (DLR, Bonn, Germany); and from Grant treatment in pollen tubes observed in the present study would Agency Vega (Grant No. 2031), Bratislava, Slovakia. We are similarly inhibit the polymerization of actin microfilaments, sincerely grateful to Dr. Mathew Benson for his careful indicating that balanced [Ca2+] is necessary for the proper correction of the manuscript.
actin microfilament dynamics.
Two proteins related to cell wall formation, reversibly Supporting Information Available: Supplementary
glycosylated polypeptide (RGP) and UDP-glucose dehydroge- Table 1 shows the proteins from pollen tube proteome identi- Journal of Proteome Research • Vol. 7, No. 10, 2008
Calcium Involved in Pollen Tube Growth
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Journal of Proteome Research • Vol. 7, No. 10, 2008

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