Vacuolar Sorting Receptor (VSR) Proteins Reach the Plasma Membrane in Germinating Pollen Tubes

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1 Molecular Plant Volume 4 Number 5 Pages September 2011 RESEARCH ARTICLE Vacuolar Sorting Receptor (VSR) Proteins Reach the Plasma Membrane in Germinating Pollen Tubes Hao Wang a,b, Xiao-Hong Zhuang a,b, Stefan Hillmer c, David G. Robinson c and Li-Wen Jiang a,b,1 a School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China b State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China c Department of Cell Biology, Heidelberg Institute for Plant Science, University of Heidelberg, Germany ABSTRACT Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP VSR. In contrast, the lack of a prominent PM localization of GFP VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type. Key words: Plasma membrane; pollen tube; vacuolar sorting receptor. INTRODUCTION The polarized and rapidly growing pollen tube is an excellent single cell system for studying protein trafficking and organelle dynamics, vesicle-mediated secretion, and endocytosis in plants (Franklin-Tong, 1999; Hepler et al., 2001; Cheung et al., 2008; Zonia and Munnik, 2008). The fast-growing pollen tube expands its membrane surface at the tip through vigorous and continuous secretion of newly synthesized proteins, cell membrane, and cell wall components. At the same time, the excess secretory membrane is recycled via endocytosis back into the cell for another round of trafficking, this process being essential for maintaining pollen tube growth polarity (Bosch et al., 2005; Cheung and Wu, 2007, 2008; Cai and Cresti, 2009; Liesche et al., 2010). However, much of the molecular machinery of vesicle trafficking and signal communication in the growing pollen tube remains unclear. Multivesicular bodies (MVBs) are spherical endosomal organelles with small internal vesicles (Piper and Katzmann, 2007). In all eukaryotic cells, MVBs mediate the transport of newly synthesized proteins in the secretory pathway destined for vacuole/lysosome or receive internalized cargo from the endocytic pathway for lysosomal/vacuolar degradation. Proteins specifically destined for degradation are sorted into the internal vesicles of the newly forming MVB, which will subsequently fuse with the pre-existing lysosomes, thus allowing the cell to remove transmembrane proteins and excessive membrane (Morales et al., 1999; Foresti et al., 2008; Gruenberg and Stenmark, 2004; Hurley, 2008; Phan et al., 2008). MVBs can also fuse with the plasma membrane (PM), leading to the release of the internal vesicles into the extracellular space. This constitutes a secretion process of the endosomal system (Simons and Raposo, 2009). In mammalian hematopoietic cells, MVBs also participate in removal of PM proteins such as transferrin receptors that are redirected to the PM via 1 To whom correspondence should be addressed. ljiang@cuhk.edu.hk, fax , tel ª The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: /mp/ssr011, Advance Access publication 23 March 2011 Received 3 December 2010; accepted 16 January 2011

2 846 Wang et al. d PM Localization of VSRs in Pollen Tube exosomes (Stoorvogel et al., 2002; Keller et al., 2006; Piper and Katzmann, 2007). It remains elusive whether plant cells also secrete exosome-derived MVBs (An et al., 2007); however, a recent study demonstrated a novel exocytosis pathway mediated by a newly discovered organelle termed Exocyst-positive organelle (EXPO) distinct from the MVBs (Wang et al., 2010b). Vacuolar sorting receptors (VSR) are type I integral membrane family proteins with a single transmembrane domain (TMD) and short cytosolic tail (CT) that are believed to mediate the vacuolar transport of cargo proteins containing vacuolar sorting determinants (VSDs) in the plant secretory pathway (Paris and Neuhaus, 2002; Jiang and Rogers, 2003). Both confocal immunofluorescent and immunogold EM studies have shown that VSR proteins are localized to prevacuolar compartments (PVCs) (Paris et al., 1997; Li et al., 2002), identified as multivesicular bodies (MVBs) in tobacco BY-2 cells (Tse et al., 2004), as well as the trans-golgi network (TGN) in various plant cell types, including tobacco BY-2 and Arabidopsis suspension culture cells, tobacco, and Arabidopsis root tip cells, developing and germinating seeds of mungbean and Arabidopsis (Tse et al., 2004; Miao et al., 2006; Hinz et al., 2007; Wang et al., 2007). By expressing GFP fusion constructs with the TMD/CT of VSRs in transgenic BY-2 or Arabidopsis cells, the TMD and CT have been shown to be essential and sufficient for the correct targeting of VSRs to PVCs in plant cells (Jiang and Rogers, 1998; Tse et al., 2004; Miao et al., 2006). Thus, these GFP VSR fusions have been commonly used as PVC markers for studies of protein trafficking, sub-cellular localization, and organelle dynamics in various plant cell systems (Saint-Jean et al., 2010; Tse et al., 2004; dasilva et al., 2005, 2006; Miao et al., 2006; Lam et al., 2009; Niemes et al., 2010). We have recently extended our studies of VSRs into the single-cell pollen system (Wang et al., 2010a; Wang and Jiang, 2011). Consistent with previous results, VSRs were found in both PVC/MVB and vacuole-like structures in lily pollen tubes prepared by high-pressure freezing/frozen substitution, the vacuolar localization presumably reflecting VSR turnover (Wang et al., 2010a). Interestingly, when transiently expressed in lily pollen tubes, the PVC reporter GFP LIVSR was found distributed throughout the whole germinating pollen tube except at the tip region, where it was enriched with the endocytic marker FM4-64 or GFP SCAMP (Wang et al., 2010a), a secretory carrier membrane protein shown to label an early endosomal compartment and cell plate in tobacco BY-2 cells (Lam et al., 2007, 2008; Cai et al., 2010). A closer look at the dynamics of GFP VSR in growing pollen tube indicated that some of the GFP-tagged vesicles or organelles may reach the plasma membrane (PM) in germinating pollen tubes. Here, we carry out further confocal immunofluorescent and immunogold EM studies with specific VSR antibodies in both lily and tobacco pollen tubes. We confirm that VSRs also localize to PM in lily and tobacco pollen tube samples prepared from both chemically fixed as well as high-pressure freezing/frozen substitution samples. Furthermore, 4-dimentional studies using Spinning Disc Confocal Microscopy on growing tobacco pollen tubes co-expressing RFP AtVSR2 and GFP AtSCAMP4 show that RFP-positive PVC/vesicles fused with the GFP-tagged PM. Taken together, these results demonstrate a PM localization of VSR proteins in the pollen tube. We present a working model on the possible roles of PM-localized VSR proteins in the germinating pollen tube. RESULTS Immunofluorescent Detection of VSR Proteins at the PM in Germinating Lily and Tobacco Pollen Tube We have previously shown that GFP fusions with the lily vacuolar sorting receptor (GFP LIVSR) are found throughout the whole pollen tube except in the tip region (Wang et al., 2010a). In contrast, GFP fusions with the lily secretory carrier membrane protein (SCAMP) are concentrated in the tip region. In addition, in growing pollen tubes expressing GFP LIVSR, numerous GFP-positive vesicles or organelles were frequently seen moving towards and leaving the PM, suggesting that VSR proteins in growing pollen tubes may reach the PM and then be internalized. To test this hypothesis, we first performed immunofluorescent studies with both VSRat-1 and BP- 80 CT antibodies using lily and tobacco germinating pollen tubes. As shown in Figure 1, in chemically fixed germinating lily and tobacco pollen tubes, both antibodies labeled similar punctate structures along the PM of the whole pollen tube (Figure 1A, 1B for lily, and 1E and 1F for tobacco), as well as at the peripheral cytoplasm, possibly representing the PVCs/ MVBs (Tse et al., 2004; Wang et al., 2010a). In addition, in wortmannin-treated lily and tobacco pollen tubes, these VSRlabeled punctate structures enlarged into ring-like structures (Figure 1C, 1D for lily, and 1G and 1H for tobacco, arrows indicate examples of the enlarged PVCs), consistent with their PVC nature as described elsewhere (Tse et al., 2004; Miao et al., 2008; Robinson et al., 2008). SCAMP1-positive structures were also found to be present along the PM, especially at the growing tip, and also in the cytoplasm in both lily and tobacco (Figure 2A and 2D). The concentration of SCAMP1 at the tip was most convincingly demonstrated in a 3-D projection prepared from multiple optical sections (Figure 2B and 2E) from lily and tobacco, respectively. In pollen tubes treated with 10 lg ml 1 brefeldin A (BFA), SCAMP1-positive structures appeared as enlarged aggregates (Figure 2C and 2F). Again, this observation is in agreement with the TGN nature of these structures in response to BFA treatment as previously demonstrated in pollen tubes (Wang et al., 2010a) and tobacco BY-2 cells (Lam et al., 2007). To further compare and possibly distinguish between the VSR- and SCAMP1-labeled compartments in germinating pollen tubes, we performed double-labeling experiments using either VSRat-1 or BP-80 CT antibodies versus SCAMP1 antibody using both lily and tobacco pollen tubes. As shown in Figure 3, VSR and SCAMP antibodies labeled distinct punctate structures along the PM as well as in the cytoplasm

3 Wang et al. d PM Localization of VSRs in Pollen Tube 847 Figure 1. Immunofluorescent Detection of VSR Proteins in Germinating Lily and Tobacco Pollen Tubes. (A, B) Germinated lily pollen tubes were chemically fixed and immunolabeled with VSRat-1 and BP-80 CT antibodies, respectively, followed by confocal imaging. (C, D) Germinated lily pollen tubes were first treated with wortmannin (Wort) at 16.5 lm for 1 h, followed by chemical fixation before labeling with VSRat-1 and BP-80 CT antibodies, respectively, followed by confocal imaging. Arrows indicate examples of wortmannin-induced enlarged PVCs. (E, F) Germinated tobacco pollen tubes were chemically fixed and immunolabeled with VSRat-1 and BP-80 CT antibodies, respectively, followed by confocal imaging. (G, H) Germinated tobacco pollen tubes were first treated with wortmannin (Wort) at 16.5 lm for 1 h, followed by chemical fixation before labeling with VSRat-1 and BP-80 CT antibodies, respectively, followed by confocal imaging. Arrows indicate examples of wortmannin-induced enlarged PVCs. DIC, differential interference contrast. Scale bar = 50 lm in (A D) and 12.5 lm in (E H). (Figure 3A, 3B, and 3D). This result is consistent with PVC localization for the VSR and TGN localization for SCAMP1 (compare Figures 1 and 2). In control labeling experiments using antibodies generated against the cis-golgi marker mannosidase 1 (Man1), the labeled Golgi stacks were largely separated from the SCAMP1-positive PM and TGN structures (Figure 3C for lily and 3E for tobacco). ImmunoEM Reveals the PM Localization of VSR in Pollen Tube To further determine the possible PM localization of VSR proteins in the pollen tube, we next performed immunogold electron microscopy (IEM) on ultra-thin sections prepared from high-pressure freezing/frozen substituted lily pollen tubes. Figure 4A presents an overview ultrathin section of a lily pollen tube, showing good preservation of the organelles and transport vesicles highly enriched in the tip region. In confirmation of our fluorescence studies, both anti- VSRat-1 and anti-bp-80 CT antibodies were observed to label the PM (Figure 4B and 4C). Besides that, the SCAMP1 antibodies also labeled the PM with higher (two to three fold) labeling intensity than that of the VSR labeling (Figure 4D). Based on the virtual lack of background labeling, the immunogold EM labeling was deemed to be highly specific, as previously demonstrated for both SCAMP and VSR antibodies in pollen tubes (Wang et al., 2010a). Taken together, these results indicate that in germinating pollen tubes, VSRs localize to PM and, in comparison to SCAMP1, at a lower labeling density but higher labeling specificity (Wang et al., 2010a). With regard to the labeling of the internal structures, these results are consistent with a PVClocalization of VSR and TGN-localization of SCAMP1 as given in previous studies (Tse et al., 2004; Lam et al., 2007; Wang et al., 2010a). However, the additional localization of VSRs to the PM seems to be a pollen-specific feature, indicating a possible novel function for VSRs at the PM in pollen tubes. Dynamic Fusion of VSR-Carrying Vesicles with the PM To have a better idea about the possible VSR localization to PM and its relationship to the SCAMPs in the same living pollen tube, we next co-expressed RFP AtVSR2 and GFP AtSCAMP4 together in germinating tobacco (Nicotiana tobacum) pollen tubes under the control of the pollen-specific promoter Lat 52 (Figure 5A), followed by confocal 3-D imaging using an ultra-speed spinning disk confocal microscopy (Revolution XD, Andor) (Figure 5B, dashed box indicates the region for collecting time-lapse images shown in Figure 5C). When 4-D imaging with a time-lapse collection of 3-D images was performed for subsequent image analysis, it was obvious that some of the VSR-positive vesicles or PVCs were in the process of fusing with the PM in less than 10 s (Figure 5C, arrowheads indicate an example of such a PVC PM fusion event, where

4 848 Wang et al. d PM Localization of VSRs in Pollen Tube Figure 2. Immunofluorescent Detection of SCAMP Proteins in Germinating Lily and Tobacco Pollen Tubes. Untreated (A, B) or BFA-treated (at 10 lg ml 1 for 1 h) (C) germinated lily pollen tubes were chemically fixed and immunolabeled with SCAMP1 antibodies. (A) and (C) show confocal images collected from a single optical section, while (B) shows a 3-D projection of multiple sections. Untreated (D, E) or BFA-treated (at 10 lg ml 1 for 1 h) (F) germinated tobacco pollen tubes were chemically fixed and immunolabeled with SCAMP1 antibodies. (A) and (C) show confocal images collected from a single optical section, while (B) shows a 3-D projection of multiple sections. DIC, differential interference contrast. Scale bar = 25 lm in (A C) and 12.5 lm in (D F). the inserted boxes are the enlarged images of the indicated PVC PM region), thus providing direct evidence for VSR localization to PM in living pollen tubes. DISCUSSION Endogenous VSRs and various GFP VSR fusions have been shown to co-localize to prevacuolar compartments (PVCs) or multivesicular bodies (MVB) in various plant cell types and germinating seeds (Li et al., 2002; Tse et al., 2004; Miao et al., 2006; Wang et al., 2007; Miao et al., 2008; Niemes et al., 2010). VSRs are also found in trans-golgi network Figure 3. Double-Immunofluorescent Localization of VSR and SCAMP1 in Germinating Lily Pollen Tubes. Germinated lily pollen tubes were fixed and labeled with antibodies of VSRat-1, BP-80 CT, and SCAMP1 (A, B) or SCAMP1 and Man1 (C), followed by confocal imaging. Germinated tobacco pollen tubes were fixed and labeled with antibodies of VSRat-1, BP-80 CT, and SCAMP1 (D, E) or SCAMP1 and Man1 (F), followed by confocal imaging. DIC, differential interference contrast. Scale bar = 50 lm in (A D) or 12.5 lm in (E, F). (TGN), depending on the cell types (Ahmed et al., 1997; Hillmer et al., 2001; Hinz et al., 2007). It is generally believed that VSRs function in sorting soluble vacuolar cargo to vacuoles in plants (Paris and Neuhaus, 2002; Jiang and Rogers, 2003), although their role in post-golgi trafficking has recently been questioned (Niemes et al., 2010). In this study, using both immunofluorescent and IEM with VSR antibodies, we have demonstrated that VSR proteins are also found in the PM (but not at the tip) of germinating pollen tube. Consistent with such an unexpected observation, GFP VSR-tagged PVCs or vesicles were found to fuse with the PM in a living tobacco-growing pollen tube. PM localization of sorting receptors for acid hydrolases is not uncommon in eukaryotic cells. For example, in mammalian cells, in addition to mediating the lysosomal transport of hydrolytic enzymes from the Golgi apparatus, the mannose-6-phosphate receptor also functions in recovery of acid hydrolases from the PM (Braulke and Bonifacino, 2009). Unexpectedly and not previously reported, a large proportion of endogenous VSRs were found to locate to the PM (but not at the tip) in growing pollen tubes (this study). Whether these VSRs actually

5 Wang et al. Figure 4. Immunogold EM Localization of VSR and SCAMP on PM in Lily Pollen Tubes. Ultrathin sections prepared from high-pressure freezing/frozensubstitute pollen samples were labeled with VSRat-1, BP-80 CT, and SCAMP1 antibodies. An overview of pollen tube was shown in (A) and arrows indicate examples of gold particles on the plasma membrane with antibodies of VSRat-1 (B), BP-80 CT (C), and SCAMP1 (D). Scale bar = 5 lm in (A), while, in (B D), it is 500 nm. function in recovery of acid hydrolases at the PM is unclear. However, we must assume that a significant population of VSRs is diverted into secretory vesicles and reaches the PM in growing pollen tubes. Obviously, there is now a need for the identification of their PM-localized cargoes and interaction. Pollen tube growth is well known as one of the fastest growing cell types in the world. During tube elongation, numerous cell materials such as the plasma membrane and cell wall materials required for tube extension are carried by secretion vesicles and transported to the right place. Compared to other types of plant cells, vesicle-mediated secretion and endocytosis in the growing pollen tube is much more active and vigorous (Gao et al., 2008; Li et al., 2008). Transport vesicles are secreted and move via cytoplasmic streaming towards the pollen tip, where they fuse with the PM and then are quickly retrieved back through endocytosis for a new round of transportation. This enables fast membrane expansion to occur while maintaining the polarity of the tube (Taylor and Hepler, 1997; Franklin-Tong, 1999; Hepler et al., 2001; Samaj et al., 2006; Krichevsky et al., 2007; Cheung and Wu, 2008; Kleine-Vehn et al., 2008; Chen et al., 2010). d PM Localization of VSRs in Pollen Tube 849 Figure 5. Fusion of VSR-Positive Organelle with PM in Growing Tobacco Pollen Tube. Germinating tobacco pollen tube co-expressing RFP AtVSR2 and GFP AtSCAMP4, under the control of the pollen-specific promoter Lat 52 (A), was subjected to 3-D confocal imaging using a Spinning Disc Confocal Microscopy (B). (C) Time-lapse collections of 3-D images of region (indicated by dashed box) in (B), arrowheads indicate examples of possible VSR PM fusion in growing pollen tube, where the inserted boxes are the enlarged images of the indicated area. Scale bar in (B, C) is 25 nm. Secretory VSRs (Jiang and Rogers, 2003) and the endocytic SCAMPs (Lam et al., 2007, 2008) are highly dynamic molecules in fast-growing pollen tubes (Wang et al., 2010a). When transiently expressed in lily or tobacco pollen tubes, the GFPtagged VSRs are present throughout the whole of the pollen tube but are absent from the apical inverted cone region. This is in contrast to the SCAMP GFP proteins that are highly enriched at the tip of the tube (Wang et al., 2010a). In addition, in living tobacco pollen tubes transiently expressing RFP AtVSR2, no prominent PM localization of VSR proteins was observed in comparison to the obvious PM localization of SCAMP proteins (Figure 5), a result consistent with previous observations in lily pollen tubes (Wang et al., 2010a). However, immunofluorescent-labeling studies with VSR antibodies in chemically fixed pollen tubes have shown that many of the VSR labelings were found to be present along the PM and in the cytosol (Figure 3). Indeed, IEM studies on ultra-thin sections prepared from high-pressure frozen-freezesubstituted lily and tobacco pollen tubes using VSR and BP80 CT antibodies further confirmed the PM localization of VSR proteins (Figure 4). How can one explain the distinct difference between the patterns of GFP VSR in living pollen tube versus the PM localization of anti-vsr antibodies in fixed pollen tubes? Based on the kiss and run concept of rapid membrane secretion and recycling in neurons as well as in rapidly growing polarized cell types (Stevens and Williams, 2000; Palfrey and Artalejo,

6 850 Wang et al. d PM Localization of VSRs in Pollen Tube 2003), secretory vesicles do not completely integrate with the target membrane, but release their content through a shortlived fusion pore before detaching. Such a mechanism might account for the observation that RFP AtVSR2 is not dominantly visible on the PM of the rapidly growing pollen tube because of the short lifetime of the VSR vesicular fusion event at the PM followed by endocytosis. In fixed samples, however, the rapid sequence of exo- and endocytic events is probably interrupted, artificially leaving some proteins, such as the VSRs stranded at the PM. In conclusion, Figure 6 shows a working model of dynamics and trafficking of VSR proteins in comparison to SCAMP proteins in plant pollen tubes. SCAMPs are highly enriched in the apical region of the pollen tube, which lacks the VSRs. In addition to their previously demonstrated location at the PVC/MVB and vacuole in germinating pollen tubes (Wang et al., 2010a) and PVC/MVB and TGN localization in other plant cell types (Li et al., 2002; Tse et al., 2004; Hinz et al., 2007), VSRs also locate to the PM in rapidly grown germinating pollen tubes via immunofluorescence and IEM studies (this study), suggesting the retrieval of cargo proteins from the PM. Further immunoprecipitation to find the pollen-tube PM VSR interacting proteins will be an essential step to illustrate the functions of VSR reaching the PM. Therefore, in addition to a possible ER Golgi TGN PVC/MVB vacuole transport pathway, VSR could also reach the PM via TGN or PVC/ MVBs exocytic fusion. Similarly, SCAMP could reach the PM from either Golgi or TGN and internalize from the PM via endocytosis. The SCAMP-positive small vesicles enriched in the apical region are believed to be derived directly from the Golgi apparatus/tgn or indirectly via endocytosis from the PM. Both VSR and SCAMP were found to reach the vacuole lumen, presumably for degradation. To find out the possible functional roles of PM-localized VSR proteins in pollen tubes, VSR-interacting proteins (i.e. putative cargo molecules) can be identified from germinating pollen tubes via biochemical approaches such as pull-downs using VSR proteins. Further characterization on the newly identified putative VSR cargo proteins for their VSR cargo interaction (Suen et al., 2010), sub-cellular localization and internalization or endocytosis will illustrate the novel roles of PM-localized VSRs in pollen tubes. METHODS Plant Materials, In Vitro Pollen Tube Germination, and Chemicals Lily (Lilium longiflorum) plants are grown in the greenhouse of the Chinese University of Hong Kong according to standard conditions as previously described (Kim et al., 2007). Tobacco (Nicotiana tabacum) plantsweregrowninagreenhouseat 22 C under a light cycle of 12-h light and 12-h darkness (Fu et al., 2001). Fresh and mature pollen grains were collected from these individual plants and used for bombardment and in vitro Figure 6. VSR Dynamics and Possible Functions in Growing Pollen Tube. Shown is a working model of possible sub-cellular localization, dynamics, and functions of VSR proteins in germinating pollen tube. SCAMP is highly enriched in the PM and apical region of the pollen tube where VSR is missing. In addition to a possible ER Golgi TGN PVC/MVB vacuole transport pathway, VSR/BP-80 could also reach the plasma membrane (PM) likely from the trans-golgi network (TGN) or likely from the multivesicular body (MVB) because VSR was also found in PM in this study. VSR in PM could also internalize to reach TGN/EE for another round of sorting for PM delivery of soluble cargo proteins. pollen tube germination. For Lily pollen germination or tobacco pollen germination, pollen grains were suspended in lily-specific pollen germination medium containing 10% sucrose, 1.3 mm H 3 BO 3, 2.9 mm KNO 3, 9.9 mm CaCl 2,pH5.8, at 27.5 C for 45 min to 1 h or in tobacco-specific pollen germination medium containing 10% sucrose, 0.01% boric acid, 1 mm CaCl 2,1mMCa(NO 3 ) 2.4H 2 O, 1 mm MgSO 4.7H 2 O, ph 6.5 at 27.5 C for 2 3 h. Stock solutions of wortmannin (2.5 mm in DMSO; Sigma-Aldrich) and BFA (1 mm in DMSO; Sigma-Aldrich) were aliquoted and stored at 20 C.

7 Wang et al. d PM Localization of VSRs in Pollen Tube 851 Immunofluorescent Staining Studies The procedures of fixation and preparation of pollen tubes and their subsequent labeling and analysis by epifluorescence and confocal immunofluorescence follow the methods described previously (Jiang and Rogers, 1998; Jiang et al., 2000; Li et al., 2002). The settings for collecting confocal images within the linear range were as described (Jiang and Rogers, 1998). For immuno double labeling, two polyclonal rabbit antibodies were incubated respectively in order at 4 C overnight at 4 mg ml 1 working concentrations for anti- VSR, anti-bp-80 CT, anti-scamp1, and anti-mani. All confocal fluorescence images were collected using an Olympus FluoView FV1000 system. Images were processed using Adobe Photoshop software (San Jose, CA) as previously described (Jiang and Rogers, 1998). Immunogold EM Studies The general procedures for transmission EM sample preparation and thin sectioning of samples were essentially as previously described (Tse et al., 2004; Lam et al., 2007; Wang et al., 2010a). For high-pressure freezing, the 45-min germinating lily pollen tubes were harvested by filtering, followed by being frozen immediately in high-pressure freezing apparatus (EMP2; Leica). Immunogold labeling was carried out using antibodies (VSR, BP-80 CT, and SCAMP1) at 40 lg ml 1 and gold-coupled secondary antibodies at 1:50 dilution. Poststained sections were examined in a Hitachi H-7650 transmission EM with a CCD camera (Hitachi High-Tech) operating at 80 kv. Pollen Particle Bombardment Nicotiana tobaccum flowers were collected and transferred into 20-ml tobacco pollen-specific germination medium. After vortexing to release the pollen grains into the medium, 20 ml of the pollen suspension was vacuum-filtrated onto pre-wetted filter paper. The filter paper covered with the pollen grains was then immediately transferred surface-up onto 2% agar in an 85-mm-diameter Petri dish. For transient expression of reporter proteins in pollen tubes, pollen grains were bombarded with gold particles as previously described (Wang et al., 2010a). Pollen grains were bombarded on filter paper three times at three different positions. Immediately after the bombardment, the pollen grains were then washed down from the filter paper with germination buffer into a 50-ml conical tube. Bombarded tobacco pollen grains were allowed to germinate in a 27.5 C shaker at 85 rpm for 2 4 h before confocal imaging. Dynamic Study of GFP Fusions in Tobacco Pollen Tubes Using Spinning Disk Confocal Microscopy Tobacco pollen tubes co-expressing GFP AtSCAMP4 and RFP AtVSR2 were subjected to image collections using a spinning disk laser confocal microscopy system (Revolution XD, Andor). GFP and RFP were excited by 488 and 561-nm laser, respectively, for image collection using an EMCCD camera (Andor). 3-D images were generated from nine stacks of individual confocal images that were collected from the top to the bottom of the growing pollen tube. Time-lapse images were collected with minimal interval settings. FUNDING This work was supported by grants from the Research Grants Council of Hong Kong (CUHK488707, CUHK465708, CUHK466309, CUHK466610, and HKUST6/CRF/08), CUHK Schemes B and C, UGC/ AoE to L.-W.J., as well as from the German Research Council (Ro 440/14 1) to D.G.R. No conflict of interest declared. REFERENCES Ahmed, S.U., Bar-Peled, M., and Raikhel, N.V. (1997). Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells. Plant Physiol. 114, An, Q., Ehlers, K., Kogel, K.H., van Bel, A.J., and Huckelhoven, R. (2006a). 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