effects of brefeldin a on pollen germination and tube ... · components, cell wall assembly, and...

Effects of Brefeldin A on Pollen Germinationand Tube Growth. Antagonistic Effects onEndocytosis and Secretion1[W]

Qinli Wang, Lingan Kong, Huaiqing Hao, Xiaohua Wang, Jinxing Lin*,Jozef Samaj, and Frantisek Baluska

Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, ChineseAcademy of Sciences, Beijing 100093, China (Q.W., L.K., H.H., X.W., J.L.); Graduate School of the ChineseAcademy of Sciences, Beijing 100049, China (Q.W., X.W.); Institute of Cellular and Molecular Botany,Department of Plant Cell Biology, Rheinische Friedrich-Wilhelms-University Bonn, D–53115 Bonn,Germany (J.S., F.B.); Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences,SK–95007, Nitra, Slovak Republic (J.S); and Institute of Botany, Slovak Academy of Sciences,SK–84223, Bratislava, Slovak Republic (F.B.)

We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 asa membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infraredanalysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dose-dependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showedtypical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from theV-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited inthe presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 mg mL21)treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmissionelectron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased,together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. Anacid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fouriertransform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing inthe presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for theretarded growth of pollen tubes induced by BFA.

The pollen tube is a highly polarized plant cell witha rapidly growing tip that is specialized to delivergenetic material from the site of pollination on theflower stigma to the site of fertilization at the ovule(Hepler et al., 2001). Polarized pollen tube growthresults from continued fusion with the plasma mem-brane by secretory vesicles derived from the Golgiapparatus. This process provides new plasma mem-brane and cell wall components, and remodels the cell

wall composition (Mascarenhas, 1993). The quantity ofmembrane delivered to the cell tip by exocytosis is inexcess of that required for the pollen tube growth rate,suggesting an underlying recycling process (Partonet al., 2001; Camacho and Malho, 2003). Obviously, adelicate balance between the exocytosis of cell wallcomponents, cell wall assembly, and endocytosis isessential for pollen tube growth (Parton et al., 2001).However, the precise mechanisms involved in the reg-ulation of exocytosis, endocytosis, and vesicle recy-cling in growing pollen tubes remain speculative.

Exocytosis is a general term used to denote vesiclefusion at the plasma membrane, and it is the final stepin the secretory pathway that typically begins in theendoplasmic reticulum (ER), passes through the Golgiapparatus, and ends at the outside of the cell (Batteyet al., 1999). Endocytosis is postulated to counterbal-ance membrane secretion (Emans et al., 2002). However,the growing number of plasma membrane proteinsand cell wall molecules, such as pectins and xyloglu-cans, accomplish recycling via endocytosis, followedby exocytosis from secretory endosomes (Baluska et al.,2002, 2005; Samaj et al., 2004, 2005). Growing pollentubes exhibit higher endocytosis and/or exocytosis

1 This work was supported by the National Science Fund of Chinafor Distinguished Young Scholars (30225005), and by grants from theEuropean Union Research Training Network TIPNET (project no.HPRN–CT–2002–00265) obtained from Brussels; from DeutschesZentrum fur Luft- und Raumfahrt (Bonn); and from Grant AgencyVega (grant nos. 2/2011/22 and 2031), Bratislava, Slovakia.

* Corresponding author; e-mail [email protected]; fax 0086–10–62590833.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Jinxing Lin ([email protected]).

[W] The online version of this article contains Web-only data.Article, publication date, and citation information can be found at

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activity in the apical region (Parton et al., 2001, 2003;Camacho and Malho, 2003). Rapid tip growth inangiosperm pollen tubes has been studied extensively(Derksen et al., 1995) and is characterized by a partic-ular type of cytoplasmic organization, i.e. the specificaccumulation of secretory vesicles, clathrin, and clathrin-coated pits at the tube tip (Derksen et al., 2002). How-ever, pollen tubes of gymnosperms differ from thoseof angiosperms in many important characteristics(Derksen et al., 1995; Wang et al., 2003). Gymnospermpollen tubes show natural ramification, and a slow,mostly Brownian-like, movement of the organelles.They have little tip-to-base zonation of large organ-elles. The cytoplasm contains numerous starch grains,and few microtubules and actin filaments are found inthe cortex. Outside the tip, the mitochondrial densityincreases toward the periphery, giving rise to rows ofmitochondria along the tube wall. Unlike in angio-sperms, the Golgi does not show any specific zonationor accumulation in the gymnosperm tube (De Winet al., 1996). In addition, the initiation of germinationand the maintenance of pollen tube elongation in gym-nosperms depend on continuous protein synthesis(Fernando et al., 2001; Hao et al., 2005). These differ-ent physical characteristics may be reflected by anothertype of cytoplasmic organization than that known inangiosperm pollen tubes.

The inhibitor brefeldin A (BFA) is a metabolite pro-duced by fungi that is used for the study of endomem-brane vesicle flow in eukaryotic cells (Rojas et al., 1999).It affects membrane traffic in the animal secretory andendocytic pathways (Lippincott-Schwartz et al., 1991).Increasing evidence documents striking and reversibleeffects of BFA on secretion from both plant Golgiapparatus and endosomes, making it a useful tool infollowing the secretory pathway in plant cells (Baluskaet al., 2002; Samaj et al., 2004). In meristematic rootcells, BFA inhibits exocytosis but allows endocytosis(Baluska et al., 2002, 2005). Using fluorescent markerFM1-43, Emans et al. (2002) reported that BFA stimu-lated temperature-dependent endocytosis in BY-2 sus-pension cells. Moreover, studies on tobacco (Nicotianatabacum) pollen tubes established that BFA blocks thesecretion of cell wall material, resulting in growtharrest (Rutten and Knuiman, 1993). Additionally, BFAinhibits root hair tip growth, accompanied by the

disappearance of the clear zone, depletion of secretoryvesicles, and the simultaneous relocation of actin andmitogen-activated protein kinase (Samaj et al., 2002).However, there is controversy regarding the effects ofBFA on endocytosis (Prydz et al., 1992; Baluska et al.,2002), and no study has systematically compared dif-ferences in the endocytic pathways to changes in thecell wall components in response to BFA treatment.Such findings may be important for bridging existinginformation among the biochemical, physiological, andcellular levels.

The goal of this investigation was to evaluate theeffects of BFA on the endocytosis and the abundanceand distribution of secretory vesicles at the apicalregion during pollen tube development in the gym-nosperm Picea meyeri using several independent meth-ods. In addition, the chemical components of the tubewall and the ultrastructure of the pollen tubes wereanalyzed to gain insight into the structural basis of theobserved effects.

RESULTS

Pollen Germination and Pollen Tube Growth

Much variation was observed in the germinationrate of pollen grains incubated in medium containingvarious concentrations of BFA. Pollen began to germi-nate after 6 h in control culture medium and reachedits maximum germination ratio of 85% after 36 h (TableI; Fig. 1A). Little difference was observed in thegermination rate with the addition of 1 mg mL21 BFAto the culture medium. However, with the addition of5 mg mL21 BFA, the average pollen germination ratewas significantly lower, at 48% (P , 0.05). The pollengrain germination process was severely retardedwhen treated with higher concentrations of BFA (TableI; Fig. 1B).

When cultured in standard medium, pollen tubeswere long, with uniform diameter and a clear zone atthe apical region (Fig. 1C). The addition of BFA led toobvious morphological changes, including changes ingrowth direction (wavy growth pattern), an increase inthe tube diameter, and variable changes in the tube tip,such as more pointed tips or tip swelling (Fig. 1, D–F).

Table I. Dose-dependent effects of BFA on P. meyeri pollen germination and pollen tube growth

The germinated pollen grains were counted after 36-h culture, and only those pollen tubes with their length longer than the diameter of pollen grainwere considered germinated.

Pollen Tube LengthBFA Germination Rate

6 h 12 h 18 h 24 h 30 h 36 h

mg mL21 % mm

0 84.5 6 3.4 45.0 6 1.8 91.5 6 3.6 187.7 6 5.6 262.5 6 8.4 300.9 6 9.1 315.9 6 9.71 83.0 6 3.3 43.0 6 1.9 89.8 6 4.5 185.9 6 5.5 250.2 6 7.9 265.7 6 9.0 265.2 6 9.53 68.1 6 2.7 39.5 6 1.6 85.3 6 4.3 151.3 6 5.5 175.0 6 7.3 169.2 6 8.4 165.1 6 8.25 48.3 6 1.9 39.2 6 1.8 78.5 6 3.8 80.5 6 3.6 82.3 6 4.0 83.0 6 4.1 84.8 6 4.28 8.2 6 0.4 38.1 6 1.8 39.5 6 1.8 45.2 6 2.2 43.7 6 2.1 49.0 6 2.4 48.0 6 2.3

Brefeldin A Stimulates Endocytosis and Inhibits Secretion

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With increasing incubation durations, the BFA-treatedpollen tubes ruptured, indicating that they were morefragile. Treatment with 1 mg mL21 BFA decreased thepollen tube growth rate to 7.8 mm h21. When the BFAconcentration was increased to 5 mg mL21, the averagepollen tube growth rate dropped to 2.5 mm h21, incomparison with 16.2 mm h21 observed in the controlduring 30 h of growth after germination. With in-creasing BFA concentrations, the inhibitory effect onpollen tube growth was more pronounced (Table I).Most of the pollen grains cultured in the presenceof 8 mg mL21 BFA did not germinate. Although thepollen grains were observed to have short protrusions,

they cannot be considered as germinated since theirlength was shorter than the pollen grain diameter(Fig. 1F). Thus, BFA inhibited pollen tube growth ina dose-dependent manner. To determine whetherthe effects of BFA were reversible, pollen grains werefirst cultured in media supplemented with 5 mg mL21

or 8 mg mL21 BFA for 18 h, and were then trans-ferred into BFA-free medium. Our results showedthat for samples previously treated with 5 mg mL21

or 8 mg mL21 BFA, not only did germination rateincrease but also pollen tubes continued to elongatewhen transferred into BFA-free medium (Supplemen-tal Fig. 1).

Figure 1. Effects of BFA on pollengermination and pollen tube morphol-ogy. A, Healthy P. meyeri pollen tubescultured in standard medium for 36 h,showing good germination and manylong pollen tubes with normal shape.B, P. meyeri pollen tubes cultured inmedium containing 5 mg mL21 BFA for36 h, showing poor germination anda few short tubes with morphologicalabnormalities. C, Micrograph of a con-trol P. meyeri pollen tube cultured for20 h, showing a regularly shapedpollen tube of constant diameter anda clear zone at apical region. D to F,Typical examples of pollen tubes inthe presence of BFA for 20 h. D, Pollentube incubated in 1 mgmL21 BFA; starsindicate changes in growth direction(wavy pattern). E, Pollen tube incu-bated in 5 mg mL21 BFA, showinga swelled tube tip (arrowhead). F,Most of the pollen grains cultured in8 mg mL21 BFA, showing aberrantprotrusions, which were not regardedas germinated. G, Details of cell wallstructure in control pollen tube. Thecell wall was uniform, compact, and0.4 mm thick. H, Details of cell wallstructure in BFA-treated pollen tube.The cell wall became more looselypacked, showing the cell wall thick-ness from 0.35 to 0.6 mm in the pres-ence of BFA. cz, Clear zone; CW,cell wall. Bars in A and B 5 100 mm;in C to F 5 50 mm; and in G andH 5 0.3 mm.

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FM4-64 Staining Distribution Pattern

In median confocal optical sections (Fig. 2A), FM4-64consistently produced a distinct peripheral and brightapical staining, which was distributed in the extremeapical (15–20 mm) region in normally growing P. meyeripollen tubes. The corresponding quantitative imagerevealed a model of a sharply defined high signal in theextreme apex; however, beyond this region, FM4-64staining was generally and significantly weakened(Fig. 2B). Peripheral staining was shown to be plasmamembrane, and not associated with the cell wall, asshown by plasmolysis with 100 mM sorbitol in thepresence of FM4-64. Pretreatment of pollen tubes with500 mM sodium azide impaired dye uptake: FM4-64fluorescence could only be observed at plasma mem-brane, but the dye was not internalized over time(Supplemental Fig. 2).

For pollen tubes cultured in medium containing5 mg mL21 BFA, the FM4-64 membrane-staining pat-tern at the apical region was disrupted. Extreme apicalstaining was more diffuse and less well localized, nolonger exhibiting the bright region at the apical clearzone that is characteristic of normally growing pollentubes (Fig. 2C). The corresponding quantitative imageshowed a broader distribution of FM4-64 staining (Fig.2D). Furthermore, a typical FM4-64 staining pattern ina normal pollen tube could be rapidly dispersed byshort-term treatment with BFA (Fig. 3). Within 10 to15 min, apical FM4-64 staining was largely redistrib-uted from the apex and became continuously scatteredover more subapical regions (Fig. 3, B and C). Ulti-mately, the apical bright FM4-64 staining patterndisappeared with increasing time of exposure to BFA(Fig. 3D).

The Time Course of FM4-64 Internalization

The uptake of FM4-64 into P. meyeri pollen tubesfollowed a strict time sequence (Fig. 4, A–F). Stainingassociated with the plasma membrane became obvi-ous immediately after dye application to the germi-nation medium (Fig. 4A). This was followed by

internalization of the dye, mainly at the apical region(Fig. 4, C and D). Within several minutes, the typicalstaining pattern of FM4-64 dye was observed, i.e.bright staining of the entire apical region that extendedto the subapical region with very weak staining (Fig. 4,E and F), rather than the inverted cone shape typicallyobserved in angiosperms. The apical fluorescent re-gion corresponds to the so-called clear zone, a regionfilled with secretory vesicles but lacking any larger or-ganelles.

In BFA-treated pollen tubes, the FM4-64 internali-zation continued even more rapidly, and the dyeinternalization occurred throughout almost the wholepollen tube (Fig. 5, A–F). However, the bright apicalFM4-64 staining pattern did not occur. With increasingtime after FM4-64 dye application, only patch-like anddispersed fluorescence of stained material emergedover most of the pollen tube (Fig. 5F). To quantify theeffect of BFA on FM4-64 dye internalization, pollentubes cultured for 18 h in control medium werepretreated with BFA, latrunculin B, or sodium azidefor 60 min, and, subsequently, FM4-64 was internal-ized for 12 min. The images in Figure 6 (A–H) showthat the FM4-64 staining pattern was disrupted bytreatment with BFA (Fig. 6C), latrunculin B (Fig. 6E),and sodium azide (Fig. 6G) compared to the control(Fig. 6A). Moreover, quantitative data suggest thatBFA stimulated the total uptake of FM4-64 dye (Fig.6D). In contrast, latrunculin B inhibited the internali-zation of FM4-64 dye (Fig. 6F), and sodium azideblocked FM4-64 internalization almost completely(Fig. 6H) compared to the control (Fig. 6B). This fluo-rimetric quantification showed that FM4-64 uptakewas stimulated about 2-fold by BFA and inhibitedabout 0.48-fold by latrunculin B, while almost nodye was internalized after the sodium azide treatment(Fig. 7).

Ultrastructural Changes in Organelles and theProduction of Secretory Vesicles

A zone filled mainly with secretory vesicles (clearzone) could be distinguished from the streaming

Figure 2. Confocal images of FM4-64staining in pollen tubes of P. meyeri.A, A median focal plane confocaloptical section, showing a typicalFM4-64 staining pattern in a growingpollen tube; the bright field at one-third size appears as an insert. B, Pixelvalues along a central transect throughthe fluorescence image in A. C, Amedian focal plane confocal opticalsection, showing a dispersed and dis-rupted FM4-64 staining pattern in BFA-treated (5 mg mL21) pollen tube; thebright field at one-third size appears asan insert. D, Pixel values along a cen-tral transect through the fluorescenceimage in C.





cytoplasm at the very tip of pollen tubes growing incontrol medium. In the zones behind the very tip,many larger organelles such as Golgi stacks, ER, andmitochondria, as well as many small round-shapedvacuoles, were well resolved (Fig. 8A). Some of thesecretory vesicles fusing with plasma membrane, andparamural bodies (PBs), probably resulting from fu-sion of multivesicular bodies (MVBs) with the plasmamembrane, were observed at higher magnification(Fig. 8, C and E). Most Golgi stacks in the control pollentubes consisted of four to seven flattened cisternaewith a distinct cis-to-trans polarity, and numerous se-cretory vesicles were present around them (Fig. 8F).

Much variation was observed when pollen grainswere incubated in the medium containing BFA. Treat-ment with 5 mg mL21 BFA drastically decreased thenumber of secretory vesicles and increased the numberof small vacuoles at pollen tube tips, and other largerorganelles such as mitochondria and lipid bodies couldbe observed at the very apical region (Fig. 8, B and D).Upon BFA treatment, Golgi stacks disassembled ortended to be stacked and curved at the trans face, whilethe number of secretory vesicles surrounding themdrastically decreased (Fig. 8, G and H). The ER becameswollen with the detachment of ribosomes from therough ER (Fig. 8G). However, no significant differenceswere observed between BFA-treated and normal pollentubes in terms of mitochondria (data not shown). Inaddition, a more loosely packed cell wall, 0.35 to 0.6mmthick, was produced (Fig. 1H) in the treated than in thecontrol pollen tubes, which showed a uniform cell wallthickness of 0.4 mm (Fig. 1G).

Activity of Secreted Acid Phosphatase

The effects of BFA on the acid phosphatase (acPase)activity are plotted in Figure 9 and show the average ofthree experiments. The acPase activity increased as thewashed pollen grains began to germinate and pollentubes elongated in the germination medium (Fig. 9, A,

a, B, a, and C, a). There were almost parallel increasesin acPase activity and pollen tube length over time(Fig. 9, A, a, and C, a). When 5 mg mL21 BFA wasadded to the medium after 12 h of incubation, theexport of acPase activity (Fig. 9A, b) was immediatelyinhibited, along with the inhibition of tube elongation(Fig. 9C, b) and pollen germination (Fig. 9B, b). Pollentube growth and secretion of active acPase werealmost completely inhibited over time with BFA treat-ment (Fig. 9, A, b, and C, b), whereas pollen germina-tion was less affected (Fig. 9B, b).

Changes in Chemical Components

Fourier transform infrared (FTIR) spectra were ana-lyzed in apical, middle, and basal regions of pollentubes. FTIR spectra of pollen tubes growing in controlmedium showed similar patterns among the three re-gions analyzed, although the proportion of proteins topolysaccharides varied among different regions withinan individual pollen tube. The absorption bands oc-curred at 1,650 cm21 and 1,550 cm21, corresponding toamide I and amide II of proteins (McCann et al., 1994),while a peak at 1,744 cm21 corresponded to saturatedesters (McCann et al., 1994), and polysaccharides absor-bed at 1,200 to 900 cm21 (McCann et al., 1994; Fig. 10A).

Treatment with 5 mg mL21 BFA induced displace-ment of the peaks or changes in absorbance. Thedifference spectra generated by digital subtraction ofthe spectra of control tube walls from the spectra ofBFA-treated tube walls (Fig. 10B) showed that both theamide-stretching bands and the carbohydrate bands

Figure 3. Time course of disruption in a normal typical FM4-64staining induced by 5 mg mL21 BFA. BFA was applied directly togrowing pollen tubes on thin gel layers in 70 mL of 115% to 120%liquid medium. A, A typical FM4-64 distribution pattern in a normalgrowing pollen tube of P. meyeri. B, The changes of FM4-64 staining at3 min after the addition of BFA, showing the FM4-64 fluorescencetended to be scattered. C and D, The changed FM4-64 stainingdistribution with increasing time, showing FM4-64 fluorescence be-came more dispersed and almost distributed in the whole pollen tubeafter 12 min of BFA treatment. Bar 5 25 mm.

Figure 4. FM4-64-uptake time course in a growing P. meyeri pollentube. A to F, Median confocal fluorescence images at increasing timesafter addition of FM4-64 (2 mM in 115% standard medium). To avoidosmotic perturbation, pollen tubes were pretreated with 115% mediumbefore dye application. The rapid uptake suggests an extremely high rateof endocytosis and membrane traffic; the bright region suggests there isan accumulation of secretory vesicles in the apical zone. Bar 5 25 mm.

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were dramatically reduced after BFA treatment in allthree regions analyzed. In contrast to a small reductionin protein content, there was a marked decrease inpolysaccharide content among the three regions. Fur-ther, the reduction of polysaccharides in the newlyformed tip region was far more obvious than in thealready formed middle and basal regions.

DISCUSSION

BFA Retarded Pollen Tube Development

The fungal metabolite BFA, which has been shownto interfere with protein transfer through the endo-membrane system, is an inhibitor of the secretorypathway of intracellular protein transfer and mainlyinduces dysfunction of the Golgi stacks (Nebenfuhret al., 2002). Lanubile et al. (1997) reported that BFAaffects the synthesis and transport of cell wall poly-saccharides and proteins in pea (Pisum sativum) rootseedlings. Additionally, the comparison of FM1-43 up-take in normal and BFA-treated tobacco suspensioncells showed that BFA can stimulate endocytosis (Emanset al., 2002). However, the reliability of FM1-43 as anendocytic tracer for plant cells has not been confirmed(Meckel et al., 2004; Samaj et al., 2005).

Little information about the effects of BFA on thesecretory pathway during pollen germination andpollen tube growth has been reported. Rutten andKnuiman (1993) reported that the secretion and pollentube growth were two separate processes. In this study,BFA not only reduced pollen germination rates (Table I;Fig. 1B) and retarded pollen tube growth (Table I), butalso resulted in abnormalities in pollen tube shape (Fig.1, D–F). The recovery experiments indicated that theeffects of BFA were reversible, clearly documenting the

concentrations of BFA used in this study are withina physiological range (Supplemental Fig. 1). The resultspresented here indicated that the pollen tube growthwas closely linked to the cellular process of the secre-tory pathway, the disruption of which led to the pollentube growth arrest. However, it should be noted that,for P. meyeri, only concentrations of BFA greater than1mg mL21 exerted a significant effect. This concentrationis much higher than that reported for angiosperms, e.g.0.1 mg mL21 of BFA in the pollen tubes of tobacco(Rutten and Knuiman, 1993). This large discrepancymay be due to differences in the plant species examined,and may also reflect alterations in the activity of the Golgiapparatus.

BFA Disrupted Secretory Vesicle Accumulation at thePollen Tube Apex

Regulated secretory vesicle delivery, distribution,vesicle fusion, and rapid membrane recycling can betracked in living cells using FM4-64 dye because thisdye is nontoxic and water soluble (Samaj et al., 2005).For studies of plant and fungal cells, FM4-64 is usuallypreferred to FM1-43 because of its superior brightness,greater contrast, and higher photostability, and, moreimportantly, unlike FM1-43, the dye is not retained incell walls (Bolte et al., 2004). Because the dye stainsmembranes in an activity-dependent manner, it hasbeen found increasingly useful in exploring the endo-cytosis and secretory mechanisms in a variety ofbiological models (Smith and Betz, 1996). The presentexperiment of plasmolysis with sorbitol and the effectof sodium azide on FM4-64 uptake (Supplemental Fig.2) confirmed earlier reports that FM4-64 staining wasplasma membrane rather than cell wall associated,and its internalization occurred via an endocyticmechanism instead of passive diffusion (Parton et al.,2001, 2003; Camacho and Malho, 2003). The medianconfocal optical images of FM4-64 fluorescence innormally growing P. meyeri tubes consistently revealedbright peripheral and apical staining (Fig. 2A). TheFM4-64 staining pattern detected in P. meyeri wasdifferent from the V-shaped apical staining reportedin angiosperm species (Parton et al., 2001, 2003). Inaddition to the staining pattern, the proportion of theapical region stained by FM4-64 to the whole pollentube in P. meyeri was much lower than that in lily(Lilium longiflorum) pollen tubes. In P. meyeri, the ratioof the length of the FM4-64-labeled apex region (15–20 mm) to the pollen tube diameter (35–40 mm) wasabout 1:2 in comparison with the corresponding param-eter (1:1) in lily pollen tubes, in which both the lengthof the FM4-64 staining region and the pollen tubediameter were about 15 to 20 mm (Parton et al., 2001,2003). Because FM4-64 staining corresponds strikinglyto the location and distribution of secretory vesicles atthe apical region (Lancelle and Hepler, 1992; Derksenet al., 1995), we may conclude that the slow growthrate of P. meyeri pollen tubes was largely caused by thesmaller region of secretory vesicles at the tip.

Figure 5. Time course of changes in dye distribution of FM4-64-loadedP. meyeri pollen tubes treated with 5 mg mL21 BFA. A to F, Confocalfluorescence images of a BFA-treated pollen tube at different times,showing BFA treatment did not inhibit the internalization of FM4-64dye through endocytosis but the dispersed FM4-64 distributing patternindicating BFA inhibited secretory vesicle accumulation at tube tip.Bar 5 25 mm.





Previous studies have established that treatmentwith BFA can result in a particular reorganization ofthe cytoplasm at the pollen tube apex (Parton et al.,2003). Our experiment with FM4-64 indicated that thetypical bright FM4-64 staining pattern that appeared atthe apical region in normal pollen tubes is lost in BFA-treated pollen tubes, and is replaced by a scattered anddispersed distribution of the dye (Fig. 2, C and D). Thisimplies that the distribution and number of secretoryvesicles at the tip region varied after BFA treatment.The time course of dissociation and dissipation ofa normal apical FM4-64 staining pattern after theaddition of BFA in P. meyeri revealed a dynamic pro-cess; BFA can rapidly disperse and reduce the abun-dance of secretory vesicles located in the tube apex(Fig. 3). The decreased number and dispersed distri-bution of secretory vesicles in the apical region in-dicated that vesicle secretion became less active afterBFA treatment, which likely resulted from the effect ofBFA on the production of secretory vesicles and/orvesicle delivery to the apical region. Vesicle trafficking

in plants is dependent on the actin cytoskeleton, andprevious reports have shown that BFA affects bothactin organization and actin-dependent endosomalmotility within the growing tips of root hairs (Samajet al., 2002; Voigt et al., 2005). The phenotype of wavypollen tubes with increased diameter produced uponBFA treatment (Fig. 1, D–F) that was observed in ourexperiment was possibly caused by the improperdelivery to and depletion of secretory vesicles fromthe clear zone of the pollen tubes. Nevertheless, we didnot detect BFA-induced vesicular aggregation, as wasfound in BFA-treated lily pollen tubes (Parton et al.,2003), probably because of the different type of cyto-plasmic streaming found in conifer and angiospermpollen tubes (De Win et al., 1996; Lazzaro et al., 2003).

BFA Stimulated Endocytosis

There have been reports of endocytosis associatedwith tip growth in the development of pollen tubes,root hairs, and fungal hyphae (Derksen et al., 2002;

Figure 6. Confocal images showing uptake of 2 mM FM4-64 into control and inhibitor-treated pollen tubes of P. meyeri within12 min. All the pixel values did not include the peripheral region. A, A median focal plane confocal optical section of a controlpollen tube; the bright field at one-third size appears as an insert. B, Pixel values along a central transect through the fluorescenceimage in A. C, A median focal plane confocal optical section of a pollen tube pretreated with 5 mg mL21 BFA for 60 min; thebright field at one-third size appears as an insert. D, Pixel values along a central transect through the fluorescence image in C,showing that BFA stimulated the internalization of FM4-64 dye. E, A median focal plane confocal optical section of a pollen tubepretreated with 1 mM latrunculin B for 60 min; the bright field at one-third size appears as an insert. F, Pixel values along a centraltransect through the fluorescence image in E, showing latrunculin B inhibited the internalization of FM4-64 dye. G, A medianfocal plane confocal optical section of a pollen tube pretreated with 500 mM sodium azide for 60 min; the bright field at one-thirdsize appears as an insert. H, Pixel values along a central transect through the fluorescence image in G, showing sodium azideblocked internalization of FM4-64 dye almost entirely. Bar 5 25 mm.

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Read and Kalkman, 2003; Voigt et al., 2005). Studies onyeast (Saccharomyces cerevisiae), tobacco BY-2 cells, andlily pollen tubes have indicated that FM dyes weretaken up by the endocytic pathway (Parton et al., 2001;Emans et al., 2002). In this study, we found that FM4-64dye was internalized to induce a typical bright stain-ing pattern at the apical region (Fig. 4) within 30 min,indicating that endocytosis occurred during normalpollen tube growth in P. meyeri, similar to reports fromother species (Parton et al., 2001; Camacho and Malho,2003). In BFA-treated pollen tubes, FM4-64 dye uptakewas more rapid, probably because the dye internali-zation occurred at broader range, in spite of dispersedstaining (Fig. 5), implying that BFA treatment stimu-lated endocytosis in contrast to untreated pollen tubes.Furthermore, latrunculin B and sodium azide inhibi-ted and blocked FM4-64 dye internalization, respec-tively, while BFA obviously stimulated endocytosis(Fig. 6, A–H). This conclusion was also strongly sup-ported by a fluorimetric quantitative analysis (Fig. 7).Because endocytosis is postulated to counterbalancemembrane secretion (Emans et al., 2002), we concludethat decreased tube growth is likely a direct conse-quence of increased endocytosis and inhibited secre-tion from both Golgi stacks and endosomes. Stimulatedapical endocytosis after BFA treatment, through itsaction on an ADP ribosylation-guanine nucleotide ex-change factor involved in endocytosis, was indeedreported earlier in tobacco BY-2 suspension cellsexposed to FM1-43 (Emans et al., 2002).

BFA Disassembled the Golgi Apparatus

Tip growth in pollen tubes requires the integrity ofthe secretory system (Moscatelli and Cresti, 2001).During tube growth, secretory vesicles derived fromthe Golgi apparatus and/or from the early/recycling

endosomes (Geldner et al., 2003; Murphy et al., 2005)transport components needed for cell wall expansion(Camacho and Malho, 2003). In this experiment, weobserved that secretory vesicles dominated the apex inP. meyeri pollen tubes, and their distribution corre-sponded strikingly to the FM4-64 staining pattern in

Figure 7. FM4-64 internalization in control and inhibitor-treated pollentubes showing that dye uptake is stimulated by BFA (5 mg mL21) inhib-ited by latrunculin B (1 mM) and sodium azide (500 mM). Cell-associatedFM4-64 fluorescence was quantified after a 12-min internalization of thedye and normalized to control pollen tubes labeling. Data shown aremeans6 SD and are representative of three experiments, each containingthree individual measurements. CK, Control; LTB, latrunculin B; SAD,sodium azide.

Figure 8. Electron micrographs in control and BFA-treated (5 mg mL21)pollen tubes. A, Tip region of a control pollen tube, showing the apicalclear zone. B, Tip region of a BFA-treated pollen tube, showing theapical clear zone was occupied by many organelles, e.g. mitochondriaand vacuoles. C, Magnified picture of a control pollen tube tip region,from where many vesicles could be observed; some of them werefusing with, or releasing from, the plasma membrane. D, Tip region ofa BFA-treated pollen tube. No PBs and secretory vesicles could beobserved, but many other types of vesicles and large organelles, such asmitochondria, vacuoles, and lipid bodies, appeared. E, Another apicalzone in control pollen tube, indicating a typical PB (arrow). F, Golgiapparatus in control pollen tubes, showing the Golgi stacks containfour flattened cisternae with a distinct cis-trans polarity with numerousvesicles attached to them. G, Abnormal arrangements of the Golgistacks in BFA-treated pollen tubes, indicating the disassembly of theGolgi apparatus accompanied by vesiculation of trans-side (arrow), lessGolgi-derived vesicles, as well as the ER dilations (arrow). H, Anotherabnormal arrangement of Golgi apparatus induced by BFA treatment.Golgi cisternae appeared abnormally stacked and curved, engulfingsome large vesicles. Bar5 2mm in A and B; 1 mm in C andD; 0.5mm inE; and 0.2 mm in F, G, and H. SV, Secretory vesicles; M, mitochondrion;V, vacuole; L, lipid body; G, Golgi apparatus; PB, paramural body.





both location and distribution, as described previ-ously. Because many of the secretory vesicles receivedthe internalized endocytic tracer FM4-64 within a fewminutes of exposure, we propose that many of thesesecretory vesicles are derived from the early/recyclingendosomes, as reported for the apices of tip-growingroot hairs (Voigt et al., 2005). However, it is importantto note that the vesicle distribution at the extremeapical region shows a lower density (Fig. 8C) than thatfound in the V-shaped vesicle accumulation in angio-sperm pollen tubes (Parton et al., 2001, 2003) and alsodiffered from other coniferous species, in which many

other large organelles are detected in the apical regionof the pollen tube (Hao et al., 2005). Deducing from thefacts that the pollen tube growth rate in P. meyeri(16 mm h21) ranks between that of the lily (390 mm h21)and other gymnosperm species (1mm h21; De Win et al.,1996; Yang et al., 1999; Hao et al., 2005), we speculatethat the pollen tube growth rate mainly depends onthe particular cellular organization of the apical re-gion. In the presence of BFA (5 mg mL21), the secretoryvesicles rapidly vanished from the very tip, whilemany small vacuoles and mitochondria, as well as somelipid bodies and various types of vesicles, appeared inthe apical region (Fig. 8, B and D). Furthermore, thenumber of secretory vesicles at the trans-Golgi sidedecreased because of the structural disintegration ofthe Golgi apparatus (Fig. 8, G and H), suggesting thatBFA inhibited the production of secretory vesicles bydisorganizing the Golgi apparatus and trans-Golginetwork. The ER also dilated after BFA treatment(Fig. 8G). Because the pollen tube growth rate dependson the supply of new material to the cell walls

Figure 9. A typical inhibitor experiment showing the effects of BFA onacPase activity (A), germination frequency (B), and mean tube length(C). Pollen grains were incubated in germination medium. After 12 h,BFA (5 mg mL21) was added to one-half of the pollen culture (:),whereas the other half served as a control (¤). All three parameterswere determined every 6 h. The data points represent means with SDs ofthree independent experiments.

Figure 10. FTIR spectra obtained from the apical region, middleregion, and basal region of P. meyeri pollen tubes cultured for 20 h.A, FTIR spectra obtained from the tip, middle, and basal regions ofpollen tubes cultured in standard medium (CK) or in medium contain-ing 5 mg mL21 BFA (BFA) revealed that BFA treatment induceddisplacements of the peaks or changes of absorbance. B, Differencespectra generated by digital subtraction of spectra CK from spectra BFA,showing that the content of proteins and polysaccharides decreasedafter BFA treatment and that the reduction in polysaccharide was mostobvious in apical region.

Wang et al.




(Derksen et al., 1995), the disorder inflicted on the se-cretory system (ER, Golgi apparatus, trans-Golgi net-work) by treatment with BFA inevitably retarded theelongation of the pollen tubes, as discussed previously.

The PB, a general term used to describe all mem-brane systems associated with the plasma membrane,comprising a group of membranous elements situatedbetween the plant cell wall and the plasma membrane,is involved in exocytosis and cell wall deposition(Marchant and Robards, 1968). Because PBs possessedvesicles similar to the internal vesicles of MVBs, theymay eventually originate from fusions of MVBs, rep-resenting endosomes with apical plasma membranes(Tse et al., 2004). In this study, we noted that many PBsappeared in the normal growing pollen tube apex (Fig.8E), suggesting that active exocytosis of MVBs oc-curred during normal pollen tube growth. However,in the BFA-treated pollen tube apical region, no PBswere observed (Fig. 10D), providing strong evidencethat BFA disrupted the secretory activity of the MVBs,which may contribute to pollen tube wall depositionduring pollen tube elongation (Derksen et al., 1995).

BFA Lowered AcPase Activity

In plant cells, secretion can be measured by moni-toring the release of secretory products in cell cultures(Ibrahim et al., 2002). For example, the growth of yeastis accompanied by the local secretion of acPase at thegrowing site of the cell (Field and Schekman, 1980).Based on a study of lily pollen tube development,Ibrahim et al. (2002) suggested that the secreted acPaseactivity released via the secretory pathway may serveas a useful indicator of exocytotic activity in terms ofthe conspicuous correlation between acPase secretionand tube growth. The present experiment showed thatsecreted acPase activity was strongly correlated withpollen grain germination and tube growth in P. meyeri.With the development of normal pollen tubes, the acPaseactivity increased (Fig. 9), suggesting that acPase wassecreted during P. meyeri pollen tube growth, and itssecretory activity increased with the elongation of pollentubes. In the presence of BFA, the export of acPase activ-ity (Fig. 9A, b) was dramatically inhibited, along with theinhibition of pollen germination (Fig. 9B, b) and pollentube growth (Fig. 9C, b), suggesting that the cellularprocess leading to secretion was disrupted by BFA. Be-cause acPase was probably secreted together with thecell wall material at the tube tip from where it diffusesinto the medium (Ibrahim et al., 2002), it seems reason-able to conclude that the inhibition of production ofsecretory vesicles induced by BFA led to the suppressionof acPase secretion.

BFA Altered the Tube Cell Wall Compositionand Structure

Pollen tube elongation requires the insertion ofmany polysaccharides and structural proteins intothe tube walls (Mascarenhas, 1993). Cell wall proteins,

polysaccharides, and other components of the wallmatrix are synthesized in the ER-Golgi system andtransported into the apoplastic space via secretoryvesicles (Moore et al., 1991). Interference with theproduction and transport of secretory vesicles resultsin the modified secretion of cell wall components(Cheung et al., 2002). FTIR microspectroscopy is a re-liable and highly reproducible assay used to study cellwall composition (McCann et al., 1994) and pollentube walls cultured in different media (Yang et al.,1999; Wang et al., 2003). Using FITR analysis, weconfirmed that proteins were present in the pollentube walls of P. meyeri (Fig. 10A), as has been reportedfor other species (Yang et al., 1999). The differencespectra (Fig. 10B) showed that the protein and poly-saccharide contents along the whole tube walls werereduced when treated with BFA, directly demonstrat-ing the inhibitory effect of BFA on tube wall depositionduring pollen tube growth. Furthermore, the poly-saccharide content decreased more obviously thanthat of protein, particularly at the apical region (Fig.10B). This explains the differential sensitivity to BFAtreatment, at the Golgi apparatus, toward the synthe-sis of cell wall polysaccharides and the machinery ofvesicular transport of proteins to the wall. The BFA-induced modifications in the composition of pollentube walls were also reflected by the noncompact anddisordered wall structure (Fig. 1H).

It has been reported that cell walls at the apicalregion are highly enriched with pectin (Li et al., 2002).The FTIR analysis described here showed that poly-saccharides decreased more obviously in the newlyformed tip region than in the already formed regionsafter treatment with BFA. Because most of the bands inthe polysaccharide region between 1,200 and 900 cm21

are the vibrations associated with the sugar mono-mers of pectin (Chen et al., 1997), we conclude that theprominent decrease in polysaccharide content at theapical region was caused by a decrease in pectin con-tent. Cell wall pectins cross-linked with boron andcalcium are internalized into plant cells (Baluska et al.,2002, 2005) and apparently recycled via early/recy-cling endosomes (Baluska et al., 2002; Samaj et al., 2004).Thus, we propose that BFA-induced aberrant cell wallsare the result not only of inhibited secretion but also ofenhanced endocytosis of cell wall pectins in the tip re-gion. Further studies should test this scenario.

In summary, our results clearly showed that BFAretarded pollen tube development; the effects of BFAwere reversible because pollen tubes can recover theirtip growth after the removal of BFA. BFA treatmentinhibited exocytosis by disrupting the distribution andreducing the abundance of secretory vesicles at theapical region through disorganizing the Golgi appa-ratus, trans-Golgi network, and early/recycling endo-somes. Importantly, endocytosis was stimulated, asrevealed by confocal microscopy and fluorimetry mea-surements, in a manner opposite to the inhibitionof exocytosis in the presence of BFA. The resultsalso demonstrated that the distribution pattern and





abundance of secretory vesicles found at the pollentube apical region in P. meyeri differ from the V-formationof vesicle accumulation in angiosperms, using bothultrastructural examination and fluorescent markerFM4-64 labeling. FTIR analysis further demonstratedthat the changes in the cell wall composition couldclearly be attributed not only to inhibited exocytosisbut also to the enhanced uptake of cell wall compo-nents (especially pectins) through increased endocyto-sis. Based on these results, we conclude that enhancedendocytosis, together with inhibited secretion, is re-sponsible for the abnormal morphology and growthinhibition of pollen tubes induced by BFA, a phenom-enon that has not been reported previously.

MATERIALS AND METHODS

Plant Materials

Cones with mature pollen were collected from trees of Picea meyeri Rehd. et

Wils. growing in the Botanical Garden of the Institute of Botany, Chinese

Academy of Sciences, prior to the beginning of the pollination season at the

middle of April 2003, and were dried overnight at room temperature. The dry

pollen was stored at 220�C until use.

Pollen Culture

Stored pollen was equilibrated at room temperature for 30 min and care-

fully suspended in 12% (w/v) Suc medium containing 0, 1, 3, 5, 8, or 10 mg mL21

BFA, respectively. The pH of the media was adjusted to 6.4 with phosphate-

buffered saline. The cultures were incubated on a shaker (100 rpm) at 25�C. For

recovery experiments, samples exposed to the drug were gently centrifuged and

transferred to drug-free medium. For confocal images, pollen grains were

transferred to thin layers of 0.2% agar solidified culture medium after hydration

for 30 min in liquid media. Thin gel layers were prepared according to Parton

et al. (2001). Thin gel layers were sown with pollen suspension and incubated in

a dark humid environment at room temperature.

Determination of Pollen Germination and

Pollen Tube Growth

The germination rate was determined by checking at least 500 grains under

a Nikon microscope. Pollen grains were considered as germinated when the

pollen tube length was greater than the diameter of the pollen grain (Wang

et al., 2003). To measure the mean tube length, images of pollen tubes cultured

in the above media were taken at 6-h intervals, and the number of pollen tubes

examined was at least 200 for each treatment. All experiments were performed

at room temperature and in triplicate.

Confocal Microscopy

Loading cells with 2 mM FM4-64 dye was generally achieved by application

during the imbibition of pollen grains by direct addition of dye solutions in 115%

liquid medium to growing tubes on thin gel layers. BFA (5mg mL21), latrunculin

B (1 mM), and sodium azide (500 mM) were applied directly to growing pollen

tubes on thin gel layers in 70 mL of 115% to 120% liquid medium. Fluorescence

from FM4-64 staining was detected using a Bio-Rad MRC 600 laser confocal

scanning unit attached to an Optiphot microscope (Nikon). The samples were

excited at 514 nm with a 25-mW argon ion laser operated at full power at an

intensity of 3%, achieved by means of neutral-density filters, with a nearly closed

pinhole and the gain adjusted to below level 7.00.

Fluorimetry Measurements

After 18-h culture, the pollen suspension of 20 mL was divided into four

equal aliquots. Three of these aliquots were incubated with BFA (5 mg mL21),

latrunculin B (1 mM), or sodium azide (500 mM) for 60 min, whereas another

aliquot served as a control. After 60-min incubation, all of these aliquots were

supplemented with 2 mM FM4-64 for 12 min. Then, the aliquots were washed

three times with control medium by centrifugation (at 1,000g for 5 min). Cell-

associated fluorescence was quantitated by fluorimetry using an F-4500

fluorospectrometer (Japan). FM4-64 was excited at 514 nm (5-nm bandpass),

and emission was detected at 580 nm (5-nm bandpass). Cell-associated

fluorescence was normalized to the cell-associated fluorescence of control

pollen tube labeling and the results of three measurements were averaged.

Electron Microscopy

For electron microscopy, pollen tubes incubated for 20 h in media

containing 0 or 5 mg mL21 BFA were fixed in 2.5% glutaraldehyde in 100 mM

phosphate buffer, pH 7.2, for 2 h at room temperature. Pollen tubes were

washed three times with this buffer, post-fixed with 1% OsO4 for 3 h in 100 mM

phosphate buffer, pH 7.2. After washing with this buffer three times, pollen

tubes were dehydrated in ethanol series and finally embedded in Spurr resin.

Ultrathin sections of pollen tubes were mounted on Formvar coated grids and

stained with 2% aqueous uranyl acetate and lead citrate, and observed with an

electron microscope (JEOL 1210) at 80 kV.

Secreted AcPase Assay

Pollen grains of 50 mg were suspended in 50 mL of germination medium

and gently shaken for 1 min. Pollen grains were washed five times by

subsequently pelleting the grains, resuspending in fresh medium, and shaking

for 1 min to remove cell wall-bound acPase. After this washing procedure, the

pollen suspension was pipetted into petri dishes for germination. AcPase

secretion was monitored by sampling aliquots every 6 h and analyzing them for

acPase activity. After 30 h the pollen grains and tubes were pelleted and aliquots

of the supernatant containing the secreted acPase activity were frozen and

stored at 220�C. The activity of the acPase was measured according to Ibrahim

et al. (2002) by measuring the release of p-nitrophenol from p-nitrophenyl

phosphate. Samples of 100 mL were incubated with 400 mL of reaction buffer

containing 100 mM acetic acid, pH 5.0, 5 mM p-nitrophenyl phosphate for 45 min

at 30�C. The reaction was stopped by the addition of 200 mL of 500 mM NaOH,

pH 9.8, and the concentration of p-nitrophenol was determined at 405-nm

wavelength (U3000; Hitachi). All assays were performed as triplicates.

FTIR Analysis

Pollen tubes cultured either in the absence of BFA or supplemented with

5 mg mL21 BFA for 20 h were collected and washed with deionized water three

times. The samples were dried at room temperature on a barium fluoride

window (13 mm diameter 3 2 mm). Infrared spectra were obtained from the

apical region, middle region, and basal region of pollen tubes, respectively, using

a MAGNA 750 FTIR spectrometer (Nicolet) equipped with a mercury-cadmium-

telluride detector. The spectra were recorded at a resolution of 8 cm21 with 128 co-

added interferograms and were normalized to obtain the relative absorbance.

ACKNOWLEDGMENTS

We thank Drs. Mathew Benson and Shi-fu Wen for valuable discussions at

the early stages of these experiments and technical assistance with FTIR

microspectroscopy. We also thank Dr. Richard Turner for his patient correc-

tion of the draft of the manuscript. This is journal paper number 0503 of the

Institute of Botany, Chinese Academy of Sciences.

Received August 10, 2005; revised August 10, 2005; accepted September 2,

2005; published November 18, 2005.

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