Rab11 GTPase-Regulated Membrane Trafficking Is Crucial for Tip-Focused Pollen Tube Growth in Tobacco^W⃞

GFP-Rab11b Localized Predominantly to the Apical Clear Zone of Elongating Pollen Tubes

A cDNA for Rab11b (see Haizel et al., 1995) was readily isolated by RT-PCR from tobacco pollen mRNA but not amplified from seedling mRNA (data not shown; see Supplemental Figure 1 online for sequence alignment with other Rab11 GTPases), consistent with its being preferentially expressed in the male gametophyte. The expression profile of Rab11b mRNA (Figure 2A) conforms to a pattern of increasing accumulation during anther development, reaching a maximum at pollen maturity typical of many pollen mRNAs (Guyon et al., 2000; V. Di Stilio and A.Y. Cheung, unpublished data), a strategy thought to ready the pollen grains for germination and the early tube growth process (Mascarenhas, 1989).

When GFP-Rab11b (Figure 1) was expressed in transformed pollen, it localized most prominently in the apical clear zone of in vitro–grown pollen tubes (Figure 2B). A low level of punctuate, but not highly discrete, structures was also weakly labeled by GFP-Rab11b throughout the tube (see also Figure 3). The apical zone of green fluorescence was always trailed by a grainward stream of fluorescence extending along the center of the tube (Figures 2B and 3A). Accumulation of GFP-Rab11b in the inverted cone region abutting the apex was also observed in pollen tubes that had elongated through the pistil. The apical clear zone is known to be densely and exclusively occupied by transport vesicles (Lancelle and Hepler, 1992; Derksen et al., 1995a, 1995b). The apical concentration of GFP-NtRab11b thus strongly supports a transport vesicle localization of this fusion protein. The accumulation of GFP-Rab11b in the clear zone was rapidly dissipated by treatment with the actin polymerization inhibitors latrunculin B (Figure 2C) or cytochalasin D (data not shown). This observation is also consistent with GFP-Rab11b being associated with transport vesicles, which are known to track along actin filaments in plant cells (see Hepler et al., 2001). Moreover, the apical localization of GFP-Rab11b mimics those shown by GFP-tagged proteins destined for the pollen tube cell surface or secretion (Cheung et al., 2002; see Figure 7), suggesting a functional link between Rab11b to the trafficking and delivery of these proteins to the growing tube tip.

The domain occupied by GFP-Rab11b within the apical dome was highly dynamic both spatially and temporally (Figure 2F; see Supplemental Movie 1 online). The GFP-Rab11b accumulation zone cycled rapidly from spanning just a narrow distance against the apex to filling the entire clear zone and then retracting to a region more closely abutting the apex (Figure 2F, compare the 25, 175, and 275 s images; see Supplemental Movie 1 online). It seems plausible that the temporal and spatial fluctuation of GFP-Rab11b–labeled vesicles at the tube apex may correlate with the periodic fast and slow growth phases of elongating pollen tubes (see Parton et al., 2001). When observed over a broader time frame over which distances covered and growth trajectories could be clearly resolved, the apical zone of GFP-Rab11b was always maintained parallel to the pollen tube growth axis, even when these cells were reorienting to a new growth trajectory (Figures 2D and 2E). With these observations, we speculate that Rab11b is important for both apical growth and directionality of elongating pollen tubes.

Apical Localization of GFP-Rab11b Depends on Proper Regulation of Its Activity

The ability for Rab11b to regulate the shuttling between the active and inactive forms by GTP-GDP exchange is apparently critical for the targeting of Rab11b-tagged vesicles to the pollen tube apical clear zone. As shown in Figure 3, a DN mutation (S28N) completely abolished apical accumulation of GFP-Rab11b(DN), while a CA mutation (Q73L) caused more subtle effects and rendered GFP-Rab11b(CA) to be more evenly distributed within the apical dome. Moreover, GFP-Rab11b(DN) accentuated labeling of the punctuate structures, whereas GFP-Rab11b(CA) showed a plasmalemma localization not observed for its wild-type or DN counterparts.

The size, abundance, and streaming pattern of the punctuate structures labeled by GFP-Rab11b, its DN and CA derivatives were most reminiscent to pollen tube Golgi bodies (Cheung et al., 2002). Treatment by Brefeldin A (BFA), which reversibly disrupts Golgi stacks in many cell types, including pollen tubes (Rutten and Knuiman, 1993; Cheung et al., 2002; Ritzenthaler et al., 2002), resulted in the dissociation of these discrete structures and appearance of membrane patches, which were especially evident in GFP-Rab11b(DN)- and Rab11b(CA)-expressing pollen tubes (Figures 3B and 3D). As with the reversible effect of BFA on the disruption of Golgi stacks, discrete GFP-labeled organelles reappeared upon BFA removal and were most readily observed among GFP-NtRab11b(DN)–expressing tubes (Figure 3C), further supporting that this Rab11b variant has a prominent Golgi association. Moreover, BFA treatment, in addition to abolishing the GFP-Rab11b(CA)–labeled organelles, also accentuated its cell membrane association (Figure 3D).

Expression of GFP-Rab11b(DN) and GFP-Rab11b(CA) Inhibits in Vitro Pollen Tube Growth and Directionality

To determine whether altering the overall pollen tube Rab11 activity would affect the pollen tube growth process, we examined pollen from Lat52-GFP-Rab11, Lat52-GFP-Rab11(DN), or Lat52-GFP-Rab11b(CA) transformed plants (Figure 1, Table 1; see also Supplemental Figure 2 online for the level of fusion proteins in these transformed pollen populations). When germinated in vitro, transformed pollen tolerated a broad range of GFP-Rab11b accumulation and showed comparable germination frequency and growth rate as control nontransformed (data not shown) and transformed GFP-expressing pollen (Figure 4). The effect of GFP-Rab11b(DN) on germination was moderate; only grains that accumulated very high levels of this protein, as judged by their green fluorescence level, were inhibited for germination. However, pollen tube growth was reduced significantly by the expression of GFP-Rab11b(DN) (Figure 4). Expression of GFP-Rab11b(CA), on the other hand, was highly inhibitory to pollen germination. When pollen grains from transgenic plants that expressed different levels of pollen GFP-Rab11b(CA) (see Supplemental Figure 2B online) were cultured, tubes that developed from all of these pollen populations consistently showed very low levels of green fluorescence (see Figure 5), and they were significantly shorter than control pollen tubes (Figure 4). Moreover, in repeated experiments, the average lengths attained by GFP-Rab11b(CA)–expressing tubes were also noticeably shorter than those observed for GFP-Rab11b(DN)–expressing tubes (Figure 4).

In vitro–grown pollen tubes have a high propensity to follow a stable growth trajectory, giving rise to tubes with a relatively straight morphology. Notably, pollen tubes expressing GFP-Rab11b(DN) or GFP-Rab11b(CA) showed dramatic meandering phenotypes (Figure 5). Some of them zigzagged along the entire length of the tube, and this property was most prevalent among the Lat52-GFP-Rab11b(CA) transformed tubes (Figure 5B). Others showed a period of straight growth interrupted by abrupt and continuous turning, resulting in a knotted morphology along their lengths or at the tube tip (Figures 5A and 5B). When pollen tubes were examined under identical imaging conditions, it became apparent that GFP-Rab11b(CA) was considerably more active in inducing pollen tube zigzagging than GFP-NtRab11(DN) (Figure 5B). Using the level of green fluorescence as a measure for the level of these fusion proteins in individual tubes, we observed that pollen tubes accumulating very low levels of GFP-Rab11b(CA) already showed severe zigzagging, whereas it took substantially higher levels of GFP-Rab11b(DN) accumulation to induce moderate twists and turns. Furthermore, pollen tubes that developed from grain populations that accumulated higher levels of GFP-Rab11b(DN) produced more meandering tubes (data not shown), consistent with a dose-dependent phenomenon. Moreover, pollen tubes apparently could tolerate relatively high levels of GFP-Rab11b(DN) as strongly fluorescent tubes with twists and turns (Figure 5B) were quite prevalent. On the other hand, few pollen tubes with green fluorescence levels higher than those seen in Figure 5B existed among GFP-Rab11b(CA)–expressing tubes. Apparently, pollen grains with even moderate levels of this fusion protein had not germinated.

Few meandering tubes were observed among pollen that expressed GFP-Rab11b. When zigzagging did occur, the amount of GFP-Rab11b in these tubes was much higher than those observed in wavy GFP-Rab11b(CA)–expressing pollen tubes (Figure 5B, right panel). However, unlike GFP-RAb11b(DN) and GFP-Rab11b(CA), the ability of GFP-Rab11b to accumulate in the apical inverted cone region had not been compromised, though pollen tube broadening was apparently induced by increasing levels of this fusion protein (Figure 5B).

In order to enter the ovules for fertilization, pollen tubes respond to guidance cues that emanate from the ovule and target growth toward and into the ovule via an aperture known as the micropyle (Hulskamp et al., 1995; Higashiyama et al., 1998, 2001; Shimizu and Okada, 2000; Huck et al., 2003; Palanivelu et al., 2003; Marton et al., 2005). Pollen tubes from a number of plant species grown in vitro retain the capacity to grow toward and penetrate ovules (Higashiyama et al., 1998, 2001), though at efficiency substantially lower than what occurs in vivo. When ovules were included in in vitro pollen tube growth cultures, GFP- and GFP-Rab11b–expressing pollen tubes that extended close to an ovule were often (∼25%) seen to approach or have entered the ovule (Figure 5C). On the other hand, of the >100 GFP-Rab11b(DN) and GFP-Rab11b(CA) pollen tubes growing in close proximity to ovules that were observed, none penetrated the ovules. Instead, the growth path of some of these pollen tubes suggests that even when elongating in the micropylar vicinity, they would elongate past it and continued their growth onto the medium (Figure 5C). The tendency for GFP-Rab11b(DN)– and GFP-NtRab11b(CA)–expressing pollen tubes to twist, turn, and meander had apparently compromised their ability to aim their growth trajectory at the micropyle.

Expression of GFP-Rab11b(DN) or GFP-Rab11b(CA) Inhibits Pollen Tube Growth in Vivo and Reduces Male Fertility

Upon pollination on the stigma, wild-type tobacco pollen grains germinate efficiently, and a majority of their tubes reach the style-ovary junction ∼30 h after pollination (Cheung et al., 1995; Wang et al., 1996). Direct examination of pollinated pistils (Figures 6A to 6N) showed that most GFP-expressing (data not shown) and GFP-Rab11b–expressing grains germinated (Figure 6A), and many of these tubes reached the basal stylar region by 30 h after pollination (Figure 6C). As for GFP-Rab11b(DN)–expressing pollen grains, most of them germinated, except the most highly fluorescent grains (data not shown). However, by 30 h after pollination, a majority of GFP-Rab11b(DN) pollen tubes only reached one-half to two-thirds down the style (Figures 6D and 6E), and a few short wavy tubes remained in the stigmatic region (Figure 6G). Germination of GFP-Rab11b(CA)–expressing pollen grains were severely inhibited (Figure 6B). Tubes that germinated were still almost exclusively within the upper half of the style (Figure 6F) even by 48 h after pollination. Short wavy GFP-Rab11b(CA)–expressing pollen tubes could be observed on the stigmatic surface (Figure 6H).

When the placenta from pistils pollinated with Lat52-GFP, transformed pollen was excised at 30 h after pollination, GFP-expressing pollen tubes could be seen penetrating micropyles, and green fluorescence was already observed in ∼10% of the ovules, reflecting pollen tube penetration (Figure 6K). Similarly, GFP-NtRab11b–expressing pollen tubes have also penetrated ovules (Figure 6L), but the number of green fluorescent ovules was slightly lower than that seen in pistils pollinated by GFP-expressing pollen. However, we have not observed GFP-Rab11b–expressing pollen tubes in the middle of entering an ovule, possibly the restricted apical localization of this fusion protein in in vivo elongating pollen tubes (Figure 6J) had made it very difficult to capture the moment of micropyle entrance. A considerable number of Lat52-GFP-Rab11b(DN) transformed pollen tubes reached the ovary by 48 h after pollination. Ovules with green fluorescence signal were observed (Figures 6M and 6N), but they were fewer than that achieved by GFP- or GFP-Rab11b–expressing pollen tubes. The ability of GFP-Rab11b(CA)–expressing pollen tubes to penetrate ovules was not examined. Their slow growth rate would have precluded their arrival at the ovary before ovules have lost their receptivity, ∼72 h after anther dehiscence (see Cheung et al., 1995; Wang et al., 1996).

Semi–in vivo pollen tube growth assays (Cheung et al., 1995; Higashiyama et al., 1998) were used to further illustrate the differences in pollen tube growth rate within the pistil for pollen that expressed the wild-type or mutant forms of GFP-Rab11b (Figures 6O to 6T). In these experiments, pollen from heterozygous transformed plants with a single T-DNA insertion locus was used to pollinate wild-type pistils. For pistils pollinated by pollen from a Lat52-GFP-Rab11b transformed plant, both wild-type and GFP-Rab11b–expressing tubes emerged at the cut stylar end at a comparable time after culturing, although the number of green fluorescent tubes was consistently lower than half of the entire pollen tube population (Figure 6O). It is possible that the strongest fusion protein-expressing tubes were slightly retarded during in vivo pollen tube growth. On the other hand, for pistils that had been pollinated with pollen from a Lat52-GFP-Rab11b(DN) or a Lat52-GFP-Rab11b(CA) transgenic plant, no transformed pollen tube emerged even when wild-type tubes had grown some distance after their emergence from the cut styles (Figures 6P and 6Q). Instead, most of the transformed pollen tubes were in the middle or upper part of the excised pistils (Figures 6R and 6S).

Observations on pollen tube growth in the pistil described above support that pollen tube growth in the stigma and stylar transmitting tissue was substantially retarded by the expression of GFP-tagged DN or CA Rab11b mutant variants. This conclusion was further supported by segregation analysis of transformed plants (Table 1). Lat52-GFP-Rab11b transformed plants showed close to normal male fertility. The slightly reduced male inheritance observed in some of these transgenic plants (Table 1) was most probably due to highly expressed GFP-Rab11b in a minor population of the transformed pollen grains, undermining their germination and growth efficiency. On the contrary, expression of DN or CA variants of the GFP-Rab11b considerably reduced male fertility, and this effect was especially drastic among Lat52-GFP-Rab11b(CA) transformed plants (Table 1). The extent of reduced male inheritance observed in Lat52-GFP-Rab11b(DN) transformed plants correlated with the level of expression of the fusion protein (see Supplemental Figure 2 online). On the other hand, a majority of the >20 Lat52-GFP-Rab11b(CA) transformed plants examined showed a comparably strong reduced male fertile property irrespective of the level of GFP-Rab11b(CA) observed among their pollen grains (Table 1; see Supplemental Figure 2 online). Seed yield was significantly reduced among these Lat52-GFP-Rab11b(CA) transformed plants (see Supplemental Table 1 online). Together with the observations that pollen germination and tube growth were highly sensitive to GFP-Rab11b(CA) (Figures 4 to 6), the uniformly observed reduced male fertility was consistent with the possibility that in every Lat52-GFP-Rab11b(CA) transformed line, only pollen grains that expressed very low and below inhibitory levels of this fusion protein managed to contribute to fertilization. Moreover, fusion with GFP apparently had modulated the activity of these small GTPases as expression of untagged versions of DN and CA Rab11bs consistently showed stronger inhibitory effects than their GFP-tagged counterparts and significantly reduced male fertility (e.g., see line 174-6 in Table 1).

Rab11 Regulates Delivery of Cell Wall and Secreted Proteins and Membrane Recycling

Three GFP-labeled cargo proteins (Figure 1) and the fluorescent lipophilic dye FM4-64 (Bolte et al., 2004) were used to examine the functional significance of Rab11 activity in membrane trafficking. The pollen tube wall-located invertase (Inv)-GFP (Figure 7A) was derived from a tobacco pollen-expressed invertase (Greiner et al., 1995). Sec-GFP was a secreted form of GFP, whereas SynAGP-GFP was a secreted fusion protein of a synthetic arabinogalactan protein and GFP (Shpak et al., 1999) (see Supplemental Figure 2 online for a protein blot for these pollen-expressed fusion proteins). Similar to several other proteins destined to the pollen tube cell surface observed (e.g., see Cheung et al., 2002; Cheung and Wu, 2004), Inv-GFP, Sec-GFP, and SynAGP-GFP showed prominent accumulation at the inverted cone region of the pollen tube apex (Figures 7A to 7D, left-hand panels). Some intracellular labeling was also noticeable, reflecting passage of these proteins through the endomembrane system. Organelles reminiscent of Golgi bodies were more prevalent in SynAGP-GFP–expressing tubes (Figure 7D) then in tubes expressing the other two cargo molecules, probably because arabinogalactan modification takes place mainly within the Golgi compartments. Cell surface labeling was never observed for Sec-GFP or SynAGP-GFP, indicating that they were efficiently released to the medium upon vesicle fusion with the apical membrane.

In order to examine the effect of downregulated Rab11b activity on protein trafficking, Lat52-Flag-Rab11b(DN) transformed plants (see Table 1) were crossed with pollen from plants transformed by each of the GFP-labeled cargo transgenes described above. Accumulation of the GFP-labeled cargo proteins in these double transformed pollen tubes was abolished (Figures 7B to 7D, right-hand panels). Furthermore, a considerably higher level of these proteins was retained intracellularly in these double transformed pollen tubes relative to that observed in control tubes expressing only the cargo protein (cf. Figures 7B to 7D, right-hand panels and their insets), consistent with reduced efficiency in secreting these cargo molecules. Because of the highly inhibitory effect of Rab11(CA) on pollen germination, its effect on cargo trafficking was examined in transiently transformed pollen that would have germinated before an inhibitory level of this protein had accumulated. In the double transformed pollen tubes, apical targeting of SecGFP and SynAGP-GFP was also abolished, and intracellular retention of these cargo molecules was prominent (Figure 7E). Together, these observations indicate that pollen tubes with misregulated Rab11 activity were compromised in their exocytic activity.

FM dyes are taken up efficiently from the growth medium by elongating pollen tubes (O'Driscoll et al., 1993; Camacho and Malho, 2003; Parton et al., 2003). The temporal pattern of FM4-64 labeling suggests an initial internalization by endocytosis followed by membrane recycling from the endosomal system to the apex, resulting in the accumulation of FM4-64–labeled transport vesicles in the clear zone (Figure 8A; Camacho and Malho, 2003; Parton et al., 2003). When GFP-Rab11b–expressing pollen tubes were treated with FM4-64, the localization of the fusion protein in the apical clear zone and in the retrograde cytoplasmic stream overlapped with that of the fluorescent dye (Figures 8B and 8C), consistent with exocytic vesicles and vesicles derived from endocytic membranes co-occupying the apical clear zone. Expression of GFP-Rab11b(DN) or GFP-Rab11b(CA) abolished the accumulation of these FM4-64–labeled vesicles at the tip (Figures 8D and 8E), suggesting probable participation of Rab11 in the endocytic and recycling processes.

Misregulating Rab11b Activity Affects Actin Organization in the Apical and Subapical Regions of Transformed Pollen Tubes

Elongating pollen tubes characteristically maintain an elaborate actin organization (see Hepler et al., 2001 and Figure 10). Transformed pollen tubes that coexpressed Rab11b and GFP-NtADF1, an actin binding reporter protein for live cell imaging (Chen et al., 2002), showed a prominent actin mesh at the subapical region subtended by long actin cables in the shank (Figure 9A), similar to that typically seen in control GFP-NtADF1–expressing pollen tubes (Figure 10; see also Chen et al., 2002; Cheung and Wu, 2004). The subapical actin mesh is a highly dynamic structure (Chen et al., 2002). It is highly sensitive to pollen tube growth property and considerably more easily dissipated when growth is perturbed (C.Y. Chen, A.Y. Cheung, and H.-m. Wu, unpublished data; see also Cheung and Wu, 2004) than the long actin cables in the shank. When Rab11b(DN) or Rab11b(CA) were coexpressed with GFP-NtADF1, actin cables along the shank of these transformed pollen tubes were by and large maintained, but the subapical actin mesh was consistently abolished (Figures 9B to 9E). On the other hand, short actin cables could be seen in the apical region, some abutting the cell membrane and especially evident when single optical sections were examined, but which were rarely observed in control pollen tubes that maintained a vibrant actin mesh (Figure 9A).

cDNA Isolation, Mutagenesis, Construction of GFP Fusion Proteins, and Gene Expression Cassettes

cDNAs for a pollen-expressed Rab11 (Rab11b) (Haizel et al., 1995) and Inv (Greiner et al., 1995) were obtained from tobacco (Nicotiana tabacum) pollen mRNA by RT-PCR. Mutation S28N and Q73L were introduced individually into Rab11b by PCR-based mutagenesis (Ho et al., 1989; Ito et al., 1991) to produce Rab11b(DN) and Rab11b(CA), respectively. N-terminal fusion of all three forms of Rab11b with GFP (Chiu et al., 1996) or a FLAG tag and C-terminal fusion of Inv with GFP (Inv-GFP) were constructed by PCR-based methodology. Inv-GFP, Sec-GFP (with a signal peptide sequence fused to the N terminus of GFP), and SynAGP-GFP (with a signal peptide and a synthetic arabinogalactan protein fused to GFP) (Shpak et al., 1999) were used as cargo molecules for the secretory pathway. All chimeric genes were fused with the pollen-active promoter LAT52 (Twell et al., 1990) either in pBluescript (pKS+) for microprojectile bombardment to transiently transform pollen (Chen et al., 2002) or in a T-DNA vector for Agrobacterium tumefaciens–mediated transformation to obtain stable transgenic plants (Delebrese et al., 1986). Figure 1 shows a list of constructs used in this study.

Transformation, Genetic Crosses, and Analyses

Transformed tobacco plants were generated by Agrobacterium Ti plasmid transformation (Delebrese et al., 1986). Stable transformed pollen grains from these plants were used for analyses unless indicated otherwise. They were also used in genetic crosses to obtain double transformed plants that expressed various forms of Rab11b and GFP-labeled cargo molecules in their pollen. Transgene segregation analyses were performed by germinating seeds in tissue culture medium supplemented with kanamycin to score for the transformation marker gene. Transient transformation of tobacco pollen by microprojectile bombardment was performed as previously described (Chen et al., 2002).

Pollen Tube Growth

In vivo, semi–in vivo, and in vitro pollen tube growth assays follow previously described procedures (Cheung et al., 1995; Chen et al., 2002). Pollen from transformed plants lines 21a, 153-12, 173-4, 173-8, 207-2, and 207-3 (Figure 1, Table 1) were used for analyses. Treatment of in vitro–grown pollen tubes by FM4-64, BFA, and latrunculin B followed published conditions (Parton et al., 2001; Cheung et al., 2002). For in vivo and semi–in vivo pollen tube growth assays, pistils from nontransformed plants were hand-pollinated. For semi in vivo growth assays, emasculated tobacco flowers were pollinated with segregating populations of pollen from Lat52-GFP-Rab11b, Lat52-GFP-Rab11b(DN), or Lat52-GFP-Rab11b(CA) transformed plants with a single T-DNA insertion locus (lines 153-12, 173-8, and 207-2). Pollinated stigma-styles were excised at specified hours (see figure legends) after pollination, and the cut stylar ends were immersed in germination medium to allow further growth of pollen tubes through the style and onto the medium. These cut pistils were examined periodically between 8 and 24 h after culturing to observe the emergence of pollen tubes at the stylar end. Pollen tubes within the transmitting tissue were observed after bisecting the styles. All pollen tube growth experiments were repeated at least three times with comparable observations.

Microscopy

Bright-field and epifluorescence imaging were performed on a Nikon Eclipse E800 microscope equipped with a SPOT camera (Nikon). Most images were taken by autoexposure. When the level of GFP fusion protein expression was compared microscopically between individual pollen tubes, identical exposure conditions were used among the samples. Confocal microscopy was performed on a Zeiss 510 Meta system.

RNA and Protein Analyses

RNA and protein isolation and gel blot analyses followed previous procedures (Wang et al., 1993, 1996), except that chemiluminescent detection was used for some of the protein blots.

Accession Numbers

Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers: L29269 (Rab11b) and X81834 (Inv). Accession numbers for genes in the alignment are shown in Supplemental Figure 1 online.