Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

Expression of SEC3 Paralogs in Arabidopsis

In Arabidopsis, there are two SEC3 paralogs, SEC3a (At1g47550) and SEC3b (At1g47560), encoded as a tandem duplication in the genome and showing 84% and 97% nucleotide identity in their genomic and coding sequences, respectively (Cvrčková et al., 2012). The high degree of sequence identity between SEC3a and SEC3b delayed their correct annotation (Lamesch et al., 2012) and compromised the determination of their expression patterns by microarrays. Reliable expression data for each of the two genes could be obtained only using oligonucleotide primers for the 3′ untranslated region (Supplemental Fig. S1). The expression patterns of SEC3a and SEC3b were determined by reverse transcription (RT)-PCR. The SEC3a transcript was detected in seedlings, roots, and different organs of the sporophyte, including flowers (Fig. 1A). GUS expression driven by the SEC3a promoter confirmed the constitutive expression pattern detected by RT-PCR and further indicated that the expression of SEC3a is higher in primary root tips, distal tips of cotyledons, and lateral root primordia (Fig. 1, B–D). Compared with SEC3a, SEC3b expression was much lower and spatially restricted, with low transcript levels that could be detected only in flowers and siliques (Fig. 1A). Within flowers, SEC3a and SEC3b expression was detected in ovules, fully developed anthers, and in vitro-germinated pollen (Fig. 1A). The expression and promoter analysis suggested that SEC3a is the major SEC3 paralog in Arabidopsis and that SEC3b may function redundantly with SEC3a in some floral tissues.

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Figure 1.

Expression pattern of the SEC3a and SEC3b paralogs and genotyping of sec3a-1 and sec3b-1 mutants. A, RT-PCR showing the expression of SEC3a and SEC3b in seedlings at 6 and 14 d after germination (DAG), different mature plant organs, closed flowers (Fc), ovules, anthers (fully developed from closed flowers), and in vitro-germinated pollen. B to D, Expression pattern of SEC3a (PSEC3a:GUS) in seedlings. Four independent lines were used for analysis. B, A 2-DAG seedling showing the highest expression levels in cotyledons and primary root tips (arrowheads). C, A primary root of a 2-DAG seedling. dfz, Differentiation zone; dz, division zone; ez, elongation zone. The arrowhead marks expression in root hairs. D, A root with an emerging lateral root primordium. Bars = 50 µm. E, Schematic representation of SEC3a and SEC3b gene exon-intron organization. Gene-specific primers used for genotyping and determining the T-DNA insert locations are specified by arrows. Numbers indicate nucleotide positions from the initiation ATG codon. The T-DNA inserts at positions 6,673 in SEC3a and 4,529 in SEC3b are highlighted by arrowheads. F, RT-PCR showing the expression of SEC3a and SEC3b in flowers of the indicated genotypes. Fo, Open flower. A genomic DNA control was included to display primer efficiency. G, SEC3a and SEC3b expression in sec3a-1/LAT52:SEC3a complemented plants. Note that in sec3a-1^−/−/LAT52:SEC3a homozygous lines, expression of the SEC3a coding sequence (CDS) indicates that the SEC3a transgene construct was in the sporophyte (for primer details, see Supplemental Fig. S1).

Identification of T-DNA Insertion Mutants in SEC3a and SEC3b

To analyze the function of SEC3 in Arabidopsis, we employed a reverse genetics approach. To this end, we identified T-DNA insertion mutants in both SEC3a and SEC3b. The GABI_652H12 line carries a T-DNA insertion in the last exon, namely exon 25, of the SEC3a gene (sec3a-1), and the SALK_064295 line carries a T-DNA insertion in exon-17 of SEC3b (sec3b-1; Fig. 1E). Exact positions of the T-DNA inserts and fitting of the insert to the specific paralog were verified by sequencing (Supplemental Fig. S1B). It is important to mention that we also analyzed additional SEC3a mutant alleles (SALK_140590, SALK_145185, and SALK_140593, all predicted to carry an insert in the first intron), but we were unable to identify T-DNA inserts at the predicted locations, suggesting incorrect annotations of these lines.

The sec3a-1 allele shows resistance to the sulfadiazine (Sd) selection marker, which cosegregates with the T-DNA insert in SEC3a. Homozygous sec3a-1^−/− plants could not be obtained from heterozygote sec3a-1 progeny (Table I; Supplemental Fig. S2, A and B). Moreover, the progeny of sec3a-1^+/− heterozygote plants segregated at a 1:1 ratio with respect to Sd resistance, deviating from the expected 3:1 Mendelian segregation ratio (Table II), thus implying a gametophytic transmission defect. In contrast to sec3a-1, homozygous sec3b-1^−/− plants were identified using a combination of SEC3b gene-specific and T-DNA-specific primers (Supplemental Fig. S2C). The SEC3b mRNA transcript could not be detected in sec3b-1^−/− homozygous flowers, indicating that sec3b-1 is a null allele (Fig. 1F). The macroscopic phenotype of sec3b-1^−/− homozygous seedlings is similar to that of wild-type Col-0 (Supplemental Fig. S3), which is not surprising, given the low expression levels of SEC3b and its redundancy with SEC3a.

Pollen-Specific Transmission Defects in sec3a-1

The sec3a-1 heterozygote plants were outcrossed to wild-type Col-0 plants for three successive generations to reduce the chance of nonrelated mutations linked to the allele, and they always segregated at a 1:1 ratio with respect to Sd resistance. In order to verify whether transmission defects in sec3a-1^+/− plants are associated with the male or the female gametophyte, reciprocal outcrosses to Col-0 were performed. When sec3a-1^+/− pollen was used for fertilization of the wild type, the mutant allele was not transferred to the progeny (n = 408), whereas in reciprocal crosses, the mutant allele was transmitted at the predicted ratio (n = 483). These data indicate that the sec3a-1 allele has a male gametophyte transmission defect (Table II).

To confirm that the pollen-transmission defects in the sec3a-1 allele were associated with the mutation in SEC3a, gene complementation experiments were performed. Plants expressing the coding sequence of SEC3a and of SEC3a N-terminally fused to GFP (GFP-SEC3a) under the control of the pollen promoter LAT52 were crossed to the sec3a-1 heterozygote plants, thus generating sec3a-1^+/−/LAT52:SEC3a and sec3a-1^+/−/LAT52:GFP-SEC3a plants, respectively. Outcrosses to the Col-0 background indicated that both constructs successfully complemented the male gametophytic defects associated with the sec3a-1 mutation, since the mutant allele was transmitted through the pollen and cosegregated with Basta resistance, a selection marker for LAT52:SEC3a/GFP-SEC3a lines (Table II; Supplemental Table S1). In control reciprocal outcrosses, LAT52:SEC3a and LAT52:GFP-SEC3a did not affect the transmission of the sec3a-1 allele through the female gametophyte and did not cosegregate with the mutation (Table II; Supplemental Table S1).

Homozygous sec3a-1^−/− plants were identified among the progeny of self-crossed sec3a-1^+/−/LAT52:SEC3a complementation lines using PCR (Table I; Supplemental Fig. S2D), which was further confirmed by nonsegregating Sd-resistant progeny (Supplemental Table S2). Endogenous SEC3a expression was not detected in sec3a-1^−/−/LAT52:SEC3a seedlings, indicating that sec3a-1 is a null allele, while a transcript corresponding to the SEC3a coding sequence used for complementation was present in sec3a-1^−/− seedlings, indicating that, in our system, the LAT52 promoter also drove expression in the sporophyte (Fig. 1G). Moreover, SEC3b expression was not up-regulated in sec3a-1^−/−/LAT52:SEC3a seedlings. To confirm that the sec3a-1 phenotype was not associated with embryonic lethality, we counted the number of aborted seeds and found it to be similar to that in plants without the T-DNA insert (Supplemental Table S3). Taken together, these data showed that knockout of SEC3a resulted in a male gametophyte transmission defect, similar to mutants in genes encoding the SEC5, SEC6, SEC8, and SEC15 exocyst subunits (Cole et al., 2005; Hála et al., 2008).

Pollen Germination and Pollen Tube Growth Are Compromised in the sec3a-1 Mutant

Next, we examined whether the male gametophyte transmission defect in sec3a-1 plants is due to compromised pollen tube growth. To this end, sec3a-1^+/− heterozygous plants were crossed into the quartet1 (qrt1) mutant background. In qrt1, the four meiotic products of the pollen mother cell are fused, and pollen grains are released as tetrads (Preuss et al., 1994). Heterozygous sec3a-1 tetrads (sec3a-1^+/−/qrt^−/−) showed a 2:2 ratio of normal to affected pollen and allowed direct examination of the mutant phenotype. When the qrt1 pollen was germinated in vitro, approximately 60% of all tetrads (n = 1,529) had three to four pollen grains with germinated long pollen tubes. In sec3a-1 heterozygous tetrads (n = 1,769), only one or two cells formed pollen tubes, and the other cells either did not germinate or produced small protrusions that did not elongate (Fig. 2A).

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Figure 2.

Tetrad analysis of sec3a-1 heterozygotes. A, In vitro-germinated tetrads from self-crosses of qrt1^−/− and qrt1^−/−/sec3a-1^+/− plants. The chart shows the percentage from total quartets with the indicated number of germinated pollen grains in each tetrad (between zero and four) compared with the total tetrads scored for each genotype. Numbers above the bars represent actual numbers of tetrads that were counted. B, Aniline Blue staining of wild-type stigma 24 h after pollination with either qrt1^−/− or qrt1^−/−/sec3a-1^+/− tetrads. Asterisks mark germinated pollen grains in tetrads as revealed by the staining. The chart shows in vivo germination analysis of qrt1^−/− and qrt1^−/−/sec3a-1^+/− tetrads germinated on Col-0 stigma. The number of pollen tubes producing cells in each quartet (between one and four) was counted using Aniline Blue staining. Bars = 50 µm.

To further confirm that male transmission defects in sec3a-1^+/− plants were associated with pollen tube growth, in situ germination experiments were performed. Pollen from sec3a-1^+/−/qrt^−/− (n = 73) and qrt^−/− (n = 58) plants were used to pollinate wild-type Col-0 stigma, and 24 h after germination, the plants were stained with Aniline Blue, which stains the callose in the pollen tube cell wall. As expected from the in vitro germination experiments, 72 of 73 sec3a-1 heterozygous tetrads germinated only one to two pollen tubes, compared with more than 50% of tetrads with three to four pollen tubes in qrt1 (Fig. 2B).

To test whether the loss of SEC3a function does affect early stages of microspore and pollen development, nuclei of mature tetrads were stained with 4′,6-diamidino-2-phenylindole (DAPI). One vegetative nucleus and two sperm cell nuclei were detected in each pollen grain of qrt^−/− and qrt^−/−/sec3a-1^+/− tetrads, indicating that the loss of SEC3a function does not affect mitosis during male gametogenesis (Supplemental Fig. S4). Collectively, our results indicate that, similar to other exocyst complex subunits, SEC3a function is required for the proper germination of pollen grains and subsequent pollen tube growth.

SEC3a Polar Localization at the Tip Is Dynamic and Predicts the Growth Direction in the Arabidopsis Pollen Tube

Given that mutation in the SEC3a gene led to pollen transmission defects and that the GFP-SEC3a fusion protein complemented the mutant phenotype, we next focused on the localization of SEC3a in Arabidopsis pollen tubes. In transgenic plants expressing LAT52:GFP-SEC3a, enrichment of the GFP-SEC3a signal was detected at the apical region of growing pollen tubes (Fig. 3A). Imaging of a middle section of the pollen tube apex with the pinhole set to 1 Airy unit revealed the presence of a polar fraction of GFP-SEC3a localized to the tip appearing as thin lines at or near the PM (Fig. 3B). Conversion of the GFP signal to a range of intensities uncovered that GFP-SEC3a also forms a subapical inverted cone (Fig. 3C). Next, we utilized time-lapse imaging to follow the dynamics of GFP-SEC3a polar localization in elongating Arabidopsis pollen tubes. Under our experimental conditions, pollen tubes elongated with a rate of 3.5 to 4 µm min⁻¹ and occasionally changed the direction of growth, suggesting a correlation between GFP-SEC3a localization and the direction of pollen tube growth. In elongating pollen tubes, the polar fraction of GFP-SEC3a close to the tip PM was highly dynamic and shifted its position toward the future growth axis before visible changes in the direction of pollen tube elongation were detected (Fig. 3E; Supplemental Movies S1 and S2). The frequent frame acquisition used for imaging caused photobleaching of the GFP signal; thus, the last 1.5 min of the time-lapse series highlight primarily the direction of pollen tube elongation. In nonelongating pollen tubes, the GFP-SEC3a signal at the pollen tube apex and tip was reduced (Fig. 3D). Furthermore, no polar accumulation of GFP-SEC3a was detected at the tip, and the signal was distributed evenly on the tube PM apex and flanks (Fig. 3F). Time-lapse imaging revealed that, in nongrowing pollen, the GFP-SEC3a signal on the PM was less dynamic and maintained its location at the flanks (Fig. 3F; Supplemental Movie S3).

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Figure 3.

Localization of GFP-SEC3a in growing Arabidopsis pollen tubes. A, In vitro-germinated Arabidopsis pollen stably expressing LAT52:GFP-SEC3a. B, Apex of the LAT52:GFP-SEC3a pollen tube. The arrowhead marks the membrane localization at the tip. C, The same apex shown in B with GFP signal converted into an intensity scale. D, A nongrowing pollen tube stably expressing LAT52:GFP-SEC3a. Note the absence of the protein from the tip. E, Selected time-lapse images showing the localization of GFP-SEC3a in a growing pollen tube. Note that apical localization of GFP-SEC3a is dynamic and the protein accumulates at the future direction of tube growth (arrowhead). The arrow represents the direction of growth as predicted by GFP-SEC3a localization. F, Localization of GFP-SEC3a in a nongrowing pollen tube. The intensity of the GFP signal decreases with time due to high magnification and frequent imaging, and the last frame (210 s) shows the final direction of tube growth after 3.5 min. Note the cone-shape accumulation of fluorescent signals in growing pollen tubes and their absence in nongrowing tubes. G, Time-lapse images showing the localization of GFP-SEC8 in a growing pollen tube of an in vitro-germinated Arabidopsis pollen, stably expressing PSEC8:GFP-SEC8 in a sec8-1 mutant background. In A, C, and D to G, GFP fluorescence is represented as an intensity color scale. Bars = 10 µm in A and D and 5 µm in B, C, and E to G.

To compare these dynamics to those of another exocyst core subunit, we visualized PSEC8:GFP-SEC8 (Fendrych et al., 2010) in Arabidopsis pollen tubes and observed essentially identical localization and dynamics as for SEC3a (Fig. 3G). From these results, we can conclude that pollen tube elongation is characterized by the presence of a mobile polar GFP-SEC3a fraction at the tip that likely marks the sites of exocytic vesicle fusion with the PM and defines the direction of the growth axis.

GFP-SEC3a Tip Accumulation Correlates Directly with the Deposition of PI-Labeled Cell Wall Pectins

To further study the function of SEC3a, we examined its localization relative to PI, which labels esterified pectins in pollen cell walls (Rounds et al., 2014) and has been used previously as a marker for exocytosis in the pollen tube (McKenna et al., 2009). PI was applied to growing pollen at a concentration of 10 µm, and imaging was performed immediately, since under our experimental conditions, pollen tube elongation was inhibited 3 to 5 min after the addition of the dye. In agreement with published data, we observed the strongest PI labeling at the pollen tube tip (Fig. 4A). The GFP-SEC3a signal was observed at the tip PM exactly below the strongest PI staining (Fig. 4B). During pollen tube growth, the pattern of high-PI fluorescence at the tip was maintained, and GFP-SEC3a signal decorated the adjacent plasma membrane (Fig. 4C). We noted that the strongest PI fluorescence at the tip was associated with thicker cell walls at this region compared with the flanks (Fig. 4D), indicating a massive secretion of cell wall material at this location. The direct correlation between PI and GFP-SEC3a signals strongly suggests that the exocyst directs secretory vesicles with essential cell wall components, such as pectins, to the sites of polar exocytosis.

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Figure 4.

Labeling of GFP-SEC3a Arabidopsis pollen tubes with PI. GFP-SEC3a Arabidopsis pollen tubes were stained with 10 µm PI. A, Apex of a pollen tube showing the localization of GFP-SEC3a and PI with intensity scale for each channel. Asterisks mark positions on the intensity plot in B. B, Intensity plot of GFP and PI signals across the plasma membrane/cell wall of pollen apex (including flanks). Asterisks mark positions on the corresponding image in A. C, Time-lapse images of a growing pollen tube. Note that GFP-SEC3a decorates the PM beneath the strongest PI signal at the tip. D, Enlarged images of the pollen tip showing thick cell wall at the tip (arrowheads) and GFP-SEC3a signal decorating the PM at this region. a.u., Arbitrary units. Bars = 5 µm.

GFP-SEC3a Tip Accumulation Is Correlated Inversely with the PM and Endocytic Vesicle Labeling

Next, we focused on the distribution of GFP-SEC3a compared with recycling/endocytic vesicles. For the visualization of PM and endocytic vesicles, the FM4-64 dye was applied to pollen at a concentration of 1 μm. Under these experimental conditions, FM4-64 did not affect the elongation of pollen tubes (around 4 µm min⁻¹ in 13 out of 16 examined pollen tubes), allowing the observation of early stages of FM4-64 labeling. FM4-64 fluorescence reached levels compatible with imaging 3 to 5 min after the addition of the dye. Initially, the staining was detected in the shank and was significantly weaker at the tip PM, while the typical inverted cone of FM4-64-labeled vesicles was not yet observed (Fig. 5A). Importantly, weak tip labeling by FM4-64 was correlated inversely with the accumulation of GFP-SEC3a at the tip and was maintained during pollen tube growth (Fig. 5, B and C). The same pattern of FM4-64 fluorescence was observed following the labeling of wild-type Col-0 nontransgenic pollen tubes (Supplemental Fig. S5), indicating that the low FM4-64 fluorescence at the tip was not due to quenching by GFP-SEC3a. This FM4-64 labeling pattern could be explained by very fast endocytosis at the tip or, more likely, by dilution of the tip membrane by extensive exocytosis, resulting in FM4-64-labeled plasma membrane being pushed away from the tip to the shank. Under our experimental conditions, the typical inverted cone of FM4-64-labeled vesicles was observed 20 to 30 min after application of the dye. However, the distribution of highest FM4-64 and GFP-SEC3A intensities in the inverted cone area was spatially separated (Fig. 5D; compare the intensity range distribution of GFP and FM4-64). The PM at the tip was characterized by a region with low FM4-64 and high GFP-SEC3a signals, both oriented toward the growing direction of the tube, further supporting the extensive exocytosis of de novo-delivered vesicles at this region (Fig. 5, E and F). Importantly, in nongrowing pollen tubes, FM4-64 effectively labeled tip PM (Supplemental Fig. S6). Taken together, these results suggested that the FM4-64-labeled inverted cone consisted mostly of recycling vesicles and was partially separable from the site of exocyst-dependent exocytosis.

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Figure 5.

Labeling of GFP-SEC3a Arabidopsis pollen tubes with FM4-64. GFP-SEC3a Arabidopsis pollen tubes were labeled with 1 µm FM4-64 for 3 min (A–C) or 20 min (D–F) and then subjected to imaging. A and D, Apex of the pollen tube showing the localization of GFP-SEC3a and FM4-64 with intensity scale for each channel. Asterisks mark positions on the intensity plots in B and E. The arrowhead in D points to the region with high GFP-SEC3a and low FM4-64 signals. B and E, Intensity plots of GFP and FM4-64 signal across the PM of the pollen apex (including flanks). Asterisks mark positions on the corresponding images in A and D. C and F, Time-lapse images showing the localization of GFP-SEC3a and FM4-64 in growing pollen tubes. In F, the white arrows mark the direction of pollen tube elongation as predicted from tip geometry, and the yellow arrow indicates the actual growth direction. Note that the position of high GFP and low FM4-64 signals correlates with growth direction. a.u., Arbitrary units. Bars = 5 µm.

Together, the data in Figures 4 and 5 and Supplemental Movies S1 and S2 implicate SEC3a as a bona fide marker for polarized exocytosis in Arabidopsis pollen tubes and suggest that the deposition of cell wall and membrane material is guided by the exocyst complex and is likely partially separable from vesicle recycling at the tip.

Partially Complemented sec3a-1 Pollen Develop Multiple Tips

To further study the function and localization of SEC3a in pollen development, we examined the phenotype of sec3a-1 mutant pollen complemented with GFP-SEC3a (Fig. 6). As expected from the self-crosses and reciprocal crosses displaying complementation of the male transmission of sec3a-1 (Table I; Supplemental Tables S1 and S2), normal-looking pollen tubes were observed in which GFP-SEC3 was localized at the tip (Fig. 6, A and B). However, the growth rate of the sec3a-1;GFP-SEC3a pollen tubes was around 1.5 µm min⁻¹ (n = 7) compared with 3.5 to 4 µm min⁻¹ for wild-type Col-0/GFP-SEC3a pollen tubes. Interestingly, in some pollen grains, multiple tips germinated from more than one germination pore, indicating that SEC3a is required to determine the pollen tube germination site. Similar to its distribution in wild-type Col-0, in elongating sec3a-1 pollen tubes, GFP-SEC3a accumulated at the apical tip and was spatially separable from FM4-64-labeled PM (Fig. 6, C and D).

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Figure 6.

Expression of GFP-SEC3a in the sec3a-1 homozygous background results in the development of pollen with multiple tips. A, Germinating sec3a-1 pollen expressing GFP-SEC3a. The multiple tips are marked by numbers. B, In pollen tubes with one tip, GFP-SEC3a concentrates at the apex (GFP fluorescence is shown as an intensity color scale). C, Selected time-lapse images of sec3a-1/GFP-SEC3a pollen labeled with 1 µm FM4-64 for 20 min. Note that GFP-SECa effectively accumulates at the apical plasma membrane. D, Intensity plot of GFP and FM4-64 signal across the PM of pollen apex (including flanks). Asterisks mark positions on the corresponding image in C. E to H, Immunolabeling of deesterified pectins (E and F) and esterified pectins (G and H) with LM19 and LM20 monoclonal antibodies, respectively. E and G, Nontransgenic Col-0 pollen. F and H, sec3a-1/GFP-SEC3a pollen. The white rectangles in E and G highlight the enlarged regions shown in the middle image of each panel. The black arrows highlight germinated pollen tubes in F and the actively growing pollen tubes in H. The yellow arrows in H highlight the arrested small pollen tubes. I, Localization of GFP-SEC3a in multiple-tip sec3a-1/GFP-SEC3a pollen. The white arrow denotes the growing pollen tube, and the yellow arrow indicates the arrested pollen tube. See also Supplemental Movies S4 and S5. a.u., Arbitrary units. Bars = 20 µm in A, B, E, and G, 10 µm in F, H, and I, and 5 µm in C.

In the multiple-tip sec3a-1/GFP-SEC3a complemented pollen, one pollen tube continues growing while the other(s) arrests. Likely, the overexpression of GFP-SEC3 is able to induce multiple germination pore activation but is unable to maintain the multiple tip growth, resulting in only one growing tip. This feature allowed us to further characterize the function of SEC3a in pollen tube growth and correlate it with the secretion of esterified pectins. LM19 and LM20 are monoclonal antibodies that recognize deesterified and methyl-esterified homogalacturonans (pectins), respectively (Verhertbruggen et al., 2009). In wild-type Col-0, LM19-labeled deesterified pectins were detected from the early stages of pollen germination at the emerging bud and in elongated pollen tubes at the tip and along the shank (Fig. 6E). In sec3a-1/GFP-SEC3 multiple-tip pollen, deesterified pectins were observed both in the growing and in the arrested tubes (Fig. 6F). In agreement with published results on tobacco and Arabidopsis pollen tubes (Chebli et al., 2012; Leroux et al., 2015), the LM20-labeled esterified pectins were observed only at the tip of elongating pollen tubes and at the bud of germinating pollen, where they are secreted (Fig. 6G). Significantly, in sec3a-1/GFP-SEC3a pollen, the esterified pectins were observed only at the tip of the growing pollen tubes but not in the arrested pollen tubes (Fig. 6H). Taken together, the results in Figure 6, F and H, indicated that esterified pectins are actively secreted only in the growing tube of sec3a-1/GFP-SEC3a, while the arrested tube only contains pectins that have been deesterified.

Unfortunately, GFP-SEC3a signal at the tip PM was lost following the fixation and labeling of cell wall epitopes; in addition, the protocol is not compatible with antibody labeling of intracellular proteins. As an alternative, we examined the localization of GFP-SEC3a at the growing and arrested pollen tubes at high resolution. In all pollen that were examined (n = 10), the typical GFP-SEC3a tip-focused PM fraction was detected only at the tip of the growing tube, while no tip-focused PM fraction was detected in the arrested pollen tubes (Fig. 6I; Supplemental Movies S4 and S5). These data indicated that there is strong positive correlation between the localization of GFP-SEC3a at the tip of growing pollen tubes and the secretion of esterified pectins and further established GFP-SEC3a as an intracellular marker for exocytosis.

Plasma Membrane Localization of SEC3a in Tobacco Pollen Tube Requires an Intact N-Terminal PH Domain

The transient expression of fluorescent proteins in tobacco pollen tubes is a commonly used model system for studying the localization of polarity determinants, due to the larger diameter and well-defined zonation of the pollen tube apex compared with Arabidopsis. LAT52:YFP-SEC3a and LAT52:SEC3a-YFP fusion proteins were transiently expressed in tobacco pollen tubes. Interestingly, the distribution of SEC3a in tobacco pollen partially differed from that observed in Arabidopsis and depended on the mode of cell elongation. In pollen tubes showing steady nonoscillatory growth, both YFP-SEC3a and SEC3a-YFP showed cytoplasmic localization with enhanced fluorescence at the PM in the subapical region, with maximum signal located 2 to 7 μm behind the apex (Fig. 7A; Supplemental Fig. S7). It is unlikely that this localization was due to the heterologous nature of Arabidopsis SEC3a in tobacco pollen, since the tobacco SEC3a homolog localized to the same region (Supplemental Fig. S7). During steady growth, the localization of the full-length SEC3a remained essentially subapical, although some periodic changes in signal intensity could be observed (Fig. 7, B and D). In contrast, the localization of SEC3a in oscillating tobacco pollen tubes also showed an oscillatory behavior, alternating between cytoplasm and apical plasma membrane (Fig. 7, A and C). Kymograph analysis suggested that a period of fast elongation was associated with the accumulation of SEC3a in the cell apex, which was followed by a temporal growth arrest and concomitant dissociation of SEC3a from the membrane (Fig. 7E). In nongrowing tobacco pollen tubes, SEC3a was distributed evenly along the apex and subapical regions (Supplemental Fig. S7).

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Figure 7.

The N-terminal PH domain of Arabidopsis SEC3a colocalizes with PIP₂ and is responsible for the localization of SEC3a at the plasma membrane in tobacco pollen tubes. A, Localization of full-length YFP-SEC3a transiently expressed in tobacco pollen tubes under the control of the LAT52 promoter. B and C, Selected time-lapse images showing the localization of LAT52:YFP-SEC3a in steady-growing (B) and oscillating (C) pollen tubes. D and E, Kymograph analysis of growth and apical YFP-SEC3a fluorescence in steady-growing (D) and oscillating (E) pollen tubes. Arrowheads in C and E point to dynamic apical localization of SEC3a in oscillating pollen tubes. F and G, Localization (F) and kymograph analysis (G) of the SEC3a N-terminal domain (LAT52:YFP-SEC3a-N). H and I, Localization (H) and kymograph analysis (I) of SEC3a lacking its N-terminal domain (LAT52:YFP-SEC3a-ΔN). J, Colocalization of YFP-SEC3a-N with the PIP₂ marker mRFP1-PH_PLCδ1. In A to I, YFP fluorescence is represented as an intensity color scale. PLC, Phospholipase C. Bars = 5 µm.

It has been demonstrated that the N-terminal domain of the yeast Sec3p, consisting of amino acids 1 to 320, interacts with PIP₂ (Zhang et al., 2008). Additional studies showed that the 75 to 248 domain of yeast Sec3p folds into a PIP₂-binding PH domain (Baek et al., 2010; Yamashita et al., 2010). To get an evolutionary insight into SEC3 function, we identified SEC3 homologs in diverse eukaryotic species. Although the general sequence identity between SEC3 homologs is rather low, we found that most of them contain a region corresponding to the PH domain. Calculation of the phylogenetic relationship between SEC3 amino acid sequences, based on either the SEC3 domain or the PH domain, showed that the distribution of SEC3 mostly follows organismal evolution (Supplemental Fig. S8). Supplemental Figure S9 shows an alignment of N-terminal regions corresponding to the PH domain of selected SEC3 proteins. We then fused the N-terminal 180-amino acid domain of Sec3a (SEC3a-N; the region corresponding to the yeast PH domain) to YFP and used it for transient expression in tobacco pollen tubes. YFP-SEC3a-N exhibited the same subapical localization pattern that was observed for the full-length protein in steady-state growing cells (Fig. 7, F and G). Interestingly, YFP-SEC3a-N and SEC3a-N-YFP showed an even stronger association with the plasma membrane than the full-length protein (Fig. 7F; Supplemental Fig. S7).

Since the canonical role of PH domains is the interaction with membrane phosphatidylphosphoinositides (PPIs), we next tested whether SEC3a-N localization corresponds to the distribution of PIP₂ in the PM. The PIP₂ marker mRFP1-PH_PLCδ1 (Helling et al., 2006) localized to the PM with enhanced fluorescence at the subapical region of the pollen tube apex. YFP-SEC3a-N colocalized with mRFP1-PH_PLCδ1 at the pollen tube apex, suggesting that its localization involves an association with PIP₂ (Fig. 7J). This experiment suggested that the N-terminal part of SEC3a is able to bind signaling phospholipids. Interestingly, the overexpression of full-length SEC3a or SEC3a-N in pollen tubes caused a marked reduction of growth rate, induced the swelling of pollen tube tips, and was associated with a nondynamic distribution of fluorescence along the pollen tube tip and shank (Supplemental Fig. S7), similar to nongrowing tobacco and Arabidopsis tubes. To further examine whether the SEC3a N-terminal domain is involved in membrane localization, we transformed tobacco pollen tubes with truncated SEC3a lacking the N terminus (i.e. region 181–887; SEC3a-ΔN). In agreement with the colocalization of SEC3a-N with PH_PLCδ1, both YFP-SEC3a-ΔN and SEC3a-ΔN-YFP localized only to the cytoplasm (Fig. 7, H and I; Supplemental Fig. S7).

The Combination of Biochemical Approaches and Structural Modeling Reveal That SEC3a-N Specifically Binds Membrane Phosphoinositides

Given the fact that, despite their low sequence identity, PH domains form a similar fold with a core consisting of seven antiparallel β-strands capped by a terminal α-helix (Lemmon, 2008), we modeled a structure of Arabidopsis SEC3a-N using the template-based approach with crystalized yeast Sec3p-N (3A58; Yamashita et al., 2010) as a template. The resulting model was energetically relaxed by short all-atom molecular dynamics (MD) simulation (30 ns). The final model of SEC3a-N (Fig. 8A) had a ProSA score of −4.92 (Wiederstein and Sippl, 2007), which is comparable to the template score (−5.43), thus implying that, despite the low primary sequence identity, our computed model is valid.

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Figure 8.

SEC3a-N binds phosphoinositides via four positively charged residues that mediate its association with the plasma membrane in tobacco pollen tubes. A, Comparison of the final relaxed SEC3a-N homology model (At) with the yeast template structure (Sc). B, Liposome-binding assay of GST-SEC3a-N. PIP₂ binding was determined using 200-nm vesicles containing 5% PIP₂:95% PC or PC alone. After incubation of GST-SEC3a-N with the vesicles, they were recovered by ultracentrifugation, and protein bound (Pel) was analyzed by SDS-PAGE. As a negative control, GST alone was used. Representative results from three independent experiments are shown. Sup, Supernatant. C, Analysis of MD simulations displaying polar contacts of SEC3a-N with a PIP₂ molecule. The bars represent average numbers of polar contacts through the last 8 μs of the simulation. Polar contacts were defined as the number of PIP₂ head group atoms within 8 Å of protein atoms. The inset image represents a coarse-grained model of SEC3a-N coordinating the PIP₂ molecule. The interacting residues are highlighted. D, Lipid-binding properties of wild-type GST-SEC3a-N and mutated GST-SEC3a-N KRKR/A recombinant proteins, as determined using a protein-lipid overlay assay. DAG, Diacylglycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI4P, phosphatidylinositol 4-phosphate; PI4,5P₂, phosphatidylinositol 4,5-bisphosphate; PI3,4,5P₃, phosphatidylinositol 3,4,5-trisphosphate; PS, phosphatidylserine; TAG, triacylglycerol. E, Localization of wild-type YFP-SEC3a-N and the mutated variant YFP-SEC3a-N KRKR/A in tobacco pollen tubes. YFP fluorescence is represented as an intensity color scale. Bars = 5 µm.

To confirm that SEC3a-N directly binds PIP₂, we fused it to glutathione S-transferase (GST-SEC3a-N) and expressed it in Escherichia coli. The purified recombinant GST-SEC3a-N was used in two widely applied in vitro lipid-binding experiments: the large unilamellar vesicle cosedimentation assay and the protein-lipid overlay assay. In large unilamellar vesicle cosedimentation assays, GST-SEC3a-N bound to large unilamellar vesicles containing 5% PIP₂ in phosphatidylcholine (PC) but not to vesicles containing only PC (Fig. 8B). In agreement, GST-SEC3a-N bound several PPIs, including PIP₂, in protein-lipid overlay assays (Fig. 8D).

To further test the binding modes of PIP₂ by SEC3a-N, we used coarse-grained MD with the MARTINI force field (Marrink et al., 2007; Monticelli et al., 2008) and simulated the self-assembly of a lipid bilayer in the presence of the modeled structure of the SEC3a-N domain, as this approach was shown to be advantageous for the characterization of peripheral membrane protein dynamics (Pleskot et al., 2012). The simulation box contained the SEC3a PH domain, 256 lipids, water, and ions. In total, we performed 16-μs-long simulations for each system. We observed that, in simulations with one PIP₂ molecule in the PC bilayer, the Arabidopsis SEC3a PH domain was targeted rapidly to the membrane and remained bound to PIP₂, showing that the system was in equilibrium. In contrast, in a system with a PC-only membrane, SEC3a-N was never bound to the bilayer and remained in the virtual cytoplasm. Analysis of amino acid residues interacting with the PIP₂ molecule, as revealed by the coarse-grained MD simulation, showed that the PIP₂ molecule was coordinated by several positively charged amino acid residues located between loops β1/β2 and β3/β4, namely Lys-34, Arg-36, Lys-51, Arg-53, and Lys-74 (Fig. 8C).

To test the importance of positively charged residues for PIP₂ binding, we generated a mutated GST-SEC3a-N, where the residues with the greatest number of polar contacts with the PIP₂ molecule, namely Lys-34, Arg-36, Lys-51, and Arg-53 (Fig. 8C), were changed to Ala. The resulting GST-SEC3a-N KRKR/A mutant did not interact with PIPs in vitro (Fig. 8D). Moreover, the YFP-SEC3a-N KRKR/A mutant completely lost the ability to bind the plasma membrane following transient expression in tobacco pollen tubes (Fig. 8E). Interestingly, the overexpression of mutated YFP-SEC3a-N never induced the depolarization of the pollen tube tip caused by wild-type YFP-SEC3a-N (Supplemental Fig. S7). These results indicated that the N-terminal region of SEC3a contains a functional PIP₂-binding domain capable of interaction with the PM and suggested that binding to PIP₂ might have an important role in subapical shank localization of SEC3a.

The N-Terminal PH Domain Is Not Essential for SEC3a Function and Localization in Arabidopsis Pollen

Next we employed genetic analysis to examine the importance of the N-terminal PH domain for SEC3a function. To this end, we complemented the sec3a-1 mutant with LAT52:SEC3a-∆N, which lacks the first 180 amino acids, and with LAT52:SEC3a KRKR/A. Successful complementation of male transmission defects associated with the sec3a-1 allele was observed in several independent lines expressing LAT52:SEC3a-∆N, LAT52:GFP-SEC3a-∆N, LAT52:SEC3a KRKR/A, and LAT52:GFP-SEC3a KRKR/A (Supplemental Table S4). The ability of SEC3a-∆N, GFP-SEC3a-∆N, SEC3a KRKR/A, and GFP-SEC3a KRKR/A to complement the male transmission defect of the sec3a-1^+/− mutant indicates that the SEC3a N-terminal domain and a functional PH domain are not absolutely required for SEC3a function in Arabidopsis. To address whether the PM association of the N-terminal SEC3a mutants was affected, we analyzed the localization of GFP-SEC3a KRKR/A and GFP-SEC3a-∆N stably expressed in Arabidopsis transgenic pollen tubes. Both mutants exhibited enrichment of the GFP signal at the pollen tube apex similar to GFP-SEC3a (Fig. 9, A–C). Moreover, time-lapse imaging revealed that, similar to wild-type GFP-SEC3a, the GFP-SEC3a KRKR/A and GFP-SEC3a-∆N mutants are highly dynamic and decorate the PM at the pollen tube tip (Fig. 9D; Supplemental Movies S6–S9). Hence, the localization and function of SEC3a in Arabidopsis pollen tubes can take place independently of PIP₂ binding.

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Figure 9.

The N-terminal PH domain of SEC3a is not required for its accumulation at the tip of growing Arabidopsis pollen tubes. A to C, In vitro-germinated Arabidopsis pollen tubes stably expressing GFP-SEC3a (A) GFP-SEC3a KRKR/A (B), and GFP-SEC3-ΔN (C) under the regulation of the LAT52 promoter. D, Selected time-lapse images of growing GFP-SEC3a, GFP-SEC3a KRKR/A, and GFP-SEC3a-ΔN Arabidopsis pollen tubes. GFP fluorescence is represented as an intensity color scale. Bars = 10 µm in A to C and 5 µm in D.