Journal of Experimental Botany, Vol. 54, No. 380, pp. 55-63,
January 1, 2003
© 2003 Oxford University Press
Characterization and localization of the transmitting tissue-specific PELPIII proteins of Nicotiana tabacum
Received 15 March 2002; Accepted 31 May 2002
Department of Experimental Botany, Laboratory of Plant Cell Biology, Graduate School of Experimental Plant Science, University of Nijmegen, Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands
1 To whom correspondence should be addressed. Fax: +31 24 3652490. E-mail: Mariani@sci.kun.nl
2 Present address: Department of Biochemistry and Molecular Biology. J.W. Lederle Graduate Research Center, Box 34505, University of Massachusetts, Amherst, MA 01003-4505, USA.
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Abstract |
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The class III pistil-specific PELP proteins (PELPIII) of Nicotiana tabacum includes at least two members of highly soluble glycoproteins containing glucan modules that are characteristic for arabinogalactan proteins (AGPs). PELPIII accumulates in the style transmitting tissue (TT) during pistil development and, at flower anthesis, is present in the intercellular matrix (IM) of non-pollinated pistils. After pollination, PELPIII appears to be directly and completely translocated from the IM into the pollen tube callose walls, no significant accumulation was observed in the primary wall in the tip. In the spent parts of the pollen tubes these proteins become detectable against the remnants of the tube cell membrane and in the callose plugs. Different protein extraction procedures of PELPIII from pollinated tobacco pistils showed that these proteins remain in the highly soluble protein fraction and are not modified by the growing pollen tubes. These data concur with a role in IM development and pollen tube growth. In addition, the data show that the PELPIII are able to reach the cell membrane, facilitated by an already present or induced high porosity of the tube wall and an additional, yet unknown, mechanism. The differences in behaviour between the three related classes of style IM glycoproteins of Nicotiana, namely, PELPII, TTS and the120 kDa glycoprotein, are proposed to connect more to their differences in glycosylation than to major differences in amino acid sequence.
Key words: Callose, Nicotiana tabacum, PELPIII, pistil, pollen tube, transmitting tissue.
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Introduction |
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During style development of Nicotiana tabacum (tobacco), the adjacent L1 layers of the developing style differentiate into the stigma secretory zone and style transmitting tissue (TT), that are designed to aid in pollen germination and then pollen tube growth towards the ovules. By far the longest stretch traversed by the pollen tubes is the TT, where pollen tubes grow through the almost liquid intercellular matrix (IM) separating the TT cells. In particular, the IM is thought to be the active element in aiding pollen tube growth (Sanders and Lord, 1992; de Graaf et al., 2001). In the TT, a number of pistil-specific glycoproteins belonging to various classes of proline- and hydroxyproline-rich glycoproteins accumulate, but no general function could be assigned to either of these glycoprotein classes (Sommer-Knudsen et al., 1997; Cheung and Wu, 1999; de Graaf et al., 2001; Wu et al., 2001). Thus, besides similarities, differences appear to exist, even between proteins from a single class. For example, comparison of the TT-specific glycoproteins or TTS-glycoproteins (Cheung et al., 1993) with the closely related galactose-rich style glycoprotein (GaRSGP) (Sommer-Knudsen et al., 1996) showed a number of differences (Wu et al., 1995, 2000). TTS and GaRSGP are very similar at the AA level, but their sugar moieties are different and TTS-glycoproteins react with the Yariv reagent whereas GaRSGP does not. The TTS-glycoproteins of N. tabacum and N. alata are present in the IM of the TT and are directly involved in pollen tube growth (Cheung et al., 1995; Wu et al., 1995, 2000), whereas GaRSGP was associated with the TT cell walls in N. alata, and no influence on pollen tube growth was detected. Therefore, GaRSGP was suggested to be a structural cell wall protein (Sommer-Knudsen et al., 1996, 1998).
Besides TTS-glycoproteins, three classes of specifically pistil-expressed genes from tobacco coding for proline-rich proteins (PELPI, II and III) have been characterized at the mRNA level (Goldman et al., 1992). PELPIII proteins were shown to have extremely large AGP-like glucan moieties (Bosch et al., 2001).
Here the distribution and accumulation of PELPIII in the TT during tobacco pistil development and after pollen tube growth in the style are described. The results show that the walls of in vivo growing tobacco pollen tubes do not form any barrier for the high molecular weight PELPIII. The possible involvement of PELPIII in IM formation and pollen tube growth is discussed.
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Plant material
Nicotiana tabacum SR1 plants were raised in the greenhouse and transferred to a climate chamber for a maximal constant environment, temperature 18–20 °C, relative humidity 65% and a 15/9 h light/dark rhythm. For the pollination experiments, flowers at stage 11 of development (Goldberg, 1988) were emasculated and pollinated with fresh pollen 1 d later at stage 12 (at anthesis). The samples were collected after different time intervals, according to the type of experiment.
Production of polyclonal antibodies against PELPIII
The QIAEXPRESS (QIAGEN) recombinant expression system in E. coli was used to produce a fusion protein of 39 kDa comprizing the DHFRS protein together with 163 AA of the C-terminal, PELPIII specific domain of MG15, one of the two sequences MG14 and MG15 coding for PELPIII (Goldman et al., 1992). After purification of the insoluble recombinant protein (BG22) according to the manufacturer’s instructions, final purification occurred by SDS-PAGE. The band with the recombinant protein was excised and used for the production of polyclonal antibodies (I-C3P) in rabbits (EUROGENTEC, Belgium). A sample of rabbit serum (pre-immune serum, PI-C3P) was taken before immunization and used as a control in all experiments.
Protein isolation
To analyse the tissue specificity of PELPIII, various plant tissues were dissected and immediately frozen in liquid nitrogen. The frozen tissues were ground to a fine powder and extracted in 50 mM phosphate buffer pH 7.0 with 0.6% PVPP, 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, 25 µg ml–1 PMSF, and 10 mM ß-mercaptoethanol. The extraction mixtures were kept on ice for 30 min., vortexed for 5 min, and centrifuged to pellet cellular debris. In addition several types of extraction buffers (Table 1) were used to analyse the accumulation profiles of PELPIII during pistil maturation and upon pollination. The insoluble fractions were analysed by extractions in buffer 3, 4 or 5 (Table 1) followed by a sonication (2x20 s 16 000 amplitude) or a boiling step of 10 min. After centrifugation, the protein fraction in the supernatant was precipitated with 5 vols of ice-cold acetone, washed with 70% EtOH, air-dried, and dissolved in a small volume of 0.1 N NaOH. The protein concentrations of the different fractions were estimated either by the Bradford method (Biorad), or by v/w-ratio estimations of equal volumes of total protein extracts.
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Pollination experiments were performed with mature and immature flowers (bud-pollinations). For the bud-pollinations, tobacco flowers at stages 5 and 7 were emasculated and pollinated, respectively, at stages 6 and 8. Tissue samples were collected for protein analysis at 24, 48 and 72 h after pollination. Pistils were dissected in the stigma (S), and in the lower (L) and the upper (U) halves of the style. The different parts were separately subjected to protein analysis. Non-pollinated pistils or pistil-parts were used as a control. Extraction buffer 2 was used (v:w-ratio: 1) for the isolation of soluble proteins. After centrifugation, the soluble fractions were divided into small aliquots, frozen in liquid nitrogen and stored at –20 °C. The insoluble pellet-fractions were washed 3x in 1.0 ml of buffer 2. After weighing, the pellets were re-extracted with buffer 3 (v:w-ratio: 2). Relatively short extractions with the high-salt buffer did not result in a significant additional release of PELPIII from the pellets, but overnight incubation on ice, followed by several short vortex steps appeared more efficient.
Western blot analysis of PELPIII
Protein extracts were analysed in duplo with SDS-PAGE and electroblotting, using the Biorad Mini-Protean II apparatus. Incubation of the blots occurred by gently shaking in blocking buffer (1x PBS or TBS, 5% skimmed milk powder [ELK] and 0.05% NaAz) or sera diluted in the blocking buffer. The blots were incubated for at least 2 h at room temperature or overnight at 4 °C, then the blocking buffer was replaced by the I-C3P immune serum diluted in blocking buffer, 1:1000 for native proteins and 1:5000 for recombinant PELPIII, and incubation continued for 2 h at room temperature. PI-C3P pre-immune serum served as a control. After incubation, the filters were washed with blocking buffer, three times for 15 min and incubated for 1 h with an alkaline phosphatase conjugated goat anti-rabbit antibody (Pierce ImmunoTechnology, USA) diluted 1:2000. The filters were then washed once with 1x PBS or TBS/5% ELK and next twice with only 1x PBS or TBS. The washed filters were presoaked in 1x carbonate buffer (0.1 M NaHCO3, 1.0 mM MgCl2, pH 9.4), the detection reaction occurred with freshly prepared 0.3 mg ml–1 NBT and 0.15 mg ml–1 BCIP in 1x carbonate buffer pH 9.4.
Immunogold detection of PELPIII by TEM
Stigmas and styles were collected from the plants and immediately cut into 1–2 mm pieces in freshly prepared fixative (2% glutaraldehyde (GA), 1% paraformaldehyde (PFA), 2% sucrose, 0.05% CaCl2.2H2O, and 0.1% Tween 20 in 100 mM PIPES-buffer (pH 7.0), and left in the fixative under low vacuum for 3 h at room temperature. After fixation, the samples were washed, once in a large volume of buffer without GA and PFA, and twice (30 min) in 100 mM PIPES buffer (pH 7.0). Finally, the samples were kept overnight in PIPES buffer at 4 °C. Next day, after a short rinse in fresh 100 mM PIPES buffer, the procedure was continued by dehydration in 10%, 30%, 50%, 70%, 90%, and 95% ethanol, 30 min for each step. The last two steps in 100% absolute ethanol took 60 min each. Infiltration with LR-White occurred via three intermediate steps, first a 1:1 mixture of propylene-oxide:ethanol (30 min), followed by 100% propylene-oxide (60 min) and a 1:1 mixture of propylene-oxide:LR-White (overnight at 4 °C). Finally, the mixture was replaced by LR-White, kept for 6 h at 4 °C, changed once more with fresh LR-White, and kept for 3 d at 4 °C. The samples were transferred to gelatine capsules with LR-White and cured at 55 °C for at least 24 h.
The immunogold labelling was performed on longitudinal and transverse ultra-thin sections from the upper and the lower part of tobacco styles. Formvar-coated copper grids (Stork Veco BV, Eerbeek, The Netherlands) with sections were incubated with a 1:50 dilution of the I-C3P antiserum or pre-immune serum in 50 mM phosphate buffer with 5% BSA (pH 7) for 2 h at room temperature. After washing twice on a drop of phosphate buffer/5% BSA, grids with sections were transferred to a colloidal 10 nm-gold conjugated Protein A diluted 1:50 in the same buffer and incubated for 30 min at room temperature. The samples were washed again, air-dried and treated for 2 min with OsO4-vapour to enhance contrast. Preparations were examined and photographed in a JEOL JEM 100 CX II used for transmission electron microscope (TEM).
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Production of antibodies against PELPIII
The I-C3P anti-PELPIII antibodies gave a strong signal with purified 39 kDa recombinant PELP (BG22) after Western blotting and no signal was recorded with the preimmune serum (data not shown). As the polyclonal antibodies were raised against the cysteine-rich C-terminal domain of pMG15, which is almost identical in all members of the class III PELPs (Goldman et al., 1992), the I-C3P serum should be classified as class III-specific.
Analysis of stigma and style extracts of Nicotiana tabacum SR1 plants (Fig. 1) showed that the I-C3P serum cross-reacted specifically with 110–140 kDa proteins (according to Bosch et al., 2001). The preimmune serum (PI-C3P) showed no cross-reactivity with any proteins (data not shown). Similar amounts of PELPIII were also detected in crude extracts of styles from the transgenic stigma-less plants (Goldman et al., 1994) in which the secretory zone of the stigma was ablated (Fig. 1). No cross-reactivity was found with any other protein in crude extracts of stem, leaf, and flower buds at stages 1–3 of development (Fig 1; Goldberg, 1988).
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IM-specific glycoproteins that may share putative antigenic regions within their C-terminal domains (Schultz et al., 1997; Cheung and Wu, 1999; de Graaf et al., 2001) may cross-react with the I-C3P antibodies. The TTS-proteins are known to be deglycosylated by the growing pollen tubes (Wu et al., 1995), and so might become accessible to the I-C3P serum in pollinated pistils. Therefore, Western blot analyses were performed on recombinant TTS-protein (C-terminal domain), a crude extract of the native glycosylated TTS-proteins, and a crude extract of HF-deglycosylated native TTS-proteins (protein blots kindly provided by Dr AY Cheung). Incubation with I-C3P anti-PELPIII serum showed an extremely weak cross-reaction, but only with large quantities of the recombinant TTS-proteins (data not shown). Therefore, it was concluded that the I-C3P antibodies specifically recognize pistil-specific PELPIII proteins of tobacco.
Developmental profile of PELPIII accumulation in non-pollinated pistils
The high salt extraction buffer (Table 1, No. 3) did not result in a substantial increase of PELPIII recovery as compared to the extraction buffers for soluble proteins (Table 1, Nos 1, 2), though a very small increase might occur at the lower side (100 kDa) of the molecular spectrum. Therefore, it was concluded that the tobacco PELPIII proteins essentially are highly soluble. Figure 2A shows that PELPIII was detected first at stage 2 and gradually accumulated till stage 8, after which PELPIII levels seemed to remain constant. However, a dilution series of the same samples indicated a further gradual accumulation of PELPIII between stages 8 and 12 of pistil development (data not shown).
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Figure 2B shows a basipetal pattern of PELPIII accumulation during style maturation. Initially, low levels of mainly high molecular weight PELPIII were seen in the lower segment of the style, whereas higher levels of mainly lower molecular weight PELPIII occurred in the upper part. The accumulation of PELPIII continued in both parts until pistil maturity (stage 12) and up to 3 d after anthesis, in pollinated as well as non-pollinated flowers. The difference in PELPIII molecular weight in the two style segments was clearly visible from stages 3 to 10 and seemed to persist throughout development (Fig. 2B, NP-72h).
To exclude possible shifts in molecular weight caused by differences in loading concentration, a series of total crude protein extracts at various concentrations were made and analysed. Figure 2C shows that such a shift in molecular weight did not occur.
PELPIII analysis in pollinated pistils
To test whether the PELPIII proteins are modified by the growing pollen tubes in vivo, several pollination experiments were performed and the low-salt (highly soluble) and high-salt (insoluble) extractable protein fractions were analysed. The 110–140 kDa PELPIII could be recovered in similar amounts in either pollinated and non-pollinated pistils using a low salt extraction buffer (Table 1, No. 2), which extracts soluble proteins (Fig. 3). Also, about equal amounts of PELPIII were recovered from lower and upper stylar parts (data not shown), from both pollinated and non-pollinated stigmas and styles of mature and immature pistils (Fig. 3A, B). From 72–96 h after pollination, the use of low salt extraction buffer resulted in a less efficient recovery of PELPIII (Fig. 3B). Successsive extraction of the sample with high salt buffer resulted in an additional release of PELPIII, especially at the lower side of the molecular weight spectrum (Fig. 3C). Apart from the possibly wall-associated PELPIII in premature pistils, it was never possible to detect any modifications of PELPIII after pollination, neither by western blot analysis after loading a large excess of protein samples, nor by the use of different extraction buffers.
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Immunolocalization of PELPIII in tobacco pistils
To study the possible function of the class PELPIII in the TT further, an immunocytological analysis was performed with mature non-pollinated pistils and with specific style parts from pistils that were pollinated for 5, 16 or 24 h. Five hours after pollination the pollen tube tips just reached the TT, and 16 h after pollination they were present at about 2/3 of the length of the style. After 24 h, pollen tube tips reached the bottom of the style. The pollinated styles were dissected in parts with and without pollen tubes. Corresponding parts of non-pollinated pistils were taken as control.
Figure 4 shows a strong label of the TT of non-pollinated pistils at stage 12 incubated with the I-C3P serum (Fig. 4A), whereas no significant label could be detected in a similar section (Fig. 4B) after incubation with the preimmune serum (PI-C3P). Most PELPIII epitopes were detected in the intercellular matrix (IM) between the TT-cell walls. At pistil maturity, significant label of PELPIII was no longer detectable in the walls of the TT-cells.
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After pollination, the PELPIII epitopes disappeared from the IM and became visible in the pollen tube walls. Figures 5 and 6 show that the epitopes became gradually translocated into the secondary, callose, wall of the actively growing part of the pollen tubes, but also in the callose plugs in the spent parts of the pollen tubes (Fig. 6B). Figure 7 shows the PELPIII epitopes to accumulate in the callose plugs (Fig. 7A), but these also showed a reaction with the control serum PI-C3P, though much weaker (Fig. 7B). PELPIII epitopes were not detected in the primary, pectic outer wall of the pollen tubes, nor in pollen tube vesicles, vacuoles or cytoplasm. Similarly, it was not possible to detect PELPIII epitopes in in vitro-grown pollen tubes (Bosch, 2002). In most of the non-growing and spent parts of the collapsed pollen tubes, the distribution of the PELPIII was completely different from the distribution found in the growing part of the pollen tubes. Whereas PELPIII was equally distributed throughout the callose wall in the growing pollen tube parts (Fig. 6), in the spent pollen tubes most of the PELPIII was detected at the border between the callosic wall and the remnants of the pollen tube cytoplasm (not shown). Thus, from the results of several independent pollination experiments, different serial sections at different time-points and pollen tube positions in the pistil, it is concluded that PELPIII are directly translocated from the IM into the callose wall, and that there they are conveyed to the tube membrane and to the callose plugs. The underlying mechanism of PELPIII translocation from the IM into and through the pollen tube walls, however, has not been elucidated. The immunogold detections show that PELPIII proteins are directly and completely translocated into the pollen tube walls and that these do not constitute an impermeable barrier for a relatively large molecule such as the 110–140 kDa PELPIII.
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Discussion |
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During its development, the style TT cells are highly active in production and secretion of an almost liquid intercellular matrix (IM) which separates the longitudinally arranged files of TT cells. TT-specific products have been suggested to be involved in signalling, guidance, protection, and nutrition of the growing pollen tubes (Sanders and Lord, 1992; Kuboyama et al., 1994; Cheung et al., 1995; Sessa and Fluhr, 1995; Wu et al., 1995, 2001; Sommer-Knudsen et al., 1997; Kuboyama, 1998; Lord, 2000; de Graaf et al., 2001). From the TT-specific proline- and hydroxyproline-rich glycoproteins of tobacco (Chen et al., 1993; Cheung et al., 1993; Cheung, 1996; Sommer-Knudsen et al., 1997; Bosch et al., 2001; Bosch, 2002), only the TTS-glycoproteins in the TT of tobacco and N. alata were shown to be involved in pollen tube growth (Cheung et al., 1995; Wu et al., 1995, 2000).
The presently described PELPIII mRNA comprizes at least two members (pMG14 and pMG15) (Goldman et al., 1992). The PELPIII proteins are highly soluble and accumulates in the IM of the TT from the early stages of development until pollination, similar to other pistil-specific products (Ori et al., 1990; Cheung et al., 1993; Wang et al., 1993; Lind et al., 1996; Schultz et al., 1997). The difference in molecular weight (MW) between the predicted protein backbones of PELPIII cDNAs pMG15 (47 kDa for pMG15) and the native proteins detected by I-C3P (100–140 kDa) is caused by post-translational modification as in other proline-rich TT-specific proteins (Wang et al., 1993; Lind et al., 1994; Sommer-Knudsen et al., 1996; Bosch et al., 2001). Biochemical analysis of PELPIII showed that they contain mainly glycomodules characteristic for AGPs, whereas the proline residues in the few Ser-Pro4 motifs most probably contain extensin-like sugar moieties (Shpak et al., 1999, 2001; Goodrum et al., 2000; Bosch et al., 2001). A comparison of the predicted protein backbones of several style-specific cDNAs revealed that NaPRP5, encoding for the 120 kDa glycoprotein in N. alata, contained a bipartite level of homology with the class II PELPs (PELPII) and PELPIII from N. tabacum (Goldman et al., 1992; Lind et al., 1994; Schultz et al., 1997). The similarity between PELPIII of tobacco and NaPRP5 of N. alata lies at the C-terminus of the backbone, to which the I-C3P antiserum was raised. The I-C3P serum, however, does not recognize the PELPII proteins of tobacco (M Bosch, B McClure, personal communication). By contrast with PELPIII, the N. alata 120 kDa glycoprotein does not react with the Yariv reagent and its extraction appears more efficient with high salt than with low salt buffer (Sommer-Knudsen et al., 1998; this study). Plant proteins extracted by high salt buffers are suggested to be ionically bound to cell walls (Kieliszewski and Lamport, 1994; Sommer-Knudsen et al., 1996). Some highly conserved amino acid residues are present in the C-terminal cysteine-rich domains of PELPIII, TTS, GaRSGP, and the 120 kDa-glycoprotein (Schultz et al., 1997; Cheung and Wu, 1999). Despite their homology at the amino acid level, TTS-glycoproteins and GaRSGP differ considerably with respect to their glucan moieties, solubility, in vivo localization, and their biochemical behaviour after pollination (Chen et al., 1993; Wang et al., 1993; Wu et al., 1995, 2000; Cheung et al., 1995; Sommer-Knudsen et al., 1996, 1997, 1998; Cheung and Wu, 1999). In addition, part of the highly conserved amino acid residues of the C-terminal domain of style P/HRGPs is also found in several non-stylar proline-rich proteins with a similar chimeric character, such as the Phaseolus vulgaris proline-rich protein1 (PvPRP1) isolated from bean cell suspension cultures (Sheng et al., 1991; Davies et al., 1997; Takeichi et al., 1998; de Graaf, 1999). Thus, stylar or non-stylar, glycoproteins with homologous amino acid sequences may have different functions, apparently based on their distinct post-translational glycosylations.
As shown by means of tissue printing (Pierson et al., 1999), PELPIII starts to accumulate in the IM of tobacco styles during the early stages of pistil maturation. It appears in the upper part of the TT during the early stages of pistil development and proceeds basipetally, following the developmental pattern described for Datura stramonium (Satina, 1944). At pistil maturity, the lower part of the style shows a higher molecular weight than the upper part. Similar differences for the TTS-proteins were shown to depend on a top to base increase in glycosylation. The pattern differs from the basipetal developmental pattern seen for PELPIII, but agrees with the stimulating and guiding effect of TTS-glycoproteins (Wang et al., 1993; Wu et al., 1995, 2000).
No significant differences in quantity of soluble PELPIII was found between pollinated and non-pollinated pistils, though the PELPIII proteins are translocated almost completely from the almost liquid, intercellular matrix of the TT into the callosic walls of pollen tubes in vivo. TTS-proteins were also found in the pollen tube wall after pollination, but neither TTS, nor PELPIII were ever detected at high levels in the pollen tube cytoplasm. These patterns contrast with those of the 120 kDa style glycoprotein from N. alata (Lind et al., 1996). The 120 kDa glycoprotein appears to accumulate to high levels in the cytoplasm and inner walls of in vivo growing pollen tubes, while a considerable amount of the 120 kDa glycoprotein remains in the IM surrounding the growing pollen tubes. Though the precise mechanism remains unknown, the almost complete translocation of PELPIII from the IM into the pollen tube wall implies the participation of an active transport or binding to the wall. A pure ionic binding seems unlikely, given the unaltered extractability with low salt buffers. The translocation of PELPIII, and the other stylar glyproteins, in any case points towards a high porosity of the pollen tube wall, possibly aided by pollen tube or TT secreted polygalacturonases and 
-1,3-glucanases (Ori et al., 1990; Tebbutt et al., 1994; Sessa and Fluhr, 1995; Delp and Palva, 1999). Since most PELPIII of the pollinated pistils retained its high molecular weight and high solubility, it was concluded that the tube wall-associated PELPIII is not notably modified by the growing pollen tubes. By contrast, TTS proteins are de-glycosylated and become tightly associated with the pollen tube walls (Wu et al., 1995). The difference between PELPIII and TTS with respect to the hydrolysis of their glucan moieties, may depend on the presence or absence of the corresponding hydrolytic enzymes. The translocation into the pollen tube wall of PELPIII and related proteins with different glycosylations, shows that these translocations are independent from their typical glycosylation.
On the basis of the present data, the following conclusions were reached. (1) PELPIII of tobacco belongs to the group of glycoproteins with a clear developmental profile that concurs with involvement in IM development and pollen tube growth. (2) PELPIII is able to penetrate the pollen tube wall. The differences in behaviour between the three related classes of style IM glycoproteins of Nicotiana, namely PELPIII, TTS-glycoproteins and the 120 kDa glycoprotein, seem to connect more to their differences in glycosylation than to major differences in amino acid sequence.
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