Planta (1998) 207: 303312

In-vitro germination and growth of lily pollen tubes is aected by protein phosphatase inhibitors
Gerhard Obermeyer, Hildegard Klaushofer, Marion Nagl, Margit Hoftberger, Friedrich-Wilhelm Bentrup
Institut fur Panzenphysiologie, Universitat Salzburg, Hellbrunnerstrae 34, A-5020 Salzburg, Austria Received: 30 March 1998 / Accepted: 6 July 1998

Abstract. Inhibitors of type-2A protein phosphatase (PPase-2A), calyculin A (cal A) and okadaic acid (OA), inhibit pollen grain germination and growth of pollen tubes of Lilium longiorum Thunb. at nanomolar concentrations. Half-maximal inhibition of cytoplasmic PPase-2A activity was below 0.1 nM for cal A and at 0.7 nM for OA. Other protein phosphatase inhibitors (tautomycin, cypermethrin, and dephostatin) were less eective. The OA- and cal A-sensitive as well as dephostatin-sensitive PPase activity in the cytoplasm did not change during germination and growth of pollen tubes. Addition of cal A and OA disturbed the direction of pollen tube growth and the distribution of cytoplasmic organelles and caused cell wall thickenings as observed by light and electron microscopy. Inhibition of PPase-2A caused multiple eects at the cellular level, cytoskeletal elements being a putative target of PPase2A activity. Key words: Calyculin A Lilium Okadaic acid Pollen Protein phosphorylation Tip growth

Introduction Elongating pollen tubes serve as a model system for studying various cellular processes that are involved in cell growth, particularly in polar, tip-focussed growth. All processes involved in tip growth, including cell wall synthesis and vesicle transport (Rutten and Knuiman 1993; Geitmann et al. 1996), exocytosis (Battey and

Abbreviations: cal A calyculin A; DMSO dimethylsulfoxide; OA okadaic acid; PPase-1 type-1 protein phosphatase; PPase-2A type-2A protein phosphatase; PPase-2B type-2B protein phosphatase; tyr-PPase phospho-tyrosine protein phosphatase; ser/thr-PPase phospho-serine/phospho-threonine protein phosphatase Correspondence to: G. Obermeyer; E-mail: gerhard.obermeyer@sbg.ac.at; Fax: 43 (662) 8044 619

Blackbourn 1993), cytoskeleton (Pierson and Cresti 1992; Cai et al. 1997), ion gradients (Obermeyer and Weisenseel 1991; Miller et al. 1992; Fricker et al. 1997), ion transporters (Feijo et al. 1995), and turgor pressure (Benkert et al. 1997), must be well-regulated in space and time to produce a regularly shaped, growing pollen tube. Phosphorylation and dephosphorylation of proteins are two of the major post-translational reactions that regulate the activity of enzymes. Two dierent types of enzyme, protein kinases and protein phosphatases (PPases), are responsible for the phosphorylation status of proteins in a cell, and in plant cells a variety of enzymes regulated by reversible phosphorylation have been identied (Huber et al. 1994). Specic inhibitors of most types of PPase are available and have been used to distinguish between the dierent PPase types (Cohen 1991; MacKintosh and MacKintosh 1994; Smith and Walker 1996), e.g. okadaic acid is a very potent and specic inhibitor of type-2A PPases (Hardie et al. 1991). Inhibitor studies have shown that protein phosphorylation is involved in almost every cellular process, including cell cycle control, organization of the cytoskeleton, regulation of ion transporter activity, exocytosis, and signal transduction (Davidson et al. 1992; Li et al. 1994; Thiel and Blatt 1994; Stone and Walker 1995; Kissmehl et al. 1996; Smith and Walker 1996; Stark 1996). Most of the cellular processes that are regulated by phosphorylation have been reported to play an important role in the formation of polarity and during tip growth of pollen tubes (Derksen et al. 1995; Obermeyer and Bentrup 1996) and thus, reversible protein phosphorylation may be essential in pollen tube growth. Especially during pollination where pollen and stigma cells communicate with each other (Dzelzkalns et al. 1992) and extracellular signals have to be transduced into cytoplasmic messages (Franklin-Tong et al. 1993; Cheung 1996; Trewavas and Malho 1997), protein phosphorylation may be involved. So far, protein phosphorylation has been detected in the self-incompatibility reaction in pollen of Papaver rhoeas (Rudd et al. 1996), Nicotiana alata (Kunz et al. 1996), and in Brassica

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G. Obermeyer et al.: Protein phosphatases and pollen tube growth tips was enhanced by staining with uranyl acetate and lead citrate. Ultrathin sections of pollen tube tips were observed with an electron microscope (Phillips EM 400 T) at 80 kV. Preparation of cytoplasmic and microsomal fractions. Pollen grains from ve owers were suspended in germination medium and aliquots of this pollen suspension were incubated for 0, 10, 30, 60, and 240 min in germination medium for preparation of cytoplasmic and microsomal fractions according to Obermeyer et al. (1996). Briey, using a Teon potter, pollen was homogenized in 330 mM sucrose, 150 mM KCl, 1 mM EDTA, 50 mM Tris adjusted with Mes to pH 7.5, ltered through a 25-lm lter (Millipore, Vienna, Austria) and centrifuged at 10,000 g for 15 min. Then, the supernatant was centrifuged at 48,000 g for 60 min. The supernatant (cytoplasmic fraction) and the resulting pellet (microsomal fraction) were frozen immediately at A70 C after the pellet had been resuspended in homogenization buer. Protein concentrations in the cytoplasmic and microsomal fractions were determined with a protein assay kit (Bio-Rad, Vienna, Austria) using bovine serum albumin as a standard. Protein phosphatase activity. Assays for PPase activity were purchased from Promega (Madison, Wis., USA) and were performed according to the instructions of the manufacturer. For detection of serine/threonine protein phosphatase (ser/thr-PPase) activity 100 lm of a synthetic, phosphorylated peptide [RRA (pT)VA] served as a substrate and the released inorganic phosphate (Pi) was measured after complexing Pi with molybdate and malachite green (Ames 1966). The assay was performed in ser/ thr-PPase buer containing 50 mM Tris-Mes (pH 7.5) and 0.1 mM EDTA with various concentrations of inhibitors as indicated. The reaction was started by addition of 1.52.5 lg protein (diluted in ser/thr-PPase buer) to give a total reaction volume of 50 ll. After 20 min the reaction was stopped by adding 50 ll of molybdate/dye solution and the resulting absorbance at 630 nm was determined after 15 min using a microtiter-plate reader (SLT, Grodig, Austria). Tyrosine protein phosphatase (tyr-PPase) activity was determined as described above but using the peptide END(pY)INASL (100 lM) as a substrate. The reaction was performed in tyr-PPase reaction buer consisting of 50 nM Mes adjusted to pH 6 with Tris, and with or without 30 lM dephostatin. The dephostatin-sensitive Pi release was used to monitor the activity of tyr-PPases.

species (Hiscock et al. 1995), as well as in pistil cells of Brassica (Kandasamy et al. 1993; Rundle et al. 1993). However, the phosphorylation status of pollen proteins may not only be part of the self-incompatibility reaction but may be essential for the regulation of germination and growth of the pollen tube itself. In other tip-growing cells, specic PPase inhibitors aect the tip growth process, e.g. in Arabidopsis thaliana the growth of root hair cells is blocked (Smith et al. 1994; Baskin and Wilson 1997). Furthermore, PPases have been reported to inhibit neurite outgrowth (Giasson and Mushynski 1997) and to be involved in controlling the cell shape of budding yeast (Shimanuki et al. 1993; Kinoshita et al. 1996; Stark 1996). Recently, it has been shown that protein phosphorylation by a MAP kinase is involved in the activation of tobacco pollen after hydration (Wilson et al. 1997). So far, the involvement of protein phosphorylation has not been tested on in-vitro-growing pollen tubes. Using specic inhibitors of various types of PPase we show that serine/threonine PPases of types 1 (PPase-1) and 2A (PPase-2A) are involved in germination and tube growth of pollen grains of Lilium longiorum. Inhibition of PPases results in a decreased germination frequency and in a distorted tube morphology as seen by light and electron microscopy. The activity of PPase-2A was detected in a cytosolic fraction and monitored during germination and pollen tube growth. Materials and methods
Plant material and growth experiments. Mature, dehydrated pollen grains were collected from owers of Lilium longiorum Thunb. obtained from local ower shops and were immediately used for growth experiments. Pollen from one anther was suspended in 5 ml germination medium containing 10% (w/v) sucrose, 1.6 mM H3BO3, 1 mM KCl and 0.1 mM CaCl2. The pH of 5.6 was adjusted with Mes or Tris. Various concentrations of the PPase inhibitors okadaic acid (OA), calyculin A (cal A), tautomycin, cypermethrin, and dephostatin, all dissolved in dimethylsulfoxide (DMSO) were added to the germination medium. All pollen tubes, including the controls, were grown in the presence of 0.1% DMSO which has no eect on germination and growth of pollen tubes. Protein phosphatase inhibitors were obtained from Calbiochem (La Jolla, Calif., USA). All other chemicals were purchased from Sigma, Merck and Fluka, respectively. At least 200 pollen grains were counted for determination of germination rates. The percentage of germinated pollen grains was calculated and plotted against the incubation time. From these plots the initial slope (germination rate) was determined for various inhibitor concentrations. To measure the mean tube length, images of pollen tubes were taken after 3 h incubation in germination medium. All experiments were performed at room temperature. Microscopy. After 3 h, germinated pollen exposed to 33 nM okadaic acid and 10 or 33 nM calyculin A, respectively, was collected and observed with a microscope (Axiovert 135; Zeiss). Photomicrographs were taken on Kodak TMX 100 lms. For electron microscopy, pollen tubes incubated for 35 h in germination medium in the presence of OA (10 nM) or cal A (3.3 nM) were xed for 20 min in 2.5% (v/v) glutaraldehyde in 100 mM phosphate buer (pH 7.2) with additional 10% (w/v) sucrose. Pollen tubes were washed in 100 mM phosphate buer, xed in 2% OsO4 for 2 h, dehydrated in an ethanol series, and nally embedded in Epon. The contrast of sections of pollen tube

Results Pollen grains of Lilium longiorum incubated in germination medium containing various concentrations of specic inhibitors of ser/thr PPases showed a lower germination rate and a reduced tube length after 3 h (Fig. 1; Fig. 2A,B). Okadaic acid and cal A are wellknown antagonists of PPase-2A (Cohen 1991; MacKintosh and MacKintosh 1994), tautomycin inhibits the PPase-1 (MacKintosh and MacKintosh 1994), whereas cypermethrin blocks PPase-2B activity (Enan and Matsumura 1992). Dephostatin was used to inhibit tyr-PPase activity (Imoto et al. 1993). Calyculin A and OA were the most potent inhibitors, blocking both germination and tube growth. Typical examples of germination of pollen grains in the presence of cal A and OA are shown in Figs. 1A and 1C, respectively. Half-maximal inhibitor concentrations (IC50) for pollen tube germination were approximately 10 nM for cal A and 90 nM for OA (Table 1). Pollen tube growth was more sensitive to phosphatase inhibitors than the germination rate as reected by the lower IC50 values: 6 nM for cal A and 10 nM for OA.

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Fig. 1AD. Eect of PPase-2A inhibitors on germination and growth of pollen tubes. A Typical time course of pollen grain germination at dierent concentrations of cal A. B Eect of cal A on the germination rate (circles) and the mean tube length (squares). The average SD of ve experiments is shown. C Time course of pollen grain germination at dierent concentrations of OA. D Eect of OA on germination rate (circles) pollen tube length (squares) and (n 5). Means SD. Tube length of germinated pollen grains was measured after 3h

Fig. 2AD. Eect of protein phosphatase inhibitors on germination and pollen tube growth. A Time course of pollen grain germination at dierent concentrations of tautomycin. B Eect of tautomycin on germination rate (circles) and pollen tube length (squares). C Eect of cypermethrin on germination rate (circles) and pollen tube length (squares). D Germination rate (circles) and pollen tube length (squares) aected by dephostatin. Data are means SD (n 5 experiments)

Addition of tautomycin, a specic inhibitor of type-1 PPases, to the germination medium inhibited the germination rate and tube growth much less than OA and cal A (Fig. 2A,B). The germination rate was blocked by just 40% and mean pollen tube length was decreased by 30% at the highest applied tautomycin concentration

(100 nM). Again, very little inhibition of germination was detected when pollen grains were incubated in the presence of cypermethrin, an inhibitor of PPase-2B. The highest concentration of cypermethrin reduced the germination rate by 40%. In contrast to the germination rate, tube growth was stimulated by 1520% (Fig. 2C).

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Table 1. Half-maximal inhibitor concentration (IC50) of germination rate and tube growth Okadaic acid Germination rate Tube growth 90 nM 10 nM Calyculin A 10 nM 6 nM Tautomycin > 100 nM > 100 nM Cypermethrin > 100 nM stimulates at > nM 33 Dephostatin stimulates at > lM 10 no eect

A stimulation of the germination frequency by 50% was observed after application of 33100 lM dephostatin, a specic tyr-PPase inhibitor, whereas pollen tube growth was not aected (Fig. 2D). These results indicate that mainly PPases of type 2A are involved in germination and growth of pollen tubes. However, a cross-reaction of cal A and OA with PPase-1 cannot be excluded. The fact that tautomycin inhibits germination and growth of pollen tubes also indicates that a type-1 PPase may be involved. Pollen grains incubated in the presence of cal A and OA at concentrations around the respective IC50 did germinate but only a few irregularly shaped pollen tubes grew (Fig. 3). As a control a pollen tube exposed to 0.1% DMSO but not to PPase inhibitors is shown in Fig. 3A. Growth is undisturbed with a regularly shaped pollen tube of constant diameter, a clear cap, cytoplasmic streaming, and no cell wall thickenings. Pollen tubes incubated in 1033 nM cal A or OA showed normal cytoplasmic streaming but had no constant tube diameter (Fig. 3B,F). Additionally, pollen tubes did not grow straight at the bottom of a Petri dish but changed direction, grew upwards and started to grow in circles (Fig. 3B,D). Sometimes, more than one pollen tube germinated from one pollen grain and growing pollen tubes also branched during growth (Fig. 3D,G). In most tubes, regions of thickened cell wall were observed (Fig. 3C,H). In electron-microscopical sections, further eects of cal A and OA became apparent (Fig. 4). Most evidently, a much thicker and more loosely packed cell wall was produced than in control pollen tube tips (Fig. 4A,B). An extreme example of cell wall thickening at the tube tip in the presence of 3.3 nM cal A is shown in Fig. 4G, H. Other eects of cal A on cell walls were inclusions of less electron-dense material into the cell wall (Fig. 4I). Similar eects on the cell wall were observed with addition of OA (10 nM) where the cell wall thickness varied along the pollen tube, showing thicker and thinner regions than in control tubes (Fig. 4CF). Also, OA-treated pollen tubes showed dierent staining of the cell walls than the controls (Fig. 4F). In addition to the eect on cell wall structure, long microtubules were observed in pollen tube tips treated with OA (Fig. 4E,F) or cal A (data not shown). Another eect of cal A and OA can be seen in the overview images (Fig. 4A,C,D). Secretory vesicles may still be found in the tip region but organelle distribution is disturbed. Larger organelles like mitochondria may be observed in the very tube tip (Fig. 4G,H) and secretory vesicles are still present behind the tube apex (Fig. 4C). Also, bow-like arrangements of secretory vesicles in the tube tip indicate that they may be lined up on cytoskeletal elements (Fig. 4C).

In general, the cell's program for regular tube growth and for polarity was disturbed by inhibitors of PPase-2B as indicated by the formation of irregularly shaped pollen tubes and by the generation of more than one tube per pollen grain. In order to characterize the PPases involved in pollen tube growth in more detail we measured their activity in a cytoplasmic fraction during germination and growth of pollen tubes. Figure 5 shows the inhibition by cal A and OA of cytoplasm-localized PPases of ungerminated pollen grains, giving a half-maximal inhibition of less than 0.1 nM for cal A and approximately 0.7 nM for OA. The PPase activity was monitored as the release of inorganic phosphate from a synthetic phosphopeptide. At 10 nM cal A or OA a PPase activity of ca. 300 pmol (mg protein)A1 sA1 was still detectable. To characterize this remaining activity the inhibitory eect of various other PPase inhibitors was determined in a cytoplasmic fraction of ungerminated (pollen grains) and germinated pollen (pollen tubes, Fig. 6). Inhibitors of PPase-2A (microcystin-LR, cal A and OA) blocked about 7080% of the total PPase activity in pollen grains and pollen tubes. Inhibitors of other PPases (tautomycin for PPase1, cypermethrin for PPase-2B and dephostatin for tyrPPases) blocked only 1030% of the cytoplasmic PPase activity in both pollen grains and tubes. No dierence in the inhibition pattern of PPase activity was detected between the cytoplasmic fractions of pollen grains and tubes. In some parallel experiments the PPase activity was measured in a microsomal fraction from the same pollen preparation as the cytoplasmic fraction. Microsomal PPase activity was about 25% of the cytoplasmic PPase activity (Table 2) and no specic eects of PPase inhibitors could be acertained. The microsomal PPase activity reected more or less an inhibitor-insensitive background activity of approximately 200 pmol (mg protein)A1 sA1. Therefore, either no specic type of PPase was associated with microsomal membranes, or the microsomal PPase activity might be due to cytoplasmic impurities. Although there was no dierence in PPase activity in ungerminated and germinated pollen grains, PPase activity may change during the dierent stages of pollen tube germination. Therefore, PPase activity was monitored in cytoplasmic fractions obtained from pollen grains after 0, 10, 30, 60, and 240 min incubation in germination medium (Fig. 7). A cal A-sensitive and OAsensitive PPase activity was determined corresponding to PPase-2A. Additionally, a dephostatin-sensitive PPase (tyr-PPase) was measured, too, because dephostatin stimulated germination of pollen grains (Fig. 2D). No signicant change in PPase-2A activity was observed during incubation of pollen grains although total ser/ thr-PPase activity increased slightly with incubation time (Fig. 7A). Also, total tyr-PPase and dephostatin-

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Fig. 3AH. Eect of phosphatase inhibitors on pollen tube morphology. A Dierential-interference contract (DIC) image of a control pollen tube. BE Typical examples of pollen tubes grown in the presence of cal A for 3 h. B Pollen tube incubated in 10 nM cal A (DIC image). C Pollen tube grown in 33 nM cal A (phase-contrast image). Arrowheads mark cell wall thickenings. D Branched pollen tube in 10 nM cal A (phase contrast). E Pollen tube changing its growth direction frequently (spiraling pollen tube, phase contrast). FH Pollen tubes grown in 33 nM OA for 3 h (phase contrast). F Stars mark changes in pollen tube diameter. G Branching pollen tube grown in the presence of 33 nM OA. H Cell wall thickenings at the tube tip (arrowheads). cc, clear cap; bar 15 lm

sensitive PPase activity did not change much with incubation time (Fig. 7B). Discussion The involvement of dierent types of PPase in cellular processes can be investigated by application of specic

drugs inhibiting the activity of these PPases and recording their eects on the respective cellular process. Measuring the germination rate or germination frequency reects eects of the inhibitors, including the successful establishment of polarity in pollen grains, whereas the mean tube length gives a reliable parameter for pollen tube growth. The most powerful PPase inhibitors aecting both parameters were cal A and OA, well-known specic inhibitors of PPase-2A. Inhibition of pollen tube growth was more sensitive to PPase2A inhibitors than germination and already occurred at concentrations less than 1 nM cal A or less than 10 nM OA. The inhibition at very low inhibitor concentrations as well as the application of the two dierent, structurally unrelated compounds cal A and OA exclude any non-specic eects that might be induced by one inhibitor alone. Inhibitors of other PPase types, such as tautomycin, cypermethrin or dephostatin, were less eective in inhibiting tube growth, indicating that a type-2A PPase is involved in a cellular process essential

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Fig. 4AI. Electron micrographs of control lily pollen tubes (A, B) and pollen tubes grown in the presence of 3.3 nM cal A (CF) and 10 nM OA (GI). A Overview of a control pollen tube tip. B Detail of the very tip of the tube shown in A. Note the secretory vesicle fusing with the plasma membrane (arrow). C Tip of a pollen tube grown in 10 nM OA for 3 h showing subapical accumulation of secretory vesicles. Arrows mark chains of secretory vesicles. D Detail from the same tube as in C showing an irregularly formed cell wall. E Microtubules (arrowheads) in the tip region. Note also the very thin cell wall. F Arrowheads mark microtubules in the very tip of the tube. G Overview of a pollen tube tip incubated in 3.3 nM cal A showing an extreme thickening of the cell wall. H Detail of the tip's cell wall (same tube as in G). Note the presence of mitochondria in the very tip of the tube. I Inclusion of less electron-dense material in the cell wall (stars) of a pollen tube grown in 3.3 nM cal A. cw, cell wall; sv, secretory vesicle; m, mitochondrion; mt, microtubule; bars 2 lm (A, C, G), 0.5 lm (B, D, E, H, I), 0.2 lm (F)

for tube elongation. On the other hand, germination of pollen tubes was very sensitive to tautomycin (0.33 nM), a specic inhibitor of type-1 PPase, although the maximal inhibition of the germination rate was only

30% and did not increase after addition of higher tautomycin concentrations. The following conclusion may be drawn from the in-vitro germination experiments: a cellular process

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Fig. 5. Concentration-dependent inhibition of ser/thr-PPase activity in a cytoplasmic fraction of ungerminated lily pollen grains. Activity was monitored as release of inorganic phosphate from a synthetic phospho-hexapeptide [RRA(pT)VA]. Data are means SD (n 3 experiments)

regulated by reversible protein phosphorylation via PPase-1 may be important for germination whereas PPase-2A is more important during growth of pollen tubes. But it should be noted that the inhibitor concentrations necessary to block germination and tube growth were higher than those reported from inhibitor studies using cell-free extracts (Cohen 1991; Hardie et al. 1991; Smith and Walker 1996) and one cannot clearly distinguish between inhibition of PPase-2A and PPase-1 by using OA or cal A. In a cytosolic extract from Brassica napus cells, PPase-2A was completely inhibited by 1 nM OA whereas PPase-1 was not aected only concentrations higher than 10 nM inhibited PPase-1, too (Cohen 1991). One cannot therefore exclude the possibility that PPase-1 is also inhibited by cal A and OA at concentrations used to block pollen tube germination and growth. However, much higher inhibitor concentrations

have been reported for intact plant tissues, e.g. Brassica stigmas have been incubated with micromolar concentrations of OA and microcystin, respectively, to inhibit pollination (Rundle et al. 1993). In other studies with tip-growing cells, partial inhibition of cell elongation was already observed at 320 nM cal A (root hair cells, Smith et al. 1994) and 0.51 nM OA (neuron cells, Giasson and Mushynski 1997), concentrations which were in the same range as the inhibitor concentrations partially inhibiting germination and growth of lily pollen tubes (1 nM OA or cal A, Fig. 1). Thus, single tipgrowing cells may be more sensitive to PPase inhibitors than plant tissue preparations, because less unspecic binding of the inhibitors to cell walls and PPaseinsensitive cell types occurs. However, using a cytosolic fraction from ungerminated pollen grains and pollen tubes, much lower inhibitor concentrations were necessary to inhibit the release of inorganic phosphate from a synthetic phosphopeptide (0.11 nM OA or cal A). Again, using dierent PPase inhibitors the same pattern of inhibition as detected for intact pollen was observed. Okadaic acid and cal A were the most potent inhibitors, reducing the cytosolic PPase activity by 7080% whereas tautomycin inhibited only 30%. The activities of PPase 1 and 2A were mainly localized in the cytoplasm and only a fraction of total PPase activity was associated with membranes but was insensitive to cal A and OA (Table 2). The dierent sensitivities of pollen germination and tube growth to inhibitors may indicate change in PPase activity during pollen germination. Also, the stimulation of tube growth by high dephostatin concentrations may reect the activity of another PPase type during tube growth but almost no dephostatin-sensitive tyr-PPase activity was detected during pollen tube germination and growth. No dierence in the activity of PPase 2A during pollen germination was observed either, indicating that the cellular process which is regulated by PPase 2Adependent dephosphorylation is active throughout pollen tube germination and elongation.

Fig. 6AB. Eect of PPase inhibitors on ser/thr-PPase activity in A ungerminated (n 7 (control), 4 (OA), 5 (cal A), 3 (microcystin), 3 (cypermethrin), 3 (dephostatin)) and B germinated (n 3 for all inhibitors) pollen grains. Concentration of PPase inhibitors as indicated. A cytoplasmic fraction of pollen tubes (germinated pollen grains) was obtained after 3 h incubation. Data are given as mean SD for the indicated number of experiments

310 Table 2. Eect of specic inhibitors on PPase activity. Protein phosphatase activity was monitored as phosphate released and was measured in the cytoplasmic and microsomal fractions. The mean value SD was determined in n (numbers in parentheses) dierent preparations from pollen grains Inhibitor PPase activity [pmol Pi (mg protein)A1 sA1] Cytoplasmic fraction None 1 nM Okadaic acid 1 nM Calyculin A 100 nM Microcystin-LR 5 nM Cypermethrin 10 lM Dephostatin 1 nM Tautomycin 1112 367 381 173 889 759 672 115 (7) 56 (4) 194 (5) 115 (3) 151 (3) 201 (3) 144 (3) Microsomal fraction 276 203 188 176 242 220 195 239 243 204 202 256 256 250 (3) (3) (3) (3) (3) (3) (3)

G. Obermeyer et al.: Protein phosphatases and pollen tube growth

Fig. 7AB. Protein phosphatase activity during pollen germination. A Type-2A PPase activity was monitored as cal A- or OA-sensitive PPase activity. Data are means SD (n 5 experiments). Inhibitor concentrations were 10 nM. B Dephostatin (33 lM) was used to detect tyr-PPase activity. Data are means SD (n 3 experiments)

But which essential cellular process or processes are regulated by PPase-2A-dependent dephosphorylation? The presented light and electron-microscopical investigations provide little evidence for a single eect caused by cal A and OA; rather, multiple eects were observed at the cellular level. Most dramatically a loss of orientation during tube growth was detected resulting in branching and spiraling pollen tubes accompanied by occasional cell wall thickenings. Therefore, by changing the phosphorylation state of some proteins the cellular program for generating a straight-growing pollen tube is

disturbed. Putative targets for the PPase-2A-dependent dephosphorylation may be cytoskeletal elements, ion transporters, and components involved in localized fusion of secretory vesicles; all of them are known to be involved in pollen tube growth and are regulated by reversible phosphorylation. In neuronal cells, phosphorylation of microtubuleassociated proteins (MAP-2, s factor) and tubulin inhibits microtubule assembly (Jameson et al. 1980; Arias et al. 1993), and type-1 as well as type-2A PPases are involved in the regulation of the phosphorylation state of microtubule proteins (Yamamoto et al. 1988). In contrast to neurons, the function of microtubules during pollen tube growth is still controversal (Franke et al. 1972; Heslop-Harrison et al. 1988; Joos et al. 1994), and compared to actin laments, they play a minor role in polar tube growth (Cai et al. 1997). Nonetheless, interactions between actin laments and microtubules have been documented (Lancelle and Hepler 1991). Changing the phosphorylation status of microtubular proteins may disturb the organization of the cytoskeleton as it does in neurons (Selden and Pollard 1983) and thus contribute to the observed eects on tube growth after PPase inhibitor treatment. Disorganization of microtubules has been observed in Arabidopsis root cells after treatment with PPase-2A inhibitors, and root elongation stopped in inhibitor-exposed plants (Baskin and Wilson 1997). Additionally, another cellular process closely linked with the cytoskeleton, namely exocytosis including transport and fusion of secretory vesicles, was also aected by PPase inhibitors as shown by the subapical accumulation of secretory vesicles in the tube tip (Fig. 4C). Disturbing the localized fusion of vesicles at the very tip of the tube may cause the cell wall thickenings. Ion transporters in the plasma membrane of pollen grains and tubes are important for regular tube growth, and are another putative target for PPases. The activity of the plasma-membrane H+-ATPase is regulated by reversible protein phosphorylation (Schaller and Sussman 1988), and an OA-dependent dephosphorylation of the H+-ATPase was observed in pump activation during interaction of tomato cells with the pathogenic fungus Cladosporium fulvum (Xing et al. 1996). The activity of ATPase is a factor determining the growth rate of pollen tubes because a stimulator of the plasma-membrane H+ATPase, fusicoccin, increased the growth rate of lily pollen tubes (Fricker et al. 1997). Additionally, we note that the action of fusicoccin is mediated by 14-3-3 proteins which are also regulated by reversible protein phosphorylation (DeBoer 1997). In summary, our data support the view of a pleiotropic eect of cal A and OA rather than inhibition of a single cellular event. The conspiciously disturbed ultrastructural organization of the pollen tube tip suggests that cytoskeletal elements are one of the major targets for type-2A PPases. The action of PPase inhibitors on in-vitro-growing pollen tubes presented in this paper should be kept in mind when the role of reversible protein phosphorylation in cell-cell communication

G. Obermeyer et al.: Protein phosphatases and pollen tube growth

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