Planta 135, 183
Planta
190 (1977)
9 by, Springer-Verlag 1977
Ultrastructural Localization of Acid Phosphatase in the Pollen Tube of Prunus avium L. (Sweet Cherry) J. Lin, W.J. Uwate, and V. Stallman* Department of Pomology, University of California, Davis, CA 95616, USA
Abstract. The pollen tube of Prunus avium (cherry)
consists of a growth zone of vesicles at the tip and an assemblage of organelles typical of an actively metabolizing cell. Electron opaque globules are closely associated with the plasma membrane and fibrillar cell wall layer at the tip. Acid phosphatase (EC 3.1.3.2) activity is localized in the membranes of 120 nm vesicles and ER system, the lumen of 50 nm vesicles, the plasma membrane and the tube nucleus. Key words: Acid phosphatase- Pollen t u b e - Prunus.
Introduction
The fine structure of pollen tubes growing in vitro has been the subject of a number of studies in the last 15 years (Crang and Miles, 1969; Franke et al., 1972; Larson, 1965; Rosen, 1971; Rosen and Gawlik, 1966; Sassen, 1964; Van Der Woude etal., 1971). These studies have shown that at the very tip of the tube is a growth zone characterized almost exclusively by secretory vesicles involved in cell wall synthesis and that proximally a diversity of organelles exists. Structural variations within the distal end of an actively elongating tube doubtless are related to differences in functional capacities and thus in enzyme distribution. Among the many enzymes which have been identified in pollen, acid phosphatase (EC 3.1.3.2) has been the most widely investigated (Brewbaker, 1971; Knox and Heslop-Harrison, 1970; Stanley and Linskens, 1974; Vithanage and Knox, * We would like to thank Dr. Richard Falk, Department of Botany, U.C.D., for his helpful suggestions and critical review of the manuscript
Abbreviations: AP = acid phosphatase; R E R = rough endoplasmic reticulum
1976). Biochemical analyses have demonstrated a decrease in AP after germination (Roggen, 1967). Within the growing tube, acid phosphatase activity reportedly decreases distally (Malik et al., 1969) or is absent in the tip region where spherosomes are lacking (Gorska-Brylass, 1965). Petrovskaya-Baranova and Tsinger (1962) on the other hand, found pronounced activity along the entire length of the tube. The ultrastructural localization of acid phosphatase has been reported for a number of plant cells and tissues (Charvat and Esau, 1975; Noguchi, 1976; Bentwood and Cronshaw, 1976), but to our knowledge, fine structural distribution of the enzyme has not been demonstrated in a pollen tube. This paper reports on the ultrastructure of the pollen tube of Prunus avium and on the distribution of acid phosphatase activity. Materials and Methods Flowers of Prunus avium, cultivar " V a n " , were collected during March, 1976, and anthers were allowed to dehisce in petri plates. Pollen was immediately frozen and used as needed during a seven month period. For optimum growth, pollen was cultured in the following medium (Brewbaker and Kwack, 1963): 100 mg/l boric acid, 100 mg/l potassium nitrate, 300 rag/1 calcium nitrate, 200 rag/1 magnesium sulfate, 10% sucrose and 0.8% agar. Sterilized medium was drawn into "9 inch" pasteur pipettes and allowed to solidify. The tips of pipettes (about 1 mm in diameter) were cut into 1 cm segments and placed upright in a holder. Cultures were started first by touching the point of a sharp needle with pollen and then inserting the needle tip approximately 1/2 mm into the agar cylinder. This prevented the loss of pollen tubes during subsequent procedures. Cultures were maintained a constant temperature (25~ and light (3500 ix). Because active growth of most pollen tubes took place between 21/2 to 31/2 h after initiation of the culture, most of the tubes studied were sampled during this time period. Lengths of pollen tubes ranged from 0.15-0.30 mm. Earlier stages were also studied. The agar cylinders with the germinated pollen were pushed out of the glass tubes and excess agar was removed. For localization of acid phosphatase, the lead capture method (Etherton and Botham, 1970) was used. This procedure was slightly modified for the present study. The agar embedded pollen tubes were pre-fixed for 30 rain in 4% glutaraldehyde in
184 0.05 M cacodylate buffer (pH 7.2) at room temperature. They were rinsed 3 times (10 min each) in a 0.05 M acetate buffer (pH 5.7) containing 7.5 % sucrose. The tubes were then incubated for 30 rain at 37~ C in a complete incubation medium containing 0.4 mg/ml c~-naphthyl phosphate, 1 mg/ml lead acetate and 7.5% sucrose in 0.05 M acetate buffer, pH 5.7. Controls consisted of incubation in a medium without the substrate, without lead acetate, or with the addition of 0.1 M sodium fluoride as an inhibitor. Lead precipitates in the incubation media were filtered out prior to use. Following incubation, pollen tubes were rinsed three times (15 min each) in 0.05 M acetate buffer, and post-fixed for 1 h in 1% OsO4 in 0.05 M cacodylate buffer at pH 7.2. They were dehydrated in a graded ethanol series and embedded in Spurr's plastic. Sections of materials incubated for enzyme localization and controls were not post-stained. Non-incubated pollen tubes were fixed in glutaraldehyde for l h and acetate buffer was not used. In some of the non-incubated tubes, 0.05 M phosphate buffer was used in place of cacodylate buffer. The latter consistently yielded superior results. Sections of non-incubated material were post-stained with uranyl acetate and lead citrate (Reynolds, 1963). All materials were observed with a Zeiss EM9 electron microscope.
Results
Ultrastructure Cultured cherry pollen germinated within 1/2 h after inoculation and tube elongation continued for about 6 h under conditions indicated above. Incipient swelling of the tube tip and subsequent bursting was observed about 8 h after germination. During active growth (i.e., 2~/2 to 3~/z h after inoculation), the distal end of the tube was dense with an assemblage of organelles typical of a rapidly metabolizing system. The growth zone (GZ) where components of the tube wall were being laid down consisted most conspicuously of nearly spherical vesicles ranging in diameter from 60 to 220 nm (Fig. 1). This zone encompassed roughly 5 pm of the distal-most portion of the tip. In the region immediately proximal to the growth zone, rod-shaped mitochondria aggregated in abundance. Mitochondria in this region appeared to be oriented randomly whereas those farther removed from the G Z tended to be aligned in the plane of the tube axis. R o u g h endoplasmic reticulum (RER) was similarly oriented reflecting cytoplasmic streaming which occurred in the living state (Fig. 2). Groups of highly anastomosed tubular smooth endoplasmic reticulum (SER) were present in the distal region, often in close proximity to the growth zone (Fig. 3). Large numbers of vesicles averaging 50 nm in diameter were interspersed a m o n g the other organelles and m a n y of them possessed a similar degree o f electron opacity as the lumen of the endoplasmic reticulum. It was not ascertained if structural continuity existed between the two components due to difficulty in distinguishing transverse sections of S E R tubules from 50 n m vesicles in the immediate vicinity
J. Linet al. : Ultrastructural Localization of Acid Phosphatase (see Discussion). Ribosomes occurred either freely or as components of the RER. Surrounding the distal 25 pm of the tube tip (including the growth zone) were highly electron opaque globules that occurred in close association with the plasma membrane. In the early stages of tube growth, these globules intergraded with the fibrillar cell wall layer at or near the growth zone and were periodically taken into the cytoplasm (Fig. 4). Immediately preceding germination, the globules existed as a continuous layer in contact with the intine (Fig. 5). It appears that those in the vicinity of the pore have been transported with the emerging tube. As the tube elongated they gradually diminished (in the tip depicted in Figure 1, most of the globules have been depleted). Thus, no globules were evident in the proximal region of the tube where an inner, relatively homogeneous, cell wall layer was present. This inner cell wall layer was continuous with the intine. Dictyosomes and amyloplasts were numerous throughout the growing tube, but were lacking in the vicinity of the growth zone. Amyloplasts exhibited close relationship with the R E R and often were partially surrounded by the latter (Fig. 6). Lipid bodies and vacuoles increased in numbers proximally. Sequestration of cytoplasmic materials was evident particularly in the proximal portion of the tube (Figs. 7 and 8).
Distribution of Acid Phosphatase Using ~-naphthyl phosphate as the substrate provided reproducible and satisfactory results for the localization of acid phosphatase activity in cherry pollen tubes. Little, if any, noticeable cytological damage was observed and thorough penetration of uncomplexed substrate was achieved within a 30 to 50 rain incubation period. These advantages are consistent with those pointed out by Etherton and Botham (1970). In tubes incubated in the complete medium, fine deposits of lead phosphate were present throughout and denser aggregations were found complexed with certain organelles. The amount and localization of reaction product was variable along the length of the tube. In the proximal region of the tube, lead phosphate deposits were bound heavily to membranes of vesicles averaging 120 nm in diameter. However, not all vesicles falling into this size category exhibited labeling (Fig. 9).The deposits appeared to be complexed with both faces of the membrane. Labeled vesicles were often seen to merge with the plasma membrane. In some cases, enzyme activity was found only on the outer face of membranes of
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
185
Fig. 1. Median longisection of the very tip of a cherry pollen tube during active growth. Note the growth zone (GZ) with its component secretory vesicles and the mitochondria (M) immediately proximal to the GZ. • 9400 Fig. 2. Rough endoplasmic reticulum (RER) in a proximal region of an elongating tube. x 14,400 Fig.3. A distal region of a pollen tube showing tubular smooth endoplasmic reticulum (SER) and 50nm vesicles (arrows). x 27,900 Fig. 4. A portion of the distal 25 pm of the tip. Note the highly electron opaque globules (G) intergrading with the plasma membrane (arrows) and the fibrillar cell wall (FCW). x 42,700
186
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
Fig. 5. A portion of a pollen grain showing electron opaque globules (arrows) in contact with the intine (Int). x 9,300 Fig. 6. An amyloplast (Amyl) with closely associated RER. x 27,600 Fig. 7. A proximal portion of a pollen tube showing lipid bodies (LB) and vacuoles (V). x 15,300 Fig. 8. Sequestration of cytoplasmic components (X). x 9300
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
187
Fig. 9. A proximal region of a pollen tube showing heavy deposition of the reaction product in the membranes of vesicles averaging 120 nm in diameter (Ves). Larger vesicles exhibiting AP activity (arrows). Section not post-stained, x 60,200 Fig. 10. Control. A proximal region of a tube incubated in a medium containing fluoride. Section not post-stained, x 60,000 Fig. 11. Control. A proximal region of a tube incubated in a medium without the substrate. Section not post-stained, x 27,500 Fig. 12. Acid phosphatase activity in 50 nm vesicles (arrows). Note some vesicles lacking the reaction product. Section not post-stained. x 60,100
188
J. Linet al. : Ultrastructural Localization of Acid Phosphatase
Fig. 13. A portion of a tube showing acid phosphatase activity in the tube nucleus (Nuc) and the plasma membrane. Section not post-stained, x 5850 Fig. 14. Acid phosphatase activity in the SER of the distal portion of the pollen tube. Section not post-stained, x 27,600 Fig. 15. Reaction product localized mostly on the inner face of RER. Section not post-stained, x 27,200 Fig. 16. Control. Lead deposits lacking in SER of a tube incubated in a medium minus substrate. Section not post-stained, x 28,000
slightly larger vesicles a n d l o c a l i z a t i o n was sporadic (Fig. 9). R e a c t i o n was n e g a t i v e in similar vesicles o f c o n t r o l t u b e s i n c u b a t e d in m e d i a c o n t a i n i n g the inhibitor s o d i u m f l u o r i d e or in m e d i a l a c k i n g e - n a p h t h y l p h o s p h a t e (Figs. 10 a n d 11). It s h o u l d be pointed o u t t h a t in one t u b e i n c u b a t e d in the c o m p l e t e med i u m plus fluoride, t h e r e were dense d e p o s i t s in some vacuoles. I t is n o t k n o w n w h e t h e r these represented
the r e a c t i o n p r o d u c t . I n all the o t h e r tubes studied, l e a d d e p o s i t s were l a c k i n g in vacuoles. T u b e s incub a t e d in a m e d i u m w i t h o u t l e a d e x h i b i t e d no electron dense particles. A P activity was also evident in the l u m e n o f 50 n m vesicles. L a b e l e d vesicles t e n d e d to occur close t o g e t h e r in a relatively h o m o g e n e o u s matrix (Fig. 12). T h e n u c l e o p l a s m o f the t u b e nucleus showed
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
scattered activity whereas a greater concentration of lead phosphate deposits were found within what may be a nucleolus (Fig. 13). Enzyme activity was also detected in the plasma membrane. Within the distal portion of the tube, AP was localized to a large extent in the membranes of the ER system. The reaction product was present in the tubular SER and also bound mostly to the inner face of smooth membrane regions of R E R (Figs. 14 and 15). Figure 16 shows the absence of lead phosphate in the SER of a tube incubated in a medium without the substrate.
Discussion
The fine structure of the visicles found in the growth zone of cherry pollen tubes fits the criteria set forth by Van Der Woude et al. (1971) for secretory vesicles. There is no doubt that these vesicles are involved in the transfer and deposition of cell wall materials and take their origin in the dictyosomes (Engels, 1973, 1974; Maruyama, 1974). The highly electron opaque globules at the tip of cherry pollen tubes have not been reported in other species. Differences in tissue preparation may in part account for this discrepancy. The exact function of these globules is unknown, but their participation in the early phases of tube growth is certain. These dense globules are not newly synthesized since they exist prior to germination and subsequently become depleted as the tube elongates. Their intergradation with the plasma membrane and fibrillar cell wall layer suggests a direct involvement in the growth process at the tip. The fact that these globules exist in contact with the intine during pregermination raises the possibility that they may be related to the wall proteins localized at the light microscope level for pollen of other species (Knox and Heslop-Harrison, 1969, 1970) AP activity was detected in 50 nm and 120 nm vesicles, SER, RER, nucleus and plasma membrane. Activity in the distal region of the tube is concentrated in SER tubules and smooth regions of the RER whereas proximally, the reaction product was associated with the other organelles. It has been suggested that the function of the 50 nm vesicles may be the formation of dictyosomal cisternae (Larson, 1965) and that these small vesicles may represent an intermediary in membrane transfer from endoplasmic reticulum (ER) to dictyosomes (Van Der Woude et al., 1971). The localization of AP and similarity in lumenal opacity in the endoplasmic reticulum and 50 nm vesicles may be regarded as supporting this notion. The presence of the enzyme in the secretory vesicles of dictyosomal origin would seem to further strengthen the idea of ER-dictyosome continuity. Be-
189
cause the possibility of differential organelle sensitivity to fixatives and substrates does exist (Etherton and Botham, 1970; Borgers and Thone, 1976), the lack of reaction product in the dictyosomes should not be interpreted as demonstrating absence of the enzyme. AP activity has been reported in the Golgi apparatus of developing pollen (Maruyama, 1974) and in other plant cells and tissues (Charvat and Esau, 1975; Bentwood and Cronshaw, 1976; Noguchi, 1976). Malik et al. (1969) has suggested that in pollen tubes, lysosomes (AP-positive granules) are involved in the transport of metabolites from the proximal to the distal end via cytoplasmic streaming. BorskaBrylass (1965) has pointed out that AP activity are due to spherosomes which are absent from the tip while Petrovskaya-Baranova and Tsinger (t962) reported pronounced enzyme activity throughout the tube. In cherry, little or no activity was demonstrable within the growth zone and reaction product was not found in organelles which may be classified as lysosomes or spherosomes. However, these inconsistencies are possibly due to variations in cytochemical techniques and in vitro conditions utilized. The functional significance of acid phosphatase in differentiati on remains obscure. It has been shown to be of gametophytic origin (Vithanage and Knox, 1976) and correlation between this enzyme and lytic processes has frequently been reported (Holtzman, 1975). In this connection, AP-positive vesicles may be involved with the digestion of sequestered cytoplasmic material leading to the differentiation of autophagic vacuoles (Matile, 1975). The role of hydrolases in pollen tube growth requires clarification by further study. It has been demonstrated, for example, that AP decreases in the stylar tissue during compatible pollen tube growth (Roggen, 1967) but the status of this enzyme before and during the rejection process in gametophytic self-incompatibility is not known. Studies are in progress to determine the distribution of AP during in situ pollen tube growth in cherry.
References Bentwood, B.J., Cronshaw, J. : Biochemistry and cytochemical localization of acid phosphatase in the phloem of N i c o t i a n a tabacure. Planta (Berl.) 130, 9%104 (1976) Borgers, M., Thone, F.: Further characterization of phosphatase activities using non-specific substrates. Histochem. J. 8, 301-317 (1976) Brewbaker, J, L. : Pollen enzymes and isoenzymes. In : Pollen: development and physiology, pp. 156-170 Heslop-Harrison J., ed. London: Butterworth 1971 Brewbaker, J.L., Kwack, B.Y. : The essential role of calcium ion in pollen germination and pollen tube growth. Amer. J. Bot. 50, 859 865 (1963)
190 Charvat, I., Esau, K. : An ultrastructural study of acid phosphatase localization in Phaseolus vulgaris xylem by the use of an azo-dye method. J. Cell Sci. 19, 543-561 (1975) Crang, R.E., Miles, P.G. : An electron microscope study of germinating Lychnis alba pollen. Amer. J. Bot. 56, 398 405 (1969) Engels, F.M.: Function of Golgi vesicles in relation to cell wall synthesis in germinating Petunia pollen. I. Isolation of Golgi vesicles. Acta. Bot. Neerl. 22, 6-13 (1973) Engels, F.M. : Function of Golgi vesicles in relation to ceil wail synthesis in germinating Petunia pollen. II. Chemical composition of Golgi vesicles and pollen tube wail. Acta. Bot. Neerl. 23, 81-89 (1974) Etherton, J.E., Botham, C.M.: Factors affecting lead capture methods for the fine localization of rat lung acid phosphatase. Histochem. J. 2, 50%519 (1970) Franke, W.W., Herth, W., Van Der Woude, W.J., Morr6, D.J.: Tubular and filamentous structures in pollen tubes: possible involvement as guide elements in protoplasmic streaming and vectorial migration of secretory vesicles. Planta (Berl.) 105, 317-341 (1972) Gorska-Brylass, A. : Hydrolases in potlen grains and pollen tubes. Acta. Soc. Bot. Polon. 34, 589-604 (1965) Holtzman, E. : Lysomes: a survey, p. 298. Berlin-Heidelberg-New York: Springer 1975 Knox, R.B., Heslop-Harrison, J. : Cytochemical localization of enzymes in the wall of the pollen grain. Nature 223, 92 94 (1969) Knox, R.B., Heslop-Harrison, J.: Pollen-wall proteins: localization, and enzymic activity. J. Cell Sci. 6, 1 27 (1970) Larson, D.A. : Fine structural changes in the cytoplasm of germinating pollen. Amer. J. Bot. 52, 139 159 (1965) Malik, C.P., Tewari, H.B., Sood, P.P.: On the functional significance of certain phospbatases in the germinating pollen grains of Portulaca grandiJlora. Acta. Biol. Portug. 2, 245-252 (1969) Maruyama, K. : Localization of polysaccharides and phosphatases
J. L i n e t al. : Ultrastructural Localization of Acid Phosphatase in the Golgi apparatus of Tradescantia pollen. Cytologia 39, 767-776 (1974) Matile, Ph: The lytic compartment of plant cells, 183 pp. New York: Springer 1975 Noguchi, T. : Phosphatase activities and osmium reduction in cell organelles of Micrasterias americana. Protoplasma 87, 163-178 (1976) Petrovskaya, T.P., Tsinger, N.V.: A histochemical investigation of phosphatase in pollen, polien tubes and root hairs. Bot. J. 47, 132%1333 (1962) (U.S.S.R.) Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Roggen, H.P., Jr.: Changes in enzyme activities during the progame phase in Petunia hybrida. Acta. Bot. Neerl. 6, 1 31 (1967) Rosen, W.G.: Pollen tube growth and fine structure. In: Pollen development and physiology, pp. 177-185 J. Heslop-Harrison ed. London: Butterworth 1971 Rosen, W.G., Gawlik, S.R.: Fine structure of Lily pollen tubes following various fixation and staining procedures. Protoplasma 61, 181-19l (1966) Sassen, M.M.A. : Fine structure of Petunia pollen grain and pollen tube. Acta. Bot. Neerl. 13, 175 181 (1964) Stanley, R.G., Linskens, H.F. : Pollen: biology biochemistry, management, pp. 307 Berlin-Heidelberg-New York: Springer 1974 Van Der Woude, W.J., MorrO, D.J., Bracker, C.E.: Isolation and characterization of secretory vesicles in germinated pollen of Lilium longiflorum. J. Cell Sci. 8, 331-351 (1971) Vithanage, H.I.M.V., Knox, R.B.: Pollen-wall proteins: quantitative cytochemistry of the origins of intine and exine enzymes in Brassica oleracea. J. Cell Sci. 21, 423 435 (1976)
Received 14 February; accepted 20 March 1977
Planta
190 (1977)
9 by, Springer-Verlag 1977
Ultrastructural Localization of Acid Phosphatase in the Pollen Tube of Prunus avium L. (Sweet Cherry) J. Lin, W.J. Uwate, and V. Stallman* Department of Pomology, University of California, Davis, CA 95616, USA
Abstract. The pollen tube of Prunus avium (cherry)
consists of a growth zone of vesicles at the tip and an assemblage of organelles typical of an actively metabolizing cell. Electron opaque globules are closely associated with the plasma membrane and fibrillar cell wall layer at the tip. Acid phosphatase (EC 3.1.3.2) activity is localized in the membranes of 120 nm vesicles and ER system, the lumen of 50 nm vesicles, the plasma membrane and the tube nucleus. Key words: Acid phosphatase- Pollen t u b e - Prunus.
Introduction
The fine structure of pollen tubes growing in vitro has been the subject of a number of studies in the last 15 years (Crang and Miles, 1969; Franke et al., 1972; Larson, 1965; Rosen, 1971; Rosen and Gawlik, 1966; Sassen, 1964; Van Der Woude etal., 1971). These studies have shown that at the very tip of the tube is a growth zone characterized almost exclusively by secretory vesicles involved in cell wall synthesis and that proximally a diversity of organelles exists. Structural variations within the distal end of an actively elongating tube doubtless are related to differences in functional capacities and thus in enzyme distribution. Among the many enzymes which have been identified in pollen, acid phosphatase (EC 3.1.3.2) has been the most widely investigated (Brewbaker, 1971; Knox and Heslop-Harrison, 1970; Stanley and Linskens, 1974; Vithanage and Knox, * We would like to thank Dr. Richard Falk, Department of Botany, U.C.D., for his helpful suggestions and critical review of the manuscript
Abbreviations: AP = acid phosphatase; R E R = rough endoplasmic reticulum
1976). Biochemical analyses have demonstrated a decrease in AP after germination (Roggen, 1967). Within the growing tube, acid phosphatase activity reportedly decreases distally (Malik et al., 1969) or is absent in the tip region where spherosomes are lacking (Gorska-Brylass, 1965). Petrovskaya-Baranova and Tsinger (1962) on the other hand, found pronounced activity along the entire length of the tube. The ultrastructural localization of acid phosphatase has been reported for a number of plant cells and tissues (Charvat and Esau, 1975; Noguchi, 1976; Bentwood and Cronshaw, 1976), but to our knowledge, fine structural distribution of the enzyme has not been demonstrated in a pollen tube. This paper reports on the ultrastructure of the pollen tube of Prunus avium and on the distribution of acid phosphatase activity. Materials and Methods Flowers of Prunus avium, cultivar " V a n " , were collected during March, 1976, and anthers were allowed to dehisce in petri plates. Pollen was immediately frozen and used as needed during a seven month period. For optimum growth, pollen was cultured in the following medium (Brewbaker and Kwack, 1963): 100 mg/l boric acid, 100 mg/l potassium nitrate, 300 rag/1 calcium nitrate, 200 rag/1 magnesium sulfate, 10% sucrose and 0.8% agar. Sterilized medium was drawn into "9 inch" pasteur pipettes and allowed to solidify. The tips of pipettes (about 1 mm in diameter) were cut into 1 cm segments and placed upright in a holder. Cultures were started first by touching the point of a sharp needle with pollen and then inserting the needle tip approximately 1/2 mm into the agar cylinder. This prevented the loss of pollen tubes during subsequent procedures. Cultures were maintained a constant temperature (25~ and light (3500 ix). Because active growth of most pollen tubes took place between 21/2 to 31/2 h after initiation of the culture, most of the tubes studied were sampled during this time period. Lengths of pollen tubes ranged from 0.15-0.30 mm. Earlier stages were also studied. The agar cylinders with the germinated pollen were pushed out of the glass tubes and excess agar was removed. For localization of acid phosphatase, the lead capture method (Etherton and Botham, 1970) was used. This procedure was slightly modified for the present study. The agar embedded pollen tubes were pre-fixed for 30 rain in 4% glutaraldehyde in
184 0.05 M cacodylate buffer (pH 7.2) at room temperature. They were rinsed 3 times (10 min each) in a 0.05 M acetate buffer (pH 5.7) containing 7.5 % sucrose. The tubes were then incubated for 30 rain at 37~ C in a complete incubation medium containing 0.4 mg/ml c~-naphthyl phosphate, 1 mg/ml lead acetate and 7.5% sucrose in 0.05 M acetate buffer, pH 5.7. Controls consisted of incubation in a medium without the substrate, without lead acetate, or with the addition of 0.1 M sodium fluoride as an inhibitor. Lead precipitates in the incubation media were filtered out prior to use. Following incubation, pollen tubes were rinsed three times (15 min each) in 0.05 M acetate buffer, and post-fixed for 1 h in 1% OsO4 in 0.05 M cacodylate buffer at pH 7.2. They were dehydrated in a graded ethanol series and embedded in Spurr's plastic. Sections of materials incubated for enzyme localization and controls were not post-stained. Non-incubated pollen tubes were fixed in glutaraldehyde for l h and acetate buffer was not used. In some of the non-incubated tubes, 0.05 M phosphate buffer was used in place of cacodylate buffer. The latter consistently yielded superior results. Sections of non-incubated material were post-stained with uranyl acetate and lead citrate (Reynolds, 1963). All materials were observed with a Zeiss EM9 electron microscope.
Results
Ultrastructure Cultured cherry pollen germinated within 1/2 h after inoculation and tube elongation continued for about 6 h under conditions indicated above. Incipient swelling of the tube tip and subsequent bursting was observed about 8 h after germination. During active growth (i.e., 2~/2 to 3~/z h after inoculation), the distal end of the tube was dense with an assemblage of organelles typical of a rapidly metabolizing system. The growth zone (GZ) where components of the tube wall were being laid down consisted most conspicuously of nearly spherical vesicles ranging in diameter from 60 to 220 nm (Fig. 1). This zone encompassed roughly 5 pm of the distal-most portion of the tip. In the region immediately proximal to the growth zone, rod-shaped mitochondria aggregated in abundance. Mitochondria in this region appeared to be oriented randomly whereas those farther removed from the G Z tended to be aligned in the plane of the tube axis. R o u g h endoplasmic reticulum (RER) was similarly oriented reflecting cytoplasmic streaming which occurred in the living state (Fig. 2). Groups of highly anastomosed tubular smooth endoplasmic reticulum (SER) were present in the distal region, often in close proximity to the growth zone (Fig. 3). Large numbers of vesicles averaging 50 nm in diameter were interspersed a m o n g the other organelles and m a n y of them possessed a similar degree o f electron opacity as the lumen of the endoplasmic reticulum. It was not ascertained if structural continuity existed between the two components due to difficulty in distinguishing transverse sections of S E R tubules from 50 n m vesicles in the immediate vicinity
J. Linet al. : Ultrastructural Localization of Acid Phosphatase (see Discussion). Ribosomes occurred either freely or as components of the RER. Surrounding the distal 25 pm of the tube tip (including the growth zone) were highly electron opaque globules that occurred in close association with the plasma membrane. In the early stages of tube growth, these globules intergraded with the fibrillar cell wall layer at or near the growth zone and were periodically taken into the cytoplasm (Fig. 4). Immediately preceding germination, the globules existed as a continuous layer in contact with the intine (Fig. 5). It appears that those in the vicinity of the pore have been transported with the emerging tube. As the tube elongated they gradually diminished (in the tip depicted in Figure 1, most of the globules have been depleted). Thus, no globules were evident in the proximal region of the tube where an inner, relatively homogeneous, cell wall layer was present. This inner cell wall layer was continuous with the intine. Dictyosomes and amyloplasts were numerous throughout the growing tube, but were lacking in the vicinity of the growth zone. Amyloplasts exhibited close relationship with the R E R and often were partially surrounded by the latter (Fig. 6). Lipid bodies and vacuoles increased in numbers proximally. Sequestration of cytoplasmic materials was evident particularly in the proximal portion of the tube (Figs. 7 and 8).
Distribution of Acid Phosphatase Using ~-naphthyl phosphate as the substrate provided reproducible and satisfactory results for the localization of acid phosphatase activity in cherry pollen tubes. Little, if any, noticeable cytological damage was observed and thorough penetration of uncomplexed substrate was achieved within a 30 to 50 rain incubation period. These advantages are consistent with those pointed out by Etherton and Botham (1970). In tubes incubated in the complete medium, fine deposits of lead phosphate were present throughout and denser aggregations were found complexed with certain organelles. The amount and localization of reaction product was variable along the length of the tube. In the proximal region of the tube, lead phosphate deposits were bound heavily to membranes of vesicles averaging 120 nm in diameter. However, not all vesicles falling into this size category exhibited labeling (Fig. 9).The deposits appeared to be complexed with both faces of the membrane. Labeled vesicles were often seen to merge with the plasma membrane. In some cases, enzyme activity was found only on the outer face of membranes of
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
185
Fig. 1. Median longisection of the very tip of a cherry pollen tube during active growth. Note the growth zone (GZ) with its component secretory vesicles and the mitochondria (M) immediately proximal to the GZ. • 9400 Fig. 2. Rough endoplasmic reticulum (RER) in a proximal region of an elongating tube. x 14,400 Fig.3. A distal region of a pollen tube showing tubular smooth endoplasmic reticulum (SER) and 50nm vesicles (arrows). x 27,900 Fig. 4. A portion of the distal 25 pm of the tip. Note the highly electron opaque globules (G) intergrading with the plasma membrane (arrows) and the fibrillar cell wall (FCW). x 42,700
186
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
Fig. 5. A portion of a pollen grain showing electron opaque globules (arrows) in contact with the intine (Int). x 9,300 Fig. 6. An amyloplast (Amyl) with closely associated RER. x 27,600 Fig. 7. A proximal portion of a pollen tube showing lipid bodies (LB) and vacuoles (V). x 15,300 Fig. 8. Sequestration of cytoplasmic components (X). x 9300
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Fig. 9. A proximal region of a pollen tube showing heavy deposition of the reaction product in the membranes of vesicles averaging 120 nm in diameter (Ves). Larger vesicles exhibiting AP activity (arrows). Section not post-stained, x 60,200 Fig. 10. Control. A proximal region of a tube incubated in a medium containing fluoride. Section not post-stained, x 60,000 Fig. 11. Control. A proximal region of a tube incubated in a medium without the substrate. Section not post-stained, x 27,500 Fig. 12. Acid phosphatase activity in 50 nm vesicles (arrows). Note some vesicles lacking the reaction product. Section not post-stained. x 60,100
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J. Linet al. : Ultrastructural Localization of Acid Phosphatase
Fig. 13. A portion of a tube showing acid phosphatase activity in the tube nucleus (Nuc) and the plasma membrane. Section not post-stained, x 5850 Fig. 14. Acid phosphatase activity in the SER of the distal portion of the pollen tube. Section not post-stained, x 27,600 Fig. 15. Reaction product localized mostly on the inner face of RER. Section not post-stained, x 27,200 Fig. 16. Control. Lead deposits lacking in SER of a tube incubated in a medium minus substrate. Section not post-stained, x 28,000
slightly larger vesicles a n d l o c a l i z a t i o n was sporadic (Fig. 9). R e a c t i o n was n e g a t i v e in similar vesicles o f c o n t r o l t u b e s i n c u b a t e d in m e d i a c o n t a i n i n g the inhibitor s o d i u m f l u o r i d e or in m e d i a l a c k i n g e - n a p h t h y l p h o s p h a t e (Figs. 10 a n d 11). It s h o u l d be pointed o u t t h a t in one t u b e i n c u b a t e d in the c o m p l e t e med i u m plus fluoride, t h e r e were dense d e p o s i t s in some vacuoles. I t is n o t k n o w n w h e t h e r these represented
the r e a c t i o n p r o d u c t . I n all the o t h e r tubes studied, l e a d d e p o s i t s were l a c k i n g in vacuoles. T u b e s incub a t e d in a m e d i u m w i t h o u t l e a d e x h i b i t e d no electron dense particles. A P activity was also evident in the l u m e n o f 50 n m vesicles. L a b e l e d vesicles t e n d e d to occur close t o g e t h e r in a relatively h o m o g e n e o u s matrix (Fig. 12). T h e n u c l e o p l a s m o f the t u b e nucleus showed
J. Lin et al. : Ultrastructural Localization of Acid Phosphatase
scattered activity whereas a greater concentration of lead phosphate deposits were found within what may be a nucleolus (Fig. 13). Enzyme activity was also detected in the plasma membrane. Within the distal portion of the tube, AP was localized to a large extent in the membranes of the ER system. The reaction product was present in the tubular SER and also bound mostly to the inner face of smooth membrane regions of R E R (Figs. 14 and 15). Figure 16 shows the absence of lead phosphate in the SER of a tube incubated in a medium without the substrate.
Discussion
The fine structure of the visicles found in the growth zone of cherry pollen tubes fits the criteria set forth by Van Der Woude et al. (1971) for secretory vesicles. There is no doubt that these vesicles are involved in the transfer and deposition of cell wall materials and take their origin in the dictyosomes (Engels, 1973, 1974; Maruyama, 1974). The highly electron opaque globules at the tip of cherry pollen tubes have not been reported in other species. Differences in tissue preparation may in part account for this discrepancy. The exact function of these globules is unknown, but their participation in the early phases of tube growth is certain. These dense globules are not newly synthesized since they exist prior to germination and subsequently become depleted as the tube elongates. Their intergradation with the plasma membrane and fibrillar cell wall layer suggests a direct involvement in the growth process at the tip. The fact that these globules exist in contact with the intine during pregermination raises the possibility that they may be related to the wall proteins localized at the light microscope level for pollen of other species (Knox and Heslop-Harrison, 1969, 1970) AP activity was detected in 50 nm and 120 nm vesicles, SER, RER, nucleus and plasma membrane. Activity in the distal region of the tube is concentrated in SER tubules and smooth regions of the RER whereas proximally, the reaction product was associated with the other organelles. It has been suggested that the function of the 50 nm vesicles may be the formation of dictyosomal cisternae (Larson, 1965) and that these small vesicles may represent an intermediary in membrane transfer from endoplasmic reticulum (ER) to dictyosomes (Van Der Woude et al., 1971). The localization of AP and similarity in lumenal opacity in the endoplasmic reticulum and 50 nm vesicles may be regarded as supporting this notion. The presence of the enzyme in the secretory vesicles of dictyosomal origin would seem to further strengthen the idea of ER-dictyosome continuity. Be-
189
cause the possibility of differential organelle sensitivity to fixatives and substrates does exist (Etherton and Botham, 1970; Borgers and Thone, 1976), the lack of reaction product in the dictyosomes should not be interpreted as demonstrating absence of the enzyme. AP activity has been reported in the Golgi apparatus of developing pollen (Maruyama, 1974) and in other plant cells and tissues (Charvat and Esau, 1975; Bentwood and Cronshaw, 1976; Noguchi, 1976). Malik et al. (1969) has suggested that in pollen tubes, lysosomes (AP-positive granules) are involved in the transport of metabolites from the proximal to the distal end via cytoplasmic streaming. BorskaBrylass (1965) has pointed out that AP activity are due to spherosomes which are absent from the tip while Petrovskaya-Baranova and Tsinger (t962) reported pronounced enzyme activity throughout the tube. In cherry, little or no activity was demonstrable within the growth zone and reaction product was not found in organelles which may be classified as lysosomes or spherosomes. However, these inconsistencies are possibly due to variations in cytochemical techniques and in vitro conditions utilized. The functional significance of acid phosphatase in differentiati on remains obscure. It has been shown to be of gametophytic origin (Vithanage and Knox, 1976) and correlation between this enzyme and lytic processes has frequently been reported (Holtzman, 1975). In this connection, AP-positive vesicles may be involved with the digestion of sequestered cytoplasmic material leading to the differentiation of autophagic vacuoles (Matile, 1975). The role of hydrolases in pollen tube growth requires clarification by further study. It has been demonstrated, for example, that AP decreases in the stylar tissue during compatible pollen tube growth (Roggen, 1967) but the status of this enzyme before and during the rejection process in gametophytic self-incompatibility is not known. Studies are in progress to determine the distribution of AP during in situ pollen tube growth in cherry.
References Bentwood, B.J., Cronshaw, J. : Biochemistry and cytochemical localization of acid phosphatase in the phloem of N i c o t i a n a tabacure. Planta (Berl.) 130, 9%104 (1976) Borgers, M., Thone, F.: Further characterization of phosphatase activities using non-specific substrates. Histochem. J. 8, 301-317 (1976) Brewbaker, J, L. : Pollen enzymes and isoenzymes. In : Pollen: development and physiology, pp. 156-170 Heslop-Harrison J., ed. London: Butterworth 1971 Brewbaker, J.L., Kwack, B.Y. : The essential role of calcium ion in pollen germination and pollen tube growth. Amer. J. Bot. 50, 859 865 (1963)
190 Charvat, I., Esau, K. : An ultrastructural study of acid phosphatase localization in Phaseolus vulgaris xylem by the use of an azo-dye method. J. Cell Sci. 19, 543-561 (1975) Crang, R.E., Miles, P.G. : An electron microscope study of germinating Lychnis alba pollen. Amer. J. Bot. 56, 398 405 (1969) Engels, F.M.: Function of Golgi vesicles in relation to cell wall synthesis in germinating Petunia pollen. I. Isolation of Golgi vesicles. Acta. Bot. Neerl. 22, 6-13 (1973) Engels, F.M. : Function of Golgi vesicles in relation to ceil wail synthesis in germinating Petunia pollen. II. Chemical composition of Golgi vesicles and pollen tube wail. Acta. Bot. Neerl. 23, 81-89 (1974) Etherton, J.E., Botham, C.M.: Factors affecting lead capture methods for the fine localization of rat lung acid phosphatase. Histochem. J. 2, 50%519 (1970) Franke, W.W., Herth, W., Van Der Woude, W.J., Morr6, D.J.: Tubular and filamentous structures in pollen tubes: possible involvement as guide elements in protoplasmic streaming and vectorial migration of secretory vesicles. Planta (Berl.) 105, 317-341 (1972) Gorska-Brylass, A. : Hydrolases in potlen grains and pollen tubes. Acta. Soc. Bot. Polon. 34, 589-604 (1965) Holtzman, E. : Lysomes: a survey, p. 298. Berlin-Heidelberg-New York: Springer 1975 Knox, R.B., Heslop-Harrison, J. : Cytochemical localization of enzymes in the wall of the pollen grain. Nature 223, 92 94 (1969) Knox, R.B., Heslop-Harrison, J.: Pollen-wall proteins: localization, and enzymic activity. J. Cell Sci. 6, 1 27 (1970) Larson, D.A. : Fine structural changes in the cytoplasm of germinating pollen. Amer. J. Bot. 52, 139 159 (1965) Malik, C.P., Tewari, H.B., Sood, P.P.: On the functional significance of certain phospbatases in the germinating pollen grains of Portulaca grandiJlora. Acta. Biol. Portug. 2, 245-252 (1969) Maruyama, K. : Localization of polysaccharides and phosphatases
J. L i n e t al. : Ultrastructural Localization of Acid Phosphatase in the Golgi apparatus of Tradescantia pollen. Cytologia 39, 767-776 (1974) Matile, Ph: The lytic compartment of plant cells, 183 pp. New York: Springer 1975 Noguchi, T. : Phosphatase activities and osmium reduction in cell organelles of Micrasterias americana. Protoplasma 87, 163-178 (1976) Petrovskaya, T.P., Tsinger, N.V.: A histochemical investigation of phosphatase in pollen, polien tubes and root hairs. Bot. J. 47, 132%1333 (1962) (U.S.S.R.) Reynolds, E.S. : The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Roggen, H.P., Jr.: Changes in enzyme activities during the progame phase in Petunia hybrida. Acta. Bot. Neerl. 6, 1 31 (1967) Rosen, W.G.: Pollen tube growth and fine structure. In: Pollen development and physiology, pp. 177-185 J. Heslop-Harrison ed. London: Butterworth 1971 Rosen, W.G., Gawlik, S.R.: Fine structure of Lily pollen tubes following various fixation and staining procedures. Protoplasma 61, 181-19l (1966) Sassen, M.M.A. : Fine structure of Petunia pollen grain and pollen tube. Acta. Bot. Neerl. 13, 175 181 (1964) Stanley, R.G., Linskens, H.F. : Pollen: biology biochemistry, management, pp. 307 Berlin-Heidelberg-New York: Springer 1974 Van Der Woude, W.J., MorrO, D.J., Bracker, C.E.: Isolation and characterization of secretory vesicles in germinated pollen of Lilium longiflorum. J. Cell Sci. 8, 331-351 (1971) Vithanage, H.I.M.V., Knox, R.B.: Pollen-wall proteins: quantitative cytochemistry of the origins of intine and exine enzymes in Brassica oleracea. J. Cell Sci. 21, 423 435 (1976)
Received 14 February; accepted 20 March 1977
