POLLEN-WALL PROTEINS: ELECTRON- MICROSCOPIC LOCALIZATION OF ACID PHOSPHATASE IN THE INTINE OF CROCUS VERNUS

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1 J. Cell Sci. 8, (197O 727 Printed in Great Britain POLLEN-WALL PROTEINS: ELECTRON- MICROSCOPIC LOCALIZATION OF ACID PHOSPHATASE IN THE INTINE OF CROCUS VERNUS R.B. KNOX* AND J. HESLOP-HARRISONf Institute of Plant Development, Birge Hall, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. SUMMARY Acid phosphatase has been localized in the wall of the pollen grain of Crocus vernus Wulf at the electron-microscope level by a method using 2-naphthyl thiol phosphate as substrate in a simultaneous coupling reaction with fast blue BBN at ph 50, the product being given electron opacity by osmication. Activity was found to be concentrated mainly in the intine, and to be associated with ribbonlike or filamentous inclusions believed to be proteinaceous on the basis of other criteria. Some activity was also detectable in the interstices of the exine. The observations confirm the general interpretation of the distribution of wall-held enzyme based upon light-microscopic cytochemistry, and provide the resolution necessary to establish unambiguously that they are associated with protein layers inserted during intine growth. INTRODUCTION In an earlier paper (Knox & Heslop-Harrison, 1970) we described the use of lightmicroscopic (LM) cytochemical methods for the detection and localization of enzyme activity in the walls of the pollen grains of 50 angiosperm species, and showed that the activity was associated principally with the cellulosic intine, especially near the pores and colpi of aperturate grains. Among the methods tested for acid phosphatase were those developed by Hanker et al. (1964) using osmiophilic reagents. The results were not entirely satisfactory for LM work with pollen because the sporopollenin of the exine darkens with OsO 4, so that exine and intine cannot readily be distinguished. However, the methods were devised by the original authors for electron-microscopic (EM) application, and in this role they have proved most successful with the pollen wall enzymes. In this note we report on the localization of acid phosphatase in the wall of the pollen grain of Crocus vernus. MATERIALS AND METHODS Mature anthers were collected from garden-grown Crocus vernus Wulf, and stored over Drierite. Tests were made on samples of loose pollen, or sometimes on anther segments containing unreleased pollen. Present address; Department of Botany, Australian National University, Box 4, G.P.O., Canberra A.C.T. f Present address: Old Post, Hatfield, Herefordshire, England.

2 728 R. B. Knox and J. Heslop-Harrison In the method of Hanker et al. (1964) for acid phosphatase, 2-naphthyl thiol phosphate is used as substrate in a simultaneous coupling reaction with fast blue BBN. The insoluble yellowish diazoether formed is then exposed to OsO 4 vapour at 50 C to give 'osmium black'. In the modification developed for pollen, 5 mg 2-naphthyl thiol phosphate were dissolved in about o-i ml dimethyl sulphoxide, and the solution taken up in 10 ml 0-5 M sucrose buffered at ph 5-0 in o-i M acetate buffer. To this were added 5 mg fast blue BBN, and the mix poured into a centrifuge tube. The dry pollen was suspended in this medium and incubated for 30 min at room temperature. The pollen was then spun down, washed in buffer, and spun down once again before transfer to a small moistened filter paper wick. The wick was then suspended freely in a vial containing osmium tetroxide crystals in a drop of distilled water; the vial was sealed with a siliconed glass slide, placed with its base in a water bath at 50 C for 20 min, and then cooled in ice water. The wick bearing the osmicated pollen was removed, thoroughly washed in buffer and dehydrated through an ethanol series before embedding in an Epon-Araldite mix by standard methods. Sections were cut with a diamond knife, and examined without post-staining. OBSERVATIONS AND DISCUSSION The pollen grain of Crocus has a relatively thick intine and a thin exine, and is nonaperturate. In the earlier LM study (Knox & Heslop-Harrison, 1970) acid phosphatase activity was readily localized by the method of Barka & Anderson (1962) using naphthol AS-BI phosphate in a coupling reaction with hexazonium pararosanalin. The main site of activity was in the central zone of the intine, but some reaction product was also formed in the exine, and in many pollen samples activity was also detected at the plasmalemma of the vegetative cell (Figs. 1, 2). Although there would undoubtedly be some loss of protein from the intine during preparation, excellent localization of acid phosphatase activity was obtained in the EM study. Examples are shown in Figs. 3, 4, 6 and 7. Control material, glutaraldehyde pre-fixed and osmicated without exposure to the reaction mixture containing substrate and coupling agent, is illustrated in Figs. 5 and 8. The concentration of activity in the intine is not in doubt. Moreover, the reaction product is seen to be associated unambiguously with inclusions on the intine. Comparison of the radial and tangential sections of Figs. 6 and 7 shows that in the outer part of the intine these inclusions take the form of ribbons or filaments, with the long axis disposed periclinally. From Fig. 3 it may be seen that in the inner part they are drawn out radially. Similar inclusions have been described in the intine of the mature pollen of many species of angiosperms, and the manner of incorporation during intine growth has been described for Silene pendula (Heslop-Harrison, 1963) and Cosmos bipinnatus (Knox & Heslop-Harrison, 1970, where references to other literature on intine inclusions are given). The proteinaceous nature of the inclusions has been inferred from electron-staining properties and other cytochemical evidence, and the present observations confirm the earlier supposition that they are the actual sites of the wall-held enzymes. Figs. 3 and 4 show that reaction product is also deposited in the interstices between the irregular sporopollenin bacula of the exine. Presumably it is this site of acid phosphatase activity which is responsible for the colouring of the exine in the LM preparation of Fig. 1. The exine of Crocus and of other Iridaceae is unusual in that it is essentially porous, with no continuous tectum or nexine, a point readily confirmed from Fig. 4.

3 Acid phosphatase in Crocus inline 729 There is a distinct likelihood that exine-held enzymes in other species are injected from the outside of the grain during late maturation (e.g. Cosmos bipinnatus, Knox & Heslop-Harrison, 1970), but this cannot yet be affirmed for Croats, since the activity apparent in the exine in Figs. 3 and 4 could also be due to a leakage of enzyme from the intine during processing. The layer of acid phosphatase activity in the intine is bounded within by a wall zone which is evidently free of enzymes, as may be seen in both LM (Fig. 1) and EM (Fig. 3) preparations. This is true for other species also, and it results from the fact that insertion of protein in the wall ceases before the conclusion of polysaccharide deposition (Knox & Heslop-Harrison, 1970). In Fig. 3, there is no evident concentration of phosphatase activity just within the plasmalemma, although reaction product is present in sparsely distributed spherical organelles in the vegetative cell cytoplasm. In the LM preparation of Fig. i, however, there is a well-defined zone of activity in the peripheral cytoplasm. The difference is not especially disconcerting, since not all LM preparations do show enzyme activity at the plasmalemma. The variation may reflect differences in the developmental state of the grain, or possibly in hydration. This work was supported by N.S.F. Grant No. GB 7775 and by the Graduate School of the University of Wisconsin: we thank both these agencies. REFERENCES BARKA, T. & ANDERSON, P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanalin as coupler. J. Histochem. Cytocliem. 10, HANKER, J. B., SEAMAN, A. R., WEISS, L. P., UENO, H., BERGMAN, R. A. & SELIGMAN, A. M. (1964). Osmiophilic reagents: new cytochemical principle for light and electron microscopy. Science, N.Y. 146, HESLOP-HARRISON, J. (1963). Ultrastructural aspects of differentiation in sporogenous tissue. Symp. Soc. exp. Biol. 17, KNOX, R. B. & HESLOP-HARRISON, J. (1970). Pollen wall proteins: localization and enzymic activity. J. Cell Sci. 6, {Received 9 October 1970)

4 730 R. B. Knox and J. Heslop-Harrison Fig. i. Croats vernus, freeze-sectioned pollen grain. Naphthol AS-BI phosphate pararosanalin reaction for acid phosphatase (Knox & Heslop-Harrison, 1970). e, exine; i, intine; pm, plasmalemma. x approx. Fig. 2. Control for Fig. 1; reaction mixture without substrate, x approx. Figs Electron micrographs of the walls of mature pollen grains of Crocus vernus. Figs. 3, 4, 6 and 7 show localization of acid phosphatase by the method of Hanker et al. (1964). Figs, s and 8 are after glutaraldehyde fixation followed by osmication, but without exposure to the acid phosphatase reaction mixture. Fig. 3. Labelling as in Fig. 1. The electron-opaque granules are the reaction product. Activity is mainly associated with the intine, but some reaction product is present in the exine. x 7000 approx.

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6 R. B. Knox and J. Heslop-Harrison Fig. 4. Exine at higher magnification showing the distribution of the reaction product as fine electron-opaque granules in the spaces between the sporopollenin bacula and also on the outer surface, x approx. Fig. 5. Control for Fig. 4. x approx. Fig. 6. Intine following the reaction for acid phosphatase, radial section of the mid-zone. The reaction product is associated with inclusions of medium electron opacity, circular or slightly elliptical in section in this orientation, x approx. Fig. 7. As Fig. 6, but sectioned somewhat tangentially. The inclusions now appear elongated, showing that they are in the forms of ribbons or filaments, x approx. Fig. 8. Control for Figs. 6 and 7. Without exposure to the acid phosphatase reaction mixture or any form of post-staining the intine inclusions appear as ribbons or filaments of medium electron opacity, x approx.

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