J. Cell Sci. 38, 61-82 (1979) Printed in Great Britain © Company of Biologists Limited DEVELOPMENTAL MECHANISMS IN HETEROSPORY: CYTOCHEMICAL DEMONSTRATION OF SPORE-WALL ENZYMES ASSOCIATED WITH /?-LECTINS, POLYSACCHARIDES AND LIPIDS IN WATER FERNS J.M. PETTITT Department of Botany, British Museum (Natural History), Cromwell Road, London SW7 5BD, England SUMMARY Cytochemical methods are used to examine the distribution and localization of acid phos- phatase, non-specific esterase, ribonuclease and peroxidase activity in the walls of the spores of the heterosporous Marsileaceae before and during germination. In the quiescent spore, the principal activity is associated with the perine layer of the wall and the intine, with some acti- vity in the outer, gelatinous wall layer, but none in the exine. The microspores of Marsilea and Pilularia have non-specific esterase activity concentrated in the intine in the immediate vicinity of the germinal site; that is, above the position of the future male gametangia. The enzymes are not leached from the wall during hydration of the spores, although ribonuclease is redistributed during imbibition with a high concentration of activity remaining in or around the germinal site. The wall enzymes occur together with PAS-reactive and acidic carbohydrates, lipids, and in the microspore perine, /?-lectins. Following the enzyme pattern, the /?-lectins are found to be concentrated in the region of the germinal site. /¥-lectin activity is absent from the megaspore wall. Acidic carbohydrates are confined to the gelatinous wall layer and this layer also binds con- canavalin A. In contrast to what has been found for other plant cells, the spore-wall /9-lectins are not water-labile; the activity is not significantly diminished after hydration. This surprising stability suggests that these molecules, together with the enzymes, may be retained in position in the wall by the waterproof overlay of lipid. From the evidence of preliminary developmental studies, it appears that the enzymes as- sociated with the perine layer of the wall originate in the sporophytic tapetal periplasmodium and inclusion of the activity is concurrent with wall differentiation, while the activity associated with the intine is derived from the gametophyte. It is possible, however, in the megaspore at least, that the distribution of the activity may to some extent be influenced by a system of exine channels which communicates between the two domains of the wall during sporogenesis. No definite information is obtained concerning the utility of the enzymes and associated molecules in the life of the spore. Acting separately or in co-operation, their role could con- ceivably be connected with one or more of four processes; wall differentiation, gametophyte nutrition, gametophyte protection or reproduction. Each of these possibilities is discussed. INTRODUCTION The evidence is now quite unequivocal that the pollen walls of many species of flowering plants and some gymnosperms contain proteins with cytochemically 5 CEL 38 62 J. M. Pettitt detectable enzymic activity (Tsinger & Petrovskaya-Baranova, 1961; Knox & Heslop- Harrison, 1969, 1970, 1971a, b; Knox, 1971; Heslop-Harrison, Heslop-Harrison, Knox & Howlett, 1973; Knox, Heslop-Harrison & Heslop-Harrison, 1975; Pettitt, 1976a, 1977a; Vithanage & Knox, 1976; Ducker, Pettitt & Knox, 1978). A range of hydrolases, but principally acid phosphatase and non-specific esterase, has been found to be concentrated in the intine, the inner, cellulosic wall layer of spermatophyte pollen (Knox & Heslop-Harrison, 1970, 1971 a, b; Heslop-Harrison et al. 1973; Knox et al. 1975; Vithanage & Knox, 1976; Pettitt, 1977a), while non-specific esterase, dehydrogenase and oxidase activities have been detected in the exine, the resistant, outer wall layer (Tsinger & Petrovskaya-Baranova, 1961; Knox & Heslop-Harrison, 1969, 1970; Knox, 1971; Vithanage & Knox, 1976). There is evidence from develop- mental studies to show that the intine proteins in the flowering plants are direct pro- ducts of the haploid gametophyte inserted into the wall during deposition of the intine layer. The proteins carried in the exine are, on the other hand, derived from the anther tapetum, a tissue of the parental sporophyte, and are injected into the wall during the final maturation of the grain. A seemingly constant feature in the flowering plant species is the occurrence with the exine proteins of other classes of material, including glycoproteins and lipids (Knox, 1971; Knox & Heslop-Harrison, 19716; Heslop-Harrison et al. 1973; Knox et al. 1975; Vithanage & Knox, 1976). A number of roles have been proposed for the well-circumscribed system of wall enzymes in the biology of the angiosperm pollen grain and it would seem, both from the distribution and the speed of release when the grain is moistened, that their programme concerns germination and degradation of the stigma cuticle as well as early growth of the pollen tube; the quintessence, in fact, of siphonogamy (Knox & Heslop-Harrison, 1970; Heslop-Harrison, 1975; Knox et al. 1975). It has been sugges- ted, too, that the elaborately chambered pollen exine characteristic of flowering plants may have evolved, rather than been exploited, expressly as a conveyance of sporophytic materials (J. Heslop-Harrison, 1976). Proteins with enzymic activity have now been recognized in the spore walls of some rather distinctive heterosporous pteridophytes, members of the infelicitously called water ferns. The fern spore enzymes are, moreover, topologically associated with /Mectins, carbohydrates and lipids and the activity is retained in the wall during germ- ination. This paper reports on the cytochemical localization and characterization of these molecules in three genera of the Marsileaceae. MATERIALS AND METHODS Spores were taken from ripe, dry sporocarps of Marsilea drummondii A. Br., M. mutica Mett. M. berhautii Tardieu, Pilularia globulifera L., P. novae-hollandiae A. Br. and Regnellidium diphy- llum Lindm. and encased in a mixture of 15% (w/v) gslatin and 2% (v/v) glycerol (Knox, 1970) for freeze-sectioning at 8 /im in a cryostat. Alternatively, the dry spores were fixed in chilled 2-5 % glutaraldehyde buffered in o-i M cacodylate-HCl at pH 7'2, 450 mosM, and rinsed in buffer before being encased in gelatin-glycerol. This brief period of fixation improved the cutting quality of the spores, but exposure to the fixative solution was found not to influence the distribution of the wall enzymes. To determine the distribution of the wall enzymes and other wall-associated components during the initial stage of spore germination, ripe sporocarps were scarified and cast into dishes Spore-wall enzymes in ferns 63 of filtered pond water at room temperature. Imbibition and swelling commenced immediately and the sporangia emerged from the open sporocarp within 15-20 min. The course of hydration, the process which actuates gametogenesis in the plants, was monitored under a dissecting micro- scope and germination was allowed to continue for 2'5 h before the spores were harvested to be either fixed for 1-5 h in chilled glutaraldehyde or quenched in isopentane in liquid nitrogen and freeze-dried in a Speedivac-Pearse tissue dryer. The specimens were then encased in gelatin- glycerol for freeze-sectioning. Water was taken up rapidly by the contents of the sporocarps and the fully imbibed spores were liberated within 15-30 min of opening. The 2-5~h interval, there- fore, was more than sufficient time to ensure complete hydration in all the species. Machlis & Rawitscher-Kunkel (1967a) provide a detailed description of the course of spore hydration in Marsilea. Polysaccharide, ji-lectin, protein and lipid localization The standard periodic acid-Schiff (PAS) reaction (Pearse, 1968) was used to detect poly- saccharides containing vicinal glycol groups. Control sections were not subjected to the acid oxidation step. Polyanions were localized for light microscopy by staining cryostat sections with Alcian blue 8GX at pH 2'5 (Mowry, 1963) and for electron microscopy by the addition of puri- fied ruthenium red to the primary aldehyde and secondary osmium fixative solutions (Luft, 1971; Pettitt, 19776). Acceptor molecules for concanavalin A (Con A) were detected in section of unfixed spores with Con A conjugated to fluorescein isothiocyanate (FITC-Con A). The sections were flooded with FITC-Con A (L'Industrie Biologique Francaise) at 10 mg/ml in. phosphate-buffered saline for 15 min, rinsed in buffered saline and examined in a fluorescence microscope transmitting in the blue range. Control sections were incubated in the competitive inhibitor a-methyl-D-mannoside, 0-2 M for 15 min, before treatment with FITC-Con A con- taining 0-2 M inhibitor. The sites of /?-lectin activity were detected in sections and whole mounts of unfixed spores by staining in /?-glucosyl (/?-GLU) Yariv artificial carbohydrate antigen (Jermyn & Yeow, 1975) according to the procedure employed by Clarke, Knox & Jermyn (1975). Negative and ambi- guous results were re-investigated by observing response in whole spores or sections previously treated with hot ethanol to remove possible low molecular mass inhibitors that are known to interfere with artificial antigen-/?-lectin binding (Clarke, Gleeson, Jermyn & Knox, 1978). The specificity of the staining was assessed by treating parallel sections with the a-galactosyl (a-GAL) Yariv derivative (Clarke et al. 1975). Proteins were detected in fixed, freeze-sectioned spores with 0-25 % Coomassie blue in