Developmental Mechanisms in Heterospory: Cytochemical Demonstration of Spore-Wall Enzymes Associated with /?-Lectins, Polysaccharides and Lipids in Water Ferns

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 phosphatase, 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 activity 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 concanavalin 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 associated 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 developmental studies to show that the intine proteins in the flowering plants are direct products 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 germination. 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 diphyllum 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 microscope 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, therefore, 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 polysaccharides 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 purified 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 containing 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 me- thanol:acetic acid (Cavvood, Potter & Dickinson, 1978). Lipids were localized by staining sections in a saturated solution of Sudan black B prepared in 70% ethanol under reflux. The sections were rinsed in 70% ethanol and then in water before mounting in polyvinylpyrrolidone medium (Burstone, 1957).

Enzyme localization Acid phosphatase. Two tests for activity were applied. In the first method, naphthol AS-BI phosphoric acid dissolved in dimethylformamide (10 mg/ml) was used in a reaction mixture at pH 5-0 with hexazotized pararosanilin (Barka & Anderson, 1962). The reaction was stopped after 30 min by rinsing in water and the sections were rapidly dehydrated in ethanol, cleared in xylenc and mounted in a permanent, synthetic medium. The second method was a modification of the Gomori lead-capture reaction (Bitensky & Cheyen, 1977). Controls for both methods were: (a) the reaction mixture with the substrate omitted: (b) sections pre-incubated in 001 M NaF for 5 min before incubation in the complete reaction mixture containing 001 M NaF. Specimens intended for enzyme localization by transmission electron microscopy were encased in gelatin-glycerol, sectioned at 30-50 /«m in the cryostat and fixed for ih in chilled 2-5 % glutaraldehyde buffered in o-i M cacodylate-HCl (pH 7-2, 450 mosM), washed in several changes of buffer and transferred to the Bitensky & Cheyen (1977) medium. The medium was withdrawn after 30 min incubation at room temperature, the sections were flushed with several volumes of distilled water and then rinsed thoroughly before being immersed in 1 % aqueous 5-2 64 J. M. Pettitt osmium tetroxide for i h. The conversion treatment with H2S-saturated water, necessary when the procedure is for light microscopy (Bitensky & Cheyen, 1977), was omitted. The osmium treatment was followed by dehydration through an acetone series and embedding in Spurr's resin mixture (Spurr, 1969). Thin sections of selected areas were cut with a diamond knife and examined before and after staining in methanolic uranyl acetate and lead citrate. Esterase. Two standard tests were employed. The substrate a-naphthyl acetate was used in a coupling reaction with Fast Blue B Salt (tetrazotized o-dianisidine) at pH 6-7-4 (Pearse, 1972) and 5-bromoindoxyl acetate in 01 M Tris-HCl buffer at pH 6—8-5 was the substrate in the indigogenic method (Pearse, 1972). In both methods control sections were incubated in the appropriate reaction mixture prepared without the substrate. To compensate for the difference in reaction rates, a considerably longer reaction time was allowed in the second method than in the first. Under these conditions, the two tests gave identical results. Ribonuclease. The method developed by Knox & Heslop-Harrison (1970) for the detection of RNase activity in pollen grains was used. Controls were run by omitting the RNA from the reaction mixture and by treating the sections with 001 M NaF for 5 min before incubation in the complete reaction mixture containing o-oi M NaF. Peroxidase. Activity was detected with the benzidine and 3-amino-9-ethyl carbazole methods (Pearse, 1972). The responses to both tests corresponded, although the final blue (quinhydrone) reaction product developed in the benzidine method proved unstable. Controls were as follows: (a) the hydrogen peroxide omitted from the benzidine reaction mixture; (6)o-oi M sodiumazide included in the complete benzidine reaction mixture; (c) the substrate omitted from both reaction mixtures.

OBSERVATIONS Wall morphology. The general features of spore-wall morphology, presented dia- grammatically in Fig. 1, are similar in all the species. For the purposes of the present

Fig. 1. Diagrams of generalized radial sections through the walls of the megaspore (A) and microspore (B) of the Marsileaceae, based upon transmission electron micrographs. The sporopollenin layers (perine and exine) are shown black; the intine is dashed and stippled, and the outer, gelatinuous layers are stippled. The various features are not drawn to scale. Spore-wall enzymes in ferns 65 study, the minor differences in construction, while of morphogenetic interest and potential taxonomic value, are not of significance. The primary subdivision of the wall is clearly demarcated both in the microspore and megaspore into the outer perine, the middle exine and the inner intine. The perine and exine are composed of sporopollenin, a resistant polymer of carotenoid and carotenoid esters (Brooks & Shaw, 1968; Brooks, 1971). Two strata can be recognized in the megaspore perine, the outer 'prismatic' layer built of radially aligned chambers and the inner 'reticulate' layer constructed of anastomosing bars (Fig. 39). At the proximal pole of the spore, in the region of the germinal site, the differentiation into 2 divisions is less definite and the thickness of the perine is reduced (Fig. 32). The homologous layer on the correspond- ing microspore consists of a plicated sheet of regular thickness and the amplitude of the perine plications is diminished towards the proximal pole of the spore (Fig. 24; Chry- sler & Johnson, 1939; Pettitt, 1971, 1979)- The exine is ostensibly without structure and of uniform texture (Fig. 40), although it has been shown that the megaspore exine in Marsilea contains a system of inosculating channels during development (Pettitt, 1979). The layer is absent at the germinal suture (Fig. 26). The intine, putatively cellulosic (Feller, 1953; Boterberg, 1956) usually exhibits some stain-stratification in thin sections stained with heavy metals (Pettitt, 1979). It has been shown that the 2 sporopollenin components of the wall have different origins in development. The source of the material for the construction of the perine is the sporophytic tapetal periplasmodium, whereas the exine is formed of material eman- ating from the gametophyte (Feller, 1953; Bonnet, 1955; Boterberg, 1956; Pettitt, 1971, 1979). During the closing phase of sporogenesis, the so-called gelatinous wall layers are formed (Figs. 37, 38). There are 2 main interpretations regarding the mode of formation of these — the periplasmodium synthesizes and secretes the constituent material (Feller, 1953) or is gradually transformed into it (Boterberg, 1956). The gelatinous layers envelop the individual spores and imbibe water readily on germination. During hydration of the megaspore they elevate to form an elaborate envelope housing the sperm lake (Muenier, 1888; Bonnet, 1955; Machlis & Rawitscher-Kunkel, 1967a).

Wall-associated components: non-enzymic Polysaccharid.es. In all the species, the microspore and megaspore perine contains PAS-reacting carbohydrates. The method of localization showed a strong reaction in the reticulate layer of the megaspore wall and a moderate reaction in the prismatic layer. The most intense staining, however, was shown by the substance of the gelatinous layers of the wall and by the intine. The exine was found not to contain sugar units susceptible to periodate oxidation (Fig. 2). Alcian blue staining at low pH revealed that acidic carbohydrates in the wall are confined to the gelatinous layers which are strongly stained by this method; none of the other layers responded (Fig. 3). Fixation of the spores in glutaraldehyde and osmium solutions in the presence of purified ruthenium red augments the electron opacity of the gelatinous layers in Marsilea and Regnellidium (Figs. 37, 38). The effect of the treatment can be judged by comparing the images in Figs. 37 and 38 with those 66 J. M. Pettitt

12 Spore-wall enzymes in ferns 67 of spores fixed by the more orthodox sequential double-fixation procedure (Pettitt, 1971). Since the specificity of ruthenium red staining depends on the presence of polyanions (Luft, 1971; Martinez-Palomo, 1970), the gelatinous layers would be expected to give a result comparable to that obtained with the Alcian blue procedure. The experiments demonstrated that FITC-Con A is bound selectively to the gelatinous wall layers in both microspore and megaspore, a result which indicates that accessible saccharides containing residues for the lectin are present only in these layers (Figs. 4, 6). The specificity of the attachment was shown by the absence of fluorescence attributable to FITC in sections pre-incubated in 0-2 M a-methyl-D-mannoside and then treated with FITC-Con A containing 0-2 M a-methyl-D-mannoside (Figs. 5, 7). fi-lectins. In sections and whole mounts of unfixed spores stained with/?-GLU Yariv antigen, /?-lectins were revealed in the microspore perine of all species examined. The pattern of staining indicated that the highest concentration occurs in the region of the germinal site and the intensity of the reaction here and elsewhere in the perine

Figs. 2—14. Non-enzymic wall-associated components. Fig. 2. PAS, localization of reactive polysaccharides. The gelatinous wall layers and intine are intensely stained and there is moderate staining in the perine. Pilularia novae-hollandiae quiescent megaspore. X 180. Fig. 3. Alcian blue pH 2-5, localization of acidic carbohydrates. Only the gelatinous wall layers are stained by this method. Marsilea drummondiiquiescent microspore. X 480. Fig. 4. Con A binding in the wall of the quiescent megaspore. FITC-Con A is bound specifically to the gelatinous wall layers. The sporopollenin in the exine and perine is autofluorescent. Marsilea mutica. X 480. Fig. 5. Con A control preparation for the megaspore wall. The section was incubated in o-2 M a-methyl-D-mannoside. The weak fluorescence is attributable to sporopollenin autofluorescence. M. mutica. X 400. Fig. 6. Con A binding in the wall of the quiescent microspore. As in the megaspore, FITC-Con A is bound specifically to the gelatinous wall layers. The sporopollenin is autofluorescent. M. mutica. X 250. Fig. 7. Con A control preparation for the microspore wall. The section was incubated in 0-2 M a-methyl-D-mannoside. Only weak sporopollenin autofluorescence is detectable. M. mutica. X 250. Figs. 8,9. /?-lectin localization with /?-GLU. There is intense staining in the perine in the region of the germinal site. M. mutica quiescent microspore. Fig. 8, X 600; Fig. 9, whole mount, X 360. Fig. 10. /?-lectin localization with /?-GLU in the hydrated microspore showing the activity in the perine away from the germinal site. Marsilea berhautii. X 1000. Fig. 11. /?-lectin control preparation for the hydrated microspore. The section was stained with a-GAL. M. berhautii. X 620. Fig. 12. Coomassie, localization of proteins. Proteins are present throughout the perine and there is some staining in the intine and gelatinous wall layers. M. drummondii quiescent megaspore. X 400. Fig. 13. Sudan black B, localization of lipids. Lipids occur as droplets and large coalescent globules in the perine chambers of the mature, quiescent megaspore. M. drummondii. X 500. Fig. 14. Sudan black B staining of the wall of an immature megaspore. There is no sudanophilic material associated with the perine chambers at this stage of development. M. drummondii. X 400. 68 J. M. Pettitt

25 Spore-wall enzymes in ferns 69 could be enhanced by pre-treating the sections with hot ethanol to remove low molecular mass inhibitors (Figs. 8, 9). The specificity of the reaction was confirmed by staining specimens in a-GAL after incubation in hot ethanol (Fig. 11). Interestingly, /?-lectin activity could not be detected in the megaspore wall, either before or following treatment with hot ethanol, in any of the species investigated. Contrary to what has been found for other plant cells and tissues (Clarke et al. 1975, 1978), the /?-lectins associated with the microspore perine in the ferns are not water-labile. The activity is retained in the wall during hydration and can be detected after 2-5 h germination (Fig. 10). In so far as it is possible to assess relative intensity of the staining without recourse to photometry, none of the material in the wall precipi- tating the /?-glucosyl dye was extracted in water. Proteins. Proteins were found to be present throughout the megaspore perine and some staining was observed in the intine and in the gelatinous layers (Fig. 12). The

Figs. 15-27. Enzymic wall-associated components. Fig. 15. Acid phosphatase localization in the quiescent megaspore of M. berhautii. Naphthol AS-BI phosphoric acid-pararosanilin reaction. X 450. Fig. 16. M. berhautii quiescent megaspore. Acid phosphatase control+ o-oi M NaF as inhibitor. X 450. Fig. 17. Acid phosphatase localization in the quiescent megaspore of P. novae-hollandiae. The major activity is associated with the reticulate perine and there is some activity in the gelatinous layers. Naphthol AS-BI phosphoric acid-pararosanilin reaction. X 650. Fig. 18. P. novae-hollandiae quiescent megaspore. Acid phosphatase control+ o-oi M NaF as inhibitor. X 520. Fig. 19. Acid phosphatase localization in the hydrated microspore of M. berhautii. The wall-associated activity is in the petine and intine. Modified Gomori method. X670. Fig. 20. M. berhautii hydrated microspore. Acid phosphatase control. Modified Gomori medium without substrate. X 620. Fig. 2i. Esterase localization in the quiescent megaspote of Regnellidium diphyllum. Staining is confined to the distal region of the perine chambers and the gelatinous layers above, a-naphthol acetate—Fast Blue B Salt as coupling agent. X 400. Fig. 22. R. diphyllum quiescent megaspore. Esterase control without substrate. X 400. Fig. 23. Esterase localization in the quiescent microspore of M. drummondii. The principal activity is in the perine and activity in the intine is distinctly concentrated in the region of the germinal site, a-naphthyl acetate-Fast Blue B Salt as coupling agent. X 500. Fig. 24. M. drummondii quiescent microspore. Esterase control without substrate. X500. Figs. 25,26. Esterase localization in the quiescent microspore of Pilularia globulifera shown by the indigogenic method. The plane of focus was selected to show the activity associated with the intine at the germinal site. The activity in the perine is not clearly resolved, but discrete granules of reaction product can be discerned in the perine of the spore in Fig. 25. Both X 500. Fig. 27. P. globulifera quiescent microspore. Esterase control. Indigogenic reaction mixture without substrate. X 500. 70 J. M. Pettitt detection of protein in the gelatinous layers is perhaps not unexpected since it can be shown that cytoplasmic debris originating in the tapetal periplasmodium is incorporated into the layers as they form (Fig. 37). In the megaspore of R. diphyllum the most intense staining coincided with the occurrence of periplasmodial debris collected at the distal terminations of the perine prisms (Fig. 38). Lipids. Sudan black B staining showed an abundance of small lipid droplets and large coalescent globules in the microspore and megaspore perine of all the species (Fig. 13). The intine, exine and gelatinous layers were not sudanophilic. The character and distribution of the lipid in the wall can be explained on the assumption that it arises in the tapetal periplasmodium and accumulates in the perine during the final phase of spore maturation. Certainly, in Marsilea, the droplets and globules cannot be detected in the perine at any time prior to the dissolution of the periplasmodium (Fig. 14).

Wall-associated components: enzymic Acid phosphatase. Acid phosphatase was found associated with the microspore and megaspore perine in all species except R. diphyllum. The most intense activity in the megaspore was invariably found in the reticulate layer (Figs. 15-18). Enzyme activity was also detected in the intine and gelatinous layers in all the species, including R. diphyllum, although there were interspecific differences in the relative intensity of the reaction (Fig. 30; Table 2, p. 72). The accumulation of acid phosphatase reaction product in the megaspore wall is strikingly illustrated in electron micrographs. The electron micrograph of Marsilea in Fig. 39 shows that the electron-opaque reaction

Figs. 28-36. Enzymic wall-associated components. Fig. 28. Esterase localization in the hydrated megaspore of M. berhautii. a-naphthyl acetate—Fast Blue B Salt as coupling agent. X 400. Fig. 29. M. berhautii hydrated megaspore. Esterase control without substrate. X550. Fig. 30. Acid phosphatase localization in the gelatinous layers of the quiescent megaspore of M. drummondii. Naphthol AS-BI phosphoric acid-pararosanilin reaction. X 400. Fig. 31. RNase localization in the hydrated microspore of M. berhautii. The principal activity is in the perine and in the intine in the vicinity of the germinal site. X 1200. Fig. 32. RNase localization in the hydrated megaspore of M. berhautii. The section is through the apex of the spore in the region of the germinal suture and shows the activity associated with the perine and intine. X 400. Fig. 33. RNase activity associated with the gelatinous envelope in the hydrated megaspore of M. berhautii. The reaction is particularly intense in the innermost zone which contains cytoplasmic debris from the periplasmodium - see Fig. 37. X 600. Fig. 34. M. berhautii hydrated megaspore. RNase control without substrate. X 600. Fig. 35. Peroxidase localization in the quiescent megaspore of P. novae-hollandiae. The activity is confined to the perine layers. Aminoethyl carbazole method. X 650. Fig. 36. P. novae-hollandiae quiescent megaspore. Peroxidase control without substrate. X 650. Spore-wall enzymes in ferns 71

35 J. M. Pettitt

Table i. General distribution of the wall-associated components and their relative activities in the Marsileaceae combining the results from all the species investigated Microspore Megaspore Gela- Gela- tinous tinous Component Intine Exine Perine layers Intine Exine Perine layers Periodate-reactive polysaccharides + + + — ++ + + + + + + — ++ + + + Acid carbohydrates — — — + + + — — — -t- + + Con A acceptors — — — + + + — — — + + + /?-lectins — — + + +— — — — — Total protein + — ++ + + — + + + + Lipids — — + + + — — — + + + — Acid phosphatase ++ — + + + + ++ — + + + + Non-specific esterase + — + + + + + — + + + + RNase + + +- - + + + +- - + + Peroxidase — — + — — — + + + — —, no activity; +, slight; + +, moderate; + + +, intense.

Table 2. Taxonomic distribution of the wall-associated enzymes and their relative activities in the microspores and megaspores as determined in freeze-sectioned dry or briefly fixed specimens Acid Material phosphatase Esterase RNase Peroxidase I + + + * microspore • E P + + + Marsilea G + berhautii I + + mutica megaspore • E drummondii P + + + G + I + E microspore - Pilularia + + NT novae-hollandiae ,1 + I globulifera I + megaspore • E P + + G + I + + - microspore • E NT P + + + Regnellidium ,G + — diphyllum I + + E NT megaspore - ++*• I + + E, exine; I, intine; P, perine; G, gelatinous layers; NT, not tested; *, intense activity in the region of the germinal site; **, staining occurs only in the distal region of the perine chambers. Spore-wall enzymes in ferns 73 product is deposited in the interstices between the sporopollenin elements of the reticulate layer, while in the prismatic layer of the wall it occurs as a thin but con- tinuous precipitate within the radial chambers. The sharpness of the localization in the perine contrasts with the diffuse distribution of the lead phosphate end-product in the intine, and although the body of the exine is unstained, there is end-product occluding some of the exine channels and extending into the perine above (Fig. 40). This finding introduces the possibility that the enzymes can diffuse across the exine. The point is discussed more fully below. No change in the distribution of acid phosphatase in the wall could be detected after 2-5 h hydration, with essentially all of the enzymic activity associated with the perine and intine (Figs. 19, 20). Non-specific esterase. The distribution of non-specific esterase closely paralleled the acid phosphatase pattern. Again, the principal activity in the megaspore was associated with the reticulate layer of the perine, the prismatic layer exhibiting moderate to heavy staining. The situation in the R. diphyllum megaspore is unusual as staining here was confined to the distal region of the radial chambers and to the suprajacent gelatinous layers (Figs. 21,22). The microspore of this species, however, is not significantly different from that of the other species and has substantial esterase activity generally distributed in the perine (Figs. 23, 24). Both the a-naphthyl acetate and indigogenic method showed faint to moderate staining throughout the body of the intine, but in the microspores of Marsilea and Pilularia activity was distinctly concentrated in this layer in the vicinity of the germinal suture (Figs. 23-27). The conditions in the hydrated spores corresponded to those demonstrated for the quiescent state. Non-specific esterase remained a major component of the enzymic protein load in the wall and the activity was not redistributed (Figs. 28, 29). Ribonuclease. RNase was found to be confined to the intine and gelatinous wall layers in dry and fixed spores, the sporopollenin wall subdivisions being completely devoid of activity. Enzyme reaction product was accumulated at the interface between the prismatic layer and the gelatinous layers in the megaspore but did not penetrate into the subjacent perine chambers. Differences in respect of distribution of the enzyme were apparent following hydration. After 2-5 h imbibition the reaction end-product was detected in the microspore perine and, except at the germinal suture, staining associated with the intine was less intense (Fig. 31). Similarly, staining was noted in the perine around the germinal site in the hydrated megaspore and although the possibility of some leaching cannot be discounted, no correlative reduction in staining in the intine or gelatinous layers was discernible (Figs. 32-34). Peroxidase. Only the perine was specifically and intensely stained in the megaspore (Figs. 35, 36). Staining in the microspore perine was faint and the reaction deposit appeared to follow closely the outline of the perine plications. The distribution of peroxidase activity in the wall after hydration was not determined. The data from the cytochemical tests are summarized in Tables 1 and 2. 74. J. M. Pettitt Spore-wall enzymes in ferns 75

Wall-enzymes: developmental factors Fine-structural studies show that the sequence of events in the differentiation of the perine and gelatinous wall layers is closely correlated with changes which are occurr- ing in the periplasmodium. In the microsporangium, the perine is modelled directly against the inner face of the periplasmodium which invests the spores and the layer is therefore a sporopollenin replica of the surface configuration (Pettitt, 1979). Perine development in the megasporangium takes a different, more intricate course and presents greater difficulty in the matter of explanation. It is clear that at the time the reticulate layer is forming, the tapetal periplasmodium in the megasporangium is vesiculated (Fig. 41) and the condition increases in extent as sporogenesis proceeds until, near the conclusion, the periplasmodium appears distinctly fenestrated in light- microscope preparations (Feller, 1953; Boterberg, 1956). During the early period, the inner face of the periplasmodium, the face adjacent to the megaspore, contains deep embayments, and appearances suggest that this is due to the fusion of vesicles with the limiting membrane (Fig. 42). The contents of the vesicles - small vesicles, dense droplets, microfibrillae and wefts of membrane - are released to congregate at the surface of the megaspore (Fig. 43). It is from or amongst this aggregation of morphologically heterogeneous material that the reticulate layer of the megaspore perine begins to develop (Pettitt, 1971). Two mechanisms can be envisaged whereby the enzymes could contrive to occupy

Fig- 37- The gelatinous wall layers in the quiescent megaspore of M. drummondii. The specimen was fixed in glutaraldehyde and postfixed in osmium solutions containing ruthenium red. The cationic dye imparts increased electron-contrast to the gelatinous layers. Notice that cytoplasmic debris, originating in the tapetal periplasmodium, has been incorporated into the structure. X 3500. Fig. 38. Termination of a radial perine chamber in the megaspore wall of R. diphyllum showing the periplasmodial debris in relation to the suprajacent gelatinous layers. The specimen was fixed in glutaraldehyde and postfixed in osmium solutions containing ruthenium red. X 7000. Fig. 39. Acid phosphatase localization in the quiescent megaspore of M. drummondii. Reaction product occurs in the interstices of the reticulate perine layer and within the radial chambers of the prismatic layer. The exine is not reactive. Modified Gomori method. X 9000. Fig. 40. Acid phosphatase localization in the exine channels of the M. drummondii megaspore. Lead phosphate end-product occludes the channels and extends into the perine above. The intine, located beneath the exine, is diffusely stained. Modified Gomori method. X 15000. Fig. 41. The tapetal periplasmodium surrounding the developing megaspore of M. drummondii is vesiculated and the inner face, adjacent to the spore, contains deep embayments. X 4000. Fig. 42. Detail of the inner face of the tapetal periplasmodium showing the embayments. Material released from vesicles in the periplasmodium is beginning to collect on the surface of the megaspore. X 5000. Fig. 43. Morphologically heterogeneous material originating in the periplasmodium has aggregated at the surface of the developing megaspore. The elements of the reticulate perine layer are beginning to form. X 7000. 76 J. M. Pettitt the sites in the mature spore wall. The scheme which presents the least conceptual difficulty is that incorporation of the activity attends wall morphogenesis. It would follow from this that the enzymes found in the perine and gelatinous layers are of sporophytic origin and those in the intine of gametophytic origin. It is relevant in this connexion that intense acid phosphatase and non-specific esterase activity can be detected throughout the periplasmodium shortly after the formation of the meiotic tetrad in Marsilea, but there is not at this stage any marked concentration of the enzyme localized at the surface of the spore (author's unpublished observations). Whether quantitative concordance is a feature of redistribution, however, has not been measured. The second mechanism requires that enzyme synthesis occurs in either the gametophyte or the sporophyte and in this case the molecules are distributed to the various wall sites by means of the exine channels (Fig. 40). So far exine channels have been found to exist in only a limited number of spores and these do not include the microspores of the Marsileaceae (Pettitt, 1979). The main objection to the second mechanism, however, is not the mechanical one, but that it is difficult to reconcile the observed distribution of the wall enzymes with what would be expected on the basis of it. The evidence, therefore, as far as it goes, suggests a mode of origin for the enzymes involving both sporophyte and gametophyte. But it must be admitted that in the absence of developmentally linked cytochemical evidence, this postulate amounts, in fact, to little more than a convenient explanation of the observed pattern of distribution and association in the wall.

DISCUSSION These observations show that hydrolytic enzymes are located at specific sites in the spore wall of heterosporous ferns and that in many cases they are associated with other classes of molecules. It seems likely that a greater spectrum of activity could be demonstrated by extending the analysis, but a central feature established by the present observations is the extracellular, lysosomal nature of the enzyme fraction. The difficulty which attends a classification such as this is that the data obtained do not provide any useful information concerning the functional significance that can be attached to the occurrence. Data have been assembled which show that the ability to synthesize the enzymes during sporogenesis and to store them in the wall, and the capacity to retain the activity during germination, is a characteristic of both male and female sides in all the species investigated. From this evidence various roles can be considered for the wall-associated components in the natural life of the spore, and while in the absence of experimental examination no one explanation can be taken as definitive, they are discussed separately with respect to four processes - wall differentiation, gametophyte nutrition and defence and reproduction.

Possible roles for the wall enzymes and associated molecules Wall differentiation. The morphology of wall development in the fern spore has been studied by light and electron microscopy and the descriptions from the 2 tech- Spore-wall enzymes in ferns 77 niques agree in general. Light- and electron-microscope cytochemical methods have determined that the sporopollenin element of the wall is constructed within a fibrillar, polysaccharide-protein matrix which completely envelops the post-meiotic cell. To account for the orderly evolution of form in wall morphogenesis a mechanism has been proposed which is probably effective for the number of investigated species although its general applicability to the ferns as a whole is by no means established. It is maintained that under circumstances of normal development in this system—the stip- ulation is important—the spore coat matrix is instrumental in the determination of wall form in so far as it serves as a template for the addition of the sporopollenin material (Lugardon, 1971; Waterkeyn & Bienfait, 1971; Bienfait & Waterkeyn, 1976; Pettitt, 1971, 1979). This is inferred from the fact that once the sequence of deposition has begun, successive layers are added rapidly to the surface of the cell as the spore increases in volume, and by the termination of the process the spore is covered by a wall which is structurally and sculpturally distinctive for a particular taxonomic rank. It is probable that no essentially new mechanisms are required to account for the move- ment and disposition of the wall material after the formation of the matrix, in spite of the logistics required to fulfil the requirements of differentiation. The circumstances suggest that structural and compositional differences in the matrix zone create conditions that have structural consequences for the later-developing layer. Accordingly, differences in different sectors of the matrix zone would bring about changes in the structure or sculptural pattern of the spore, such as are observed (Pettitt, 1979). The major problem with this model at the molecular level is the need for sufficient structural or qualitative diversity in the matrix component to accommodate the range of pattern found in the different structural units of the wall. But a more convincing argument could be developed in support of the feasibility of the mechanism if it could be shown that the depositional process goes hand in hand with temporally or spatially related lytic remodelling of the matrix. However, it is recognized that speculation upon the effects of enzymic activity in the determination of wall pattern in a model does not provide evidence of validity. Gametophyte nutrition. Germination involves a change in the spore from a dormant, metabolically quiescent state to one of vigorous developmental activity towards gametogenesis. The studies of Gantt & Arnott (1965) and Raghavan (1976, 1977) have shown that very early in germination the stored protein reserves of the spore are hydro- lysed, presumably by specific enzyme proteins. The ultrastructural aspects of the process have been illustrated and discussed by Gullvag (1968), whose data correlate nicely with the biochemical findings. It would appear that extra protein production is involved. Experiments designed to inhibit proteolysis with cycloheximide in photo- induced spores clearly indicate that new hydrolytic enzymes are synthesized from endogenous precursors (Raghaven, 1977). Indeed, there is evidence to suggest that in at least one species, the first of these proteins to be synthesized on germination are actually coded by mRNA formed during sporogenesis and persisting in the quiescent spore (Raghavan, 1976). The data, therefore, argue in favour of de novo synthesis of hydrolases rather than mobilization of endogenous sources. Fertilization in the Marsileaceae occurs within a short time of imbibition, the dura- 78 J. M. Pettitt tion required to complete gametogenesis being the determining factor, and this provides for temporal coincidence of maturity between the sexes (Bierhorst, 1971; Lloyd & Klekowski, 1970). During the early term there is a rapid rise in cytochemically detectable hydrolase activity (a major component of which is acid phosphatase) in the cytoplasm, the peak concentration being reached after about 2-4 h, depending on the species (author's unpublished observations). As the present observations show, however, the increase in cytoplasmic activity does not affect the activity incorporated in the wall since the level here remains relatively constant over the period. Thus, the observations from the Marsileaceae appear to be consistent in general terms with the findings of Raghavan (1976, 1977) in the homosporous ferns. The studies of spore development in the ferns prompt a question of some importance. If a supply of metabolites is necessary to the growing gametophyte, how is the supply maintained and monitored as development proceeds? Clearly, in all but the initial stages of development before it is formed, the spore wall must mediate in the transfer of any incoming substance to the gametophyte. An in vitro incubation procedure with Lycopodium isospores and with Marsilea megaspores has demonstrated that electron-dense tracer particles can be transmitted across the developing spore wall (Pettitt, 19766, 1979). Although the response may well have been exaggerated under the conditions of the experiment, the significant aspect of the phenomenon is that the exine was shown to be traversed by a system of interconnecting channels in open communication with the sporophyte on one side and the gametophyte on the other. If in fact the enzymes are synthesized in the periplasmodium and gametophyte and latent activity is incorporated into the wall independently of differentiation, it becomes necessary to ask whether the particular qualities of the products could be utilized to provide metabolites to the gametophyte while it is contained within the parent spor- rangium. If this was the case then the exine channels might carry particular significance; they could be the means by which the products from polysaccharide and protein breakdown are delivered for the general metabolic pool. Gametophyte defence. The factors involved in arresting fungal, bacterial and viral infections of germinating spores with endosporic gametophytes have never been determined. But clearly, they must be of decisive importance for the prosperity of the gametophyte. In the present context, it could be surmised that if special provision is made for protection, then a strategy based on the wall would have obvious advantages. Since the accumulated activity endows the wall with particular properties, the particular significance of the distribution and unusual stability of the components can be explained if they are induced to behave in defence of the developing or germinating gametophyte by degrading invading pathogens. For instance, the carbohydrate-rich gelatinous envelope would seem especially vulnerable to bacterial attack in the wet environment required for spore germination and fertilization, and within the general antimicrobial system of the wall an important inhibitory role could be assigned to peroxidase (Klebanoff, 1967). Further, this explanation incorporates the intriguing phenomenon of concentrated RNase, esterase and /?-lectin activity in the region of the germinal suture; it is an incidence of expedient stategic siting. The suggestion is certainly consistent with the attributes of the molecules involved. From the pattern of Spore-wall enzymes in ferns 79 distribution of /?-lectins in plant tissues and cells and from the composition of the molecules (80% carbohydrate and 20% protein), Clarke et al. (1975) and Clarke & Knox (1978) conjecture that they could carry determinants containing disease resis- tance. Reproduction. As mentioned above, observational evidence can be adduced in support of the view that in the pollen of the siphonogams the wall-associated enzymic proteins facilitate reproduction. Indeed, in the flowering plants their active partici- pation seems essential for the process to take place (Heslop-Harrison, 1975). However, the difference which exists in the mechanics of gamete approach suggests that there is little possibility of a similar correlation being demonstrated in the ferns. Taken together, the cytochemical data (Table 1) suggest that the gelatinous envelope on the spore contains glycoproteins, and of particular interest to the role of the component in reproduction is the direct demonstration that it binds Con A. It has been shown that in the elevated state the gelatinous envelope on the megaspore of Marsilea acts as a sperm trap (Machlis & Rawitscher-Kunkel, 19766), but what has not been established is whether this process provides for interspecific discrimination. While such a method of selection seems feasible in principle, it would stand in marked contrast to the analogous situation in animal systems where selection is a responsibility relegated to the surface of the egg (Metz, 1967; Schmell, Earles, Breaux & Lennarz, 1977). Nevertheless, in view of the proven importance of Con A acceptor sites in flowering plant reproduction (Watson, Knox & Creaser, 1974; Y. Heslop-Harrison, 1976; Knox et al. 1976) and in algal reproduction (Weiss, 1974), it would be instruc- tive to examine the situation in the Marsileaceae by observing whether Con A treatment had consequences in the successful establishment of the zygote in intraspecific mating.

CONCLUSIONS The precise role of the spectrum of enzymes in the fern spore wall remains to be determined; the benefit, if any, of the association is not immediately obvious. Pre- sumably the fact that the activity of the complement is not lost during hydration is of significance and suggests that the role is likely to relate to germination, if not to reproduction. One obvious concern at this stage of the cycle is the matter of gametophyte protection, and a tangible selective advantage would be conferred on spores equipped with an effective defence mechanism. Thus, it may be no coincidence that lipid material is deposited into the wall interstices towards the final phase of spore maturation since this could be a provision to prevent diffusion of the labile molecules during the period in which they are expected to act in the interest of the haplophase cell. Whatever role is considered for the wall-associated molecules, certain qualitative individual and interspecific differences in the distribution of the activity revealed by the study need to be explained, for instance, the absence of/?-lectins in the megaspores of all the species.

6-2 80 J. M. Pettitt This work was aided by grants from the Research Fund of the University of London which are gratefully acknowledged. I wish to thank Dr M. A. Jermyn of the Division of Protein Chemistry, CSIRO, Melbourne, Australia, for the gift of Yariv antigen and Professor R. W. Home of the John Innes Institute, Norwich, for providing electron-microscope facilities.

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