Developmental mechanisms in heterospory: cytochemical demonstration of spore-wall enzymes associated with beta-lectins, polysaccharides and lipids in water ferns.

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 inthe 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, beta-lectins. Following the enzyme pattern, the beta-lectins are found to be concentrated in the region of the germinal site. beta-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 beta-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 conceivably be connected with one or more of four processes; wall differentiation, gametophyte nutrition, gametophyte protection or reproduction. Each of these possibilities is discussed.