Studies on the ultrastructure and histochemistry of plant cuticles: Isolated cuticular membrane preparations of Agave americana L. and the effects of various extraction procedures

The cuticular membrane (CM)of Agave americana with the adhering cellin wall was isolated with ammonium oxalate-oxalic acid solution, air-dried and dry-embedded without fixation. After KMnO4 staining, electron translucent lamellae are visible in the cuticle proper and cuticular layer. The fine structure of the opaque lamellae in the cuticle proper is more complex than previously observed in situ. It is more clearly observed in CM isolated at 40 °C than in those isolated at 100 °C, or in air-dried tissue, subsequently remoistened, fixed and dehydrated in acetone.Although extraction of CM with hot organic solvents removes substantial quantities of wax (mainly long chain alcohols and fatty acids), not all of the electron-lucent lamellae disappear completely. Strong sulphuric acid dissolves the cellin walls adhering to the CM and strongly diminishes the iodine/potassium iodide-sulphuric acid-silver proteinate staining reactivity of the CM, probably due to the marked reduction in epoxide content of the cutin. The acid does not completely remove the carbohydrate reticulum included in the cuticular layer.In sodium methoxide solution the CM is decutinized from the cellin wall side where the carbohydrate fibrillae included in the interior cuticular layer become completely exposed. On the outside, the lamellate cuticle proper is also lost. Major cutin monomers solubilized are 9, 10-epoxy-18-hydroxyoctadecanoic and 9, 10, 18-trihy-droxyoctadecanoic acids. Partial decutinization of the CM with methanolic HC1 produces similar but less drastic effects than methoxide apparently because the outer surface is protected by an artificial layer of lipids originating from depolymerized cutin.