ULTRASTRUCTURE OF THE ENDOCYTOTIC PATHWAY IN GLUTARALDEHYDE-FIXED ANDHIGH-PRESSURE FROZEN FREEZE-SUBSTITUTED PROTOPLASTS OF WHITE SPRUCE (PICEA-GLAUCA)

An ultrastructural study of endocytosis has been made for the first ti me in protoplasts of a gymnosperm, white spruce (Picea glauca), fixed by high-pressure freezing and freeze substitution. Protoplasts derived from the WS1 line of suspension-cultured embryogenic white spruce wer e labelled with cationized ferritin, a nonspecific marker of the plasm a membrane. The timing of cationized ferritin uptake and its subcellul ar distribution were determined by fixing protoplasts at various inter vals after labelling. To address concerns about using chemical fixatio n to study the membrane-bound transport of cationized ferritin, protop lasts were fixed both by conventional glutaraldehyde fixation and by r apid freezing in a Balzers high-pressure freezing apparatus (followed by freeze substitution). Cationized ferritin appeared rapidly in coate d pits and coated vesicles after labelling. Later it was present in un coated vesicles, and in Golgi bodies, trans-Golgi membranes and partia lly coated reticula, then subsequently in multivesicular bodies, which may ultimately fuse with and deliver their contents to lytic vacuoles . The results show that the time course and pathway of cationized ferr itin uptake in the gymnosperm white spruce is very similar to the time course and pathway elucidated for cationized ferritin uptake in the a ngiosperm soybean. High-pressure freezing yielded much better preserva tion of intracellular membranes and organelles, although plasma membra nes appeared ruffled. Protoplasts fixed by both methods possessed nume rous smooth vesicles in the cortex and smooth invaginations of the pla sma membrane. These became labelled with cationized ferritin, but appa rently did not contribute directly to the internalization of cationize d ferritin, except via the formation of coated pits and vesicles from their surfaces.