Microfibril synthesis at the surfaces of isolated tobacco mesophyll protoplasts, a freeze-etch study

Summary

Outer surfaces of the plasmalemma of freshly isolated protoplasts and of protoplasts at various stages of cell wall regeneration up to 6 days of culture were examined using deepetching. At the same stages the internal structure of the plasmalemma was observed using freeze-fracturing. Deep-etching proved to be a sensitive technique for the detection of the first-formed microfibrils of the regenerated wall which were first found after 24 hours of culture. These microfibrils have a close association with the plasmalemma and give the appearance of being synthesised within it. Microfibril density increases progressively after the initial “lag phase”. Previous findings would suggest that short microfibrils protruding from the plasmalemma or localised groups of short microfibrils were to be expected, but these were not found. The only feature of frozen fractured membranes which changed in association with microfibril biosynthesis was a marked alteration in the polarisation of membrane-associated particle segregation between the “A” (inner fracture face attached to cytoplasm) and “B” (outer fracture face attached to extracellular medium) fracture faces. In freshly isolated protoplasts the segregation is approximately equal between the two fracture faces, whereas after microfibrils arise it is roughly 4 ∶ 1 (“A” ∶ “B”). Although rows of membrane-associated particles which might be involved in microfibril biosynthesis were found on fractured plasmalemma surfaces there were not more of these after microfibril synthesis had begun. Furthermore, similar rows of particles were equally common on tonoplast membranes which are not involved in microfibril biosynthesis.