SPATIAL STRUCTURE OF MITOCHONDRIA AND ER DENOTES CHANGES IN CELL PHYSIOLOGY OF CULTURED TOBACCO PROTOPLASTS

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC(6)(3) (3,3'-dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell's physiolog y was investigated using a hormone-based model system for elongation a nd division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts sm all spherical mitochondria are clustered around the nucleus and the ch loroplasts. During the first 4 days of culture they are transformed in to long vermiform organelles which distribute evenly throughout the cy toplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increa se in quantity. In a medium with auxins and cytokinins cells start div iding. Their mitochondria typically become numerous and very small, an d are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER i s always present as a network close to the cell membrane. In freshly i solated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced comp lexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots o f plate-like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure f ound in epidermal cells of the intact plant.