Chloroplast maintenance and partial differentiation in vitro

Tissue homogenates, etioplasts, and developing chloroplasts were prepared from cucumber (Cumucis sativus L.) cotyledons in tris-sucrose. They were incubated aerobically in the dark or in the light at pH 7.7 in the presence or absence of a cofactor mixture containing coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate. These cofactors were previously shown to be essential for protochlorophyll and chlorophyll biosynthesis. Ultrastructural changes were monitored by electron microscopy. The following observations were made. (a) Crude homogenates contained agents which degraded etioplasts and developing chloroplasts. (b) Added cofactors were essential for the maintenance of the membrane structure; they were also implicated in the transformation of the prolamellar body in the absence and presence of light. (c) Light pretreatment of the cotyledons improved the maintenance of the developing chloroplast membranes during subsequent in vitro incubation. (d) In the presence of the cofactors, grana formation appeared to take place in the absence of nuclear-cytoplasmic control.