A PROCEDURE FOR ISOLATION OF PROPLASTIDS FROM ETIOLATED MAIZE LEAVES

Abstract

Proplastids in etiolated leaves develop into mature chloroplasts when the leaves are exposed to light. The structural changes which occur during this process have been described by von Wettstein and coworkers (6, 16, 17). Their electron micrographs show that the organelles undergo extensive development when they are exposed to light. Further study of these organelles should provide information concerning both the metabolic processes and the regulatory mechanisms governing chloroplast development. Biochemical studies on the development of proplastids have not been extensive. There are a few reports on protein synthesis in these organelles (5, 13), and some work has been done on changes in nucleic acid metabolism during development (4, 18). There is also some information on the development of photosynthetic enzymes in the organelles in the light (1). These studies have all been done with crude preparations, or with preparations stabilized with formaldehyde, because the organelles are unstable and are isolated with difficulty. Such preparations are of limited usefulness for further work, and it has been necessary to develop a better procedure for obtaining the organelles. This report presents a procedure which has proved successful in the isolation of proplastids from etiolated maize leaves. The procedure was developed by the modification of two published methods (12, 2); however, both the initial homogenization medium and the protocol for differential centrifugation have been changed. The modified procedure, as described below, has made it possible to obtain much larger yields of intact organelles, relatively free of nonplastid contaminants. The preparations have been characterized morphologically with both the light and the electron microscopes; the contaminants have been examined in some detail; and the yield of organelles has been measured at various stages during the isolation procedure. Although the preparations still suffer from a number of limitations, the method represents a substantial improvement over other methods and should be of use to other investigators.