Successful in vitro Synthesis of Oxygen Evolving Photosynthetic Membranes-A Milestone in Photosynthesis Research

Abstract

Interesting observations that chloroplaste posses DNA and RNA as well as the machinery for the biosynthesis of lipids and proteins (Kirk, Tilney-Basset 1968) gave a new dimension to the understanding of chloroplast autonomy. Successful demonstration of the ability of the plastids to divide in vitro (Ridley, Leech 1970) and synthesize protochlorophyllide (Pchlide) (Rebeiz, Castelfranco 1971a) and chlorophyll (Chl) (Rebeiz, Castelfranco 1971b; Well-burn, Wellburn 1971) in the last decade confirmed the status of autonomy of the chloroplast. However, further investigations to evaluate the autonomy of this organelle, by studying the process of greening of isolated etioplasts in vitro showed only signs of disorganization and rapid loss of their activi­ties, leading to the conclusion that etioplasts do not grow after isolation (Leech 1980). Since chloroplast DNA contains insufficient information to code for the complete spectrum of the chloroplast proteins and the subunits of many chloroplast proteins are known to be synthesized in different cellular locations; it was expected that any successful culture system will be a highly complex one including purified macromolecules, cellular membranes and organelles in addition to the plastids (Leech 1980). Chl synthesis was also found to be greatly limited in vitro by a structural protein synthesized in the cytoplasm (Rebeiz et al. 1973 and it is now known that the enzymes involved in the conversion of S-aminolevulinic acid (ALA) to Pchlide are in­deed synthesized in the cytoplasm (Bradbeer, 1981). However, it has recently been demonstrated that pretreatment of etiolated tissues with hormones induces excess accumulation of prothylakoid proteins and upon isolation of the plastids and incubation with ALA the later is rapidly converted into membrane bound Pchlide (Daniell, Rebeiz 1982a). Etioplasts thus enriched in proteins of chloroplastic and cytoplasmic origin and incubated in a cofactor enriched medium were capable of synthesizing Pchlide from exogenous ALA at a rate 12 to 18 fold higher than that observed in excised cotyledons incubated with ALA (Daniell, Rebeiz 1983a) and Chl at a rate twice higher than the highest rates observed in greening tissues in vivo (Daniell, Rebeiz 1982b). Electron microscopic studies of these etioplasts showed partial mobilization of prolamellar bodies to form membranes after 2 h of illumination followed by complete mobilization of the prolamellar bodies into huge macrograna after 4 h of illumination (Daniell, Rebeiz 1983b). Functional characterization of the membranes synthesized in vitro showed the commencement of photosystem I (PS I) activity 15 min after the onset of illumination (Daniell et al. 1983a). Recently we observed that the membranes synthesized in vitro were capable of evolving oxygen upon illumination and also synthesized polypeptides associated with water-splitting function (Daniell et al. 1983b). We summarize here the progress made in this field in the last 15 years and discuss its possible impact on photosynthesis research in future.