An efficient and prolonged in vitro translational system from isolated cucumber etioplasts

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursors protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immunoprecipitation with anti-spinach RuBisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.