Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts

Abstract

The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated [32P]-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast. However, both DNA uptake and binding are severely inhibited by ATP. Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts. beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of [35S]methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.