ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 235, No. 1, November 15, pp. 26-33, 1984 Transcriptional Activity of Isolated Maize Chloroplasts ARIE ALTMAN,’ BENNETT N. COHEN, HERBERT WEISSBACH, AND NATHAN BROT2 Roehe Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey, 07110 Received May 3, 1984, and in revised form July 25, 1984 Chloroplasts and etioplasts, isolated from light- or dark-grown Zea mays plants, respectively, can incorporate labeled UTP into RNA in a reaction stimulated by light or ATP. This in organello RNA synthesis proceeded at a linear rate for up to 2 h. When expressed per unit protein, plastids from dark-grown plants incorporated more UTP than those from light-grown plants, and the highest rate of UTP incorporation was found in plastids from light-stimulated leaves (grown previously in the dark). The in organello newly synthesized RNA was heterodispersed, with most transcripts smaller than 14 S. Specific transcripts were detected in organelles from both dark- and light-grown plants that contain sequences that are homologous to the mRNAs for the rbcL gene (coding for the large subunit of ribulose bisphosphate carboxylase (LS-RuBPCase)) and for the psbA gene (32-kDa thylakoid membrane protein). Quali- tatively, the newly synthesized in wrganello transcripts were similar from the dark _ - - and light organelles. Q 1984 Academic PRSS. IN. It is now well established that the chlo- roplast has all the components required for gene expression, and that a large number of genes coded on cpDNA are expressed both in vivo and in vitro. Al- though in organello chloroplast protein synthesis using chloroplasts from higher plants is well documented (l-5), there have been only a limited number of studies describing in organello transcription and how light effects this reaction. In experi- ments using lysed chloroplasts (6, 7), it was found that nucleoside triphosphates could be incorporated into RNA. In these studies, the synthesized transcripts were heterogeneous in size, and no discrete products were identified. Hartley and Ellis (8), however, using intact spinach chlo- roplasts, showed that labeled uridine could be incorporated into specific RNA products and, furthermore, that incorporation only ’ Visiting Scientist from the Department of Hor- ticulture, The Hebrew University of Jerusalem, Re- hovot. Jerusalem. - occurred when the incubation was carried out in the light and did not take place when lysed chloroplasts were used. These authors concluded that the primary effect of light in their system was to catalyze the photophosphorylation of uridine to UTP, and that this reaction only takes place in the intact chloroplast. Since no incorporation of uridine into RNA oc- curred in the dark, this system could not be used to study the effect of light on the regulation of RNA synthesis in the iso- lated chloroplast. The present study was undertaken to develop an active transcriptional system using labeled nucleoside triphosphates in isolated maize chloroplasts3 to investigate the effect of light on in organello tran- scription. We show that plastids from ’ To whom correspondence should be addressed. 3 In this study we have used the term “intact chloroplasts” to describe chloroplast preparations that are morphologically intact, as seen by phase- contrast microscopy, and also show a light stimula- tion of RNA synthesis. 0003-9861/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 26