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Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid reduction of 70-80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to be mainly transcriptional, and not restricted to tufA, as transcription of other chloroplast-encoded genes were cycloheximide-sensitive, although not all equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blocking cytoplasmic translation was obtained with the cycloheximide-resistant mutant act1, and by using another translation inhibitor, anisomycin. In synchronously growing Chlamydomonas, chloroplast transcription is regulated by the circadian clock, with the daily peak occurring during the early light period. When cycloheximide was added during this period, transcription was inhibited, but not when it was added during the trough period (late light to early dark). Moreover, in synchronized cells switched to continuous light, the drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different types of chloroplast transcription in Chlamydomonas, basal (cycloheximide-insensitive) and clock-induced (cycloheximide-sensitive), and indicate that the relative contribution of each type to the overall transcription of a given gene are not identical for all genes. The results also provide evidence for nuclear regulation of chloroplast transcription, thereby obviating the need for an organellar clock, at least for these rhythms.

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.