Requirement for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplast-encoded genes in Chlamydomonas

Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid red uction of 70-80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to b e mainly transcriptional, and not restricted to tufA, as transcription of o ther chloroplast-encoded genes were cycloheximide-sensitive, although not a ll equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blockin g cytoplasmic translation was obtained with the cycloheximide-resistant mut ant act1, and by using another translation inhibitor, anisomycin. In synchr onously growing Chlamydomonas, chloroplast transcription is regulated by th e circadian clock, with the daily peak occurring during the early light per iod. When cycloheximide was added during this period, transcription was inh ibited, but not when it was added during the trough period (late light to e arly dark). Moreover, in synchronized cells switched to continuous light, t he drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different typ es of chloroplast transcription in Chlamydomonas, basal (cycloheximide-inse nsitive) 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 fo r nuclear regulation of chloroplast transcription, thereby obviating the ne ed for an organellar clock, at least for these rhythms.