In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus, has the conserved motifs (2.1- 4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper, both suggested to be involved in protein-protein interactions. PEST sequences were also found in approximately 40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA increased 2- 3-fold in the mid-to-late dark period of light-dark cycling cells, just prior to, or coincident with, the peak in chloroplast transcription. Also, the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.
In Chlamydomonas growing under 24 h light-dark cycles, chloroplast transcription is under circadian clock control, and peaks early in the morning. The peak (but not trough) requires ongoing cytoplasmic translation, as it is sensitive to cycloheximide (CH). The chloroplast transcriptional apparatus in Chlamydomonas is simpler than in land plants, with only one type of RNA polymerase (RNAP, bacterial) and apparently only one sigma factor (RPOD). Core RNAP can be assayed in vitro with a non-sigma factor dependent template, and is sensitive to rifampicin. We developed a membrane-based assay for RNAP activity, and used it to determine that core activity is only weakly affected by pre-treating cells with CH. Moreover, core chloroplast RNAP activity was steady during a 24 h light-dark cycle. Levels of the sigma factor (RPOD) were examined using western blots, and found to fluctuate less than 25 % during light-dark cycles. These data indicate that circadian regulation of chloroplast transcription is distinct from regulation by sulfur availability, which involves significant changes in RPOD levels. The implications of this data for hypotheses that purport to explain the circadian control mechanism are discussed.
In response to food scarcity and low ambient temperatures, mice enter bouts of torpor resulting in energy conservation. During a torpor bout, mice exhibit a substantial decrease in body temperature (Tb), heart rate (HR), and metabolic rate. The neurobiology of torpor is largely unknown. It has been shown that peripheral administration of ghrelin, a stomach‐derived hormone released during energy deprivation, to calorically restricted mice deepens and lengthens torpor bouts. It is thought that ghrelin induces its physiological effects by activating Agouti‐related protein (AgRP) containing neurons in the arcuate nucleus of the hypothalamus. Therefore, we hypothesized that direct stimulation of AgRP neurons would decrease the minimum Tb of torpid mice and increase the time spent in torpor. To test this hypothesis, mice were implanted with ECG/Tb telemeters and hypothalamic AgRP neurons were selectively targeted with the light‐sensitive channelrhodopsin‐2 transgene. Mice were calorically restricted daily (65% of normal caloric intake) until regular daily torpor bouts were achieved. On baseline days, when AgRP neurons were not activated, minimum Tb was 25.6 ± 0.8 °C and time in torpor was 233 ± 34 minutes. When AgRP neurons were stimulated for one hour during entry into torpor (20 Hz for 1 second every 4 seconds for 60 minutes), minimum Tb was significantly lower (22.8 ± 0.3 °C) and torpor bouts were significantly longer (435 ± 29 min). These data support the hypothesis that AgRP neurons directly regulate torpor physiology and begin to elucidate the neural circuits responsible for torpor regulation.Support or Funding InformationSupported by 1R15HL120072‐01A1 to SJS and by 1R15DK105510 to MC
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