Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis

The transcription of CAB genes, encoding the chlorophyll a/b-binding proteins, is rapidly induced in dark-grown Arabidopsis seedlings following a light pulse. The transient induction is followed by several cycles of a circadian rhythm. Seedlings transferred to continuous light are known to exhibit a robust circadian rhythm of CAB expression. The precise waveform of CAB expression in light–dark cycles, however, reflects a regulatory network that integrates information from photoreceptors, from the circadian clock and possibly from a developmental program. We have used the luciferase reporter system to investigate CAB expression with high time resolution. We demonstrate that CAB expression in light-grown plants exhibits a transient induction following light onset, similar to the response in dark-grown seedlings. The circadian rhythm modulates the magnitude and the kinetics of the response to light, such that the CAB promoter is not light responsive during the subjective night. A signaling pathway from the circadian oscillator must therefore antagonize the phototransduction pathways controlling the CAB promoter. We have further demonstrated that the phase of maximal CAB expression is delayed in light–dark cycles with long photoperiods, due to the entrainment of the circadian oscillator. Under short photoperiods, this pattern of entrainment ensures that dawn coincides with a phase of high light responsiveness, whereas under long photoperiods, the light response at dawn is reduced.

Vagaries of the molecular clock

The hypothesis of the molecular evolutionary clock asserts that informational macromolecules (i.e., proteins and nucleic acids) evolve at rates that are constant through time and for different lineages. The clock hypothesis has been extremely powerful for determining evolutionary events of the remote past for which the fossil and other evidence is lacking or insufficient. I review the evolution of two genes, Gpdh and Sod. In fruit flies, the encoded glycerol-3-phosphate dehydrogenase (GPDH) protein evolves at a rate of 1.1 × 10−10 amino acid replacements per site per year when Drosophila species are compared that diverged within the last 55 million years (My), but a much faster rate of ≈4.5 × 10−10 replacements per site per year when comparisons are made between mammals (≈70 My) or Dipteran families (≈100 My), animal phyla (≈650 My), or multicellular kingdoms (≈1100 My). The rate of superoxide dismutase (SOD) evolution is very fast between Drosophila species (16.2 × 10−10 replacements per site per year) and remains the same between mammals (17.2) or Dipteran families (15.9), but it becomes much slower between animal phyla (5.3) and still slower between the three kingdoms (3.3). If we assume a molecular clock and use the Drosophila rate for estimating the divergence of remote organisms, GPDH yields estimates of 2,500 My for the divergence between the animal phyla (occurred ≈650 My) and 3,990 My for the divergence of the kingdoms (occurred ≈1,100 My). At the other extreme, SOD yields divergence times of 211 My and 224 My for the animal phyla and the kingdoms, respectively. It remains unsettled how often proteins evolve in such erratic fashion as GPDH and SOD.

Accumulation of a Clock-Regulated Transcript during Flower-Inductive Darkness in Pharbitis nil1

To clarify the molecular basis of the photoperiodic induction of flowering in the short-day plant Pharbitis nil cv Violet, we examined changes in the level of mRNA in cotyledons during the flower-inductive photoperiod using the technique of differential display by the polymerase chain reaction. A transcript that accumulated during the inductive dark period was identified and a cDNA corresponding to the transcript, designated PnC401 (P. nil C401), was isolated. RNA-blot hybridization verified that levels of PnC401 mRNA fluctuated with a circadian rhythm, with maxima between 12 and 16 h after the beginning of the dark period) and minima of approximately 0. This oscillation continued even during an extended dark period but was damped under continuous light. Accumulation of PnC401 mRNA was reduced by a brief exposure to red light at the 8th h of the dark period (night-break treatment) or by exposure to far-red light at the end of the light period (end-of-day far-red treatment). These results suggest that fluctuations in levels of PnC401 mRNA are regulated by phytochrome(s) and a circadian clock and that they are associated with photoperiodic events that include induction of flowering.