The Evolution of Circadian Cycles
By Bruce Scofield
Circadian rhythms are biological cycles of approximately 24 hours that are a general feature of the physiological organization of organisms on Earth. The alternation of the light-dark cycle is the most important environmental signal for circadian systems.
The history of the study of circadian rhythms began in modern times with French astronomer Jean Jacques d’Ortous de Mairan. In 1729 he observed that the sleep movements of the sensitive plant persisted in continual darkness, a D:D cycle. In 1880 Darwin published his observations of this phenomena in his book The Power of Movement in Plants. German scientist Erwin Bünning studied this phenomena, In 1936 Bünning published his hypothesis in which he stated that the timing mechanisms of photoperiodism were the same for leaf movements, and these were endogenous. In 1960 a major conference on biological clocks was held at Cold Springs Harbour, NY where many influential papers were read and published. Some of the important participants in this conference were Bünning, Sweeney, Hastings, Brown, and Pittendrigh. In 1986 circadian rhythms were discovered in cyanobacteria (Mitsui, Grobbelaar, Huang, Sweeney, Kondo, Golden, Johnson, Ishiura, and others) and in the 1990’s the emergence of a molecular model for the circadian oscillator accelerated progress in the field.
Three primary phenomena are used to define circadian rhythms.
- The persistence of the rhythm in constant conditions.
- Temperature compensation within a permissive range.
- Entrainment through phase resetting by light/dark and temperature cues.
One of the first microorganisms to be studied for its circadian rhythms was Gonyaulax polyedra, a dinoflagellate single-celled algae. It has an obvious circadian rhythm (CR) of bioluminescence that persists in constant light (L:L). Gonyaulax has been studied intensively for about 50 years (Sweeney, Hastings, Krasnow, and others) and is now known to have circadian cycles of photosynthesis, and cell division, in addition to bioluminesence. All the rhythms of Gonyaulax are entrained by cycles of light-dark. These can be either the natural day/night sequence or single short light pulses which are capable of phase-shifting the rhythm.
In Gonyaulax the period of bioluminescence becomes longer at higher temperatures and is weakened at low temperatures, but within a wide temperature range (values?) the rhythm is very stable. The biochemistry of luminescence does not seem to explain the regularity of the cycle. There does not appear to be any dependence on interaction among organisms of the same species (social activity) in maintaining the cycle. Cell-division appears to be keyed by the ending of the night phase - it is set by darkness. Cell-division does not control the clock, the clock controls the cell cycle.
The circadian rhythm of photosynthesis seems to be keyed to light, a process that is maximum at midday (O2 release). Entrainment is the beginning of the light phase (dawn). Sweeney found that entrained single cells continued the circadian rhythm of photosynthesis - but it stopped after 14 hours. Cells entrained to L:L did not follow a circadian cycle. A population of cells maintained in continuous bright light loses its rhythm because every cell in the population does so - not because rhythmic cells drift out of phase with each other.
Attempts have been made to construct a mechanism for circadian rhythms in Gonyaulax. Experiments have been done using various inhibitors, which present problems due to toxicity, but they have shown that the cell clocks are very stable. Cell metabolism has been inhibited, but this also has shown no consistent connection to the clocks. Ionophores, which alter the permeability of the cell membranes to ions, do cause small phase shifts. The amount of phase shifting by ionophores is dependent on the timing of their introduction to the cell. Phase shifts are positive at the end of the day period, negative near the middle of the night period.
Two chemical models were suggested in regard to the cell membrane as a key to the clock. One involves plasma membrane, the other organelle (which organelles?) membrane. In both cases there is a proposed two-component feedback loop composed of a potassium gradient across a membrane and a transport of potassium across a membrane.
Molecular models have been proposed to explain the mechanism of circadian cycles. (A distinction needs to be made between the circadian system and the circadian oscillator.) Well known model systems are found in Drosophila, neurospora, mice, humans, Arabidopsis, and cyanobacteria. Such models posit the following:
- An input pathway for entrainment.
- An oscillator which establishes period length from light and temperature cues.
- An output pathway which informs physiological activities.
In the cell, a photo-receptor recognizes alterations of light and dark. This information is then fed to an oscillator which is capable of being reset by the photo-receptor. Clock-controlled genes are then instrumental in the output part of the process which establish the overt rhythms that regulate the cell. The general logic of the oscilator system is that of a transcription/translation negative feedback loop. As translation continues, the protein gene product accumulates in the cell and eventually acts to inhibit further transcriptions of its own gene.The circadian oscillator thus uses loops that close within cells and do not require cell to cell interactions.
From the 1960’s to the 1990’s it was thought that circadian rhythms did not exist in prokaryotes, only in eukaryotes. The assumption was that since circadian systems appear to control the cell-division cycle, then prokaryotes, which divide in periods less than one day, could not have circadian cycles. The reasoning behind this is expressed in the Circadian-Infradian Rule. “Circadian rhythms are express only in cells that are dividing once a day or more slowly.” More rapid cell division was thought to cause uncoupling. Circadian rhythms found in Synechococcus have made this rule obsolete.
The cyanobacterium Synechococcus exhibits several circadian cycles. The first observed in the mid 1980’s (author?) were daily oscillations in nitrogenase activity. Apparently, cycanobacteria separate nitrogen fixation from photosynthesis because the nitrogenase enzyme complex is highly sensitivity to oxygen and becomes inactive. This problem is solved in other cyanobacteria (filamentous) with specialized cells called heterocycts that separate these functions spatially.
Synechococcus RF-1 exibits many circadian rhythms including nitrogen fixation, nitrogenase mRNA abundance changes, amino-acid uptake, protein synthesis, light/dark entrainment, and the cell division cycle. In order to thoroughly test this organism for circadian rhythms a new strain was created. Synechococcus PCC 7942 was transformed with luciferase genes that express luminescence. The easily observed luminsescence rhythm is thus a report on gene expression. Screening for mutations was the next step. Chemical mutagenesis and the isolation of aberrant luminescence rhythms let to the establishment of over 100 mutant populations (as of 1996) with ranges from 16 hours to 60 hours. From these populations three genes were identified that produced the rhythms. The actual modeling of the cyanobacteria clock was based on work done with Synechocystis, a cyanobacteria related to Synechococcus which has a sequenced genome.
In Synechococcus several genes, called kai (cycle in Japanese) genes, are necessary in combination for the daily cycle to persist. These kai genes are not found in eukaryotes, but two of them are present in archaeal genome databases. Three key genes, kaiA, B, and C, form a cluster. The deletion of one or all of them causes arhymicity.
We now know that circadian rhythms exist in many, if not most, organisms and serve an important purpose - maintanence of homeostasis. They have been studied extensively in a number of microorganisms including algae (protoctista) and fungi. It has been found that a wide variety of organisms use the same molecules to control their circadian rhythms. This suggests that the mechanisms for biological clocks are ancestral and that the circadian oscilling system was probably developed early in life’s history. However, the cyanobacterial mechanism is similar in gneral principles to those in eukaryotic systems, but the proteins are completely different, suggesting an independent evolution. This observation has been used to support a three-branched tree of life: Archae, Eubacteria, and Eukaryotes.
- Studies of eukaryotic clocks have led to the identification of specific clock genes. It appears that mammals and insects share at least some clock parts. The known fungal clocks are as yet unique. Plant clocks are still under investigation.
- Clocks of eukaryotes are different from those of bacteria.
- Natural selection favors clocks that are in tune with natural cycles, or at least match the L:D cycle that is established in a laboratory.
- Clocks allow organisms to anticipate changes in their environment and permit the phasing of incompatible processes.
- Clocks are either highly conserved and evolutionarily ancient, or they have evolved multiple times.
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