A Possible Second Role for Calmodulin in Biological Clock-Controlled Processes of Euglena

Abstract

ABSTRACIThe response of the Euglena gradiis (Klebs strain Z) photosynthesis circadian rhythm to three calmodulin antagonists was examined. In the presence of an antagonist, the photosynthetic reactions were uncoupled from the biological clock. Instead of the highly predictable rhythmic pattern characteristic of a biological clock-controlled circadian rhythm, the photosynthetic rate appears to be influenced by the light/dark cycle. The rate of 02 evolution increases throughout the light portion of the cycle and does not decrease until the cells are exposed to darkness. Shortterm exposure to a calmodulin antagonist (2 hour pulses) failed to cause phase shifts in the timing of the rhythm. This suggests that calmodulin is not part of the clock controlling photosynthesis and that it has a clockrelated role different from that reported for the cell division rhythm in Euglena.Numerous physiological and biochemical processes in eucaryotic cells display predictable daily changes in rate that are accomplished by coupling the process to the biological clock. The biochemical basis for the biological clock is unknown and it is not certain whether cells have one master clock controlling all circadian rhythms, or whether multiple clocks control specific subsets ofrhythmic processes. The circadian rhythms ofEuglena have been extensively studied (3) and recently a feedback loop involving Ca2" and calmodulin was proposed in an attempt to define biochemically components of the clock controlling the Euglena cell division rhythm (7). The rationale for proposing calmodulin involvement was based on the observation that exposure to a calmodulin antagonist results in phase shifting (timing advances or delays) of the monitored cell division rhythm (7). If the proposed feedback loop represents a master clock in Euglena, then all rhythms using the master clock should be phase shifted by the application of calmodulin inhibitors. This investigation reports that the photosynthetic rhythm of 02 evolution is not phase shifted by three commonly used calmodulin antagonists. Instead, it appears that the effect of inhibiting calmodulin is to uncouple the photosynthetic reactions from the clock that controls them, thereby changing the expression of this rhythm.These observations suggest that calmodulin may serve a second role in clock regulated processes in Euglena by participating in the steps used to link photosynthesis to the clock.

Plastid and nuclear mRNA fluctuations in tomato leaves ? diurnal and circadian rhythms during extended dark and light periods

“…At present no factor has been isolated which is evidentially involved in the regulation of oscillating patterns. A possible role in controlling the process of the "biological clock" in Euglena was proposed for calmodulin [10]. Further experiments and detailed analyses may bring some more insight into the molecular mechanisms of the "biological clock" in plants.…”

Steps Linking the Photosynthetic Light Reactions to the Biological Clock Require Calcium

“…These results were interpreted to mean that in this population of cells, the biological clock no longer regulated the rate of oxygen evolution in the second half of the light cycle. The uncoupling of the oxygen evolution rhythm from the clock was mimicked in control cells by exposure to calmodulin antagonists (12). This observation suggested that calmodulin participated in the steps connecting the photosynthetic reactions to the biological clock and promoted a further examination of the role of calcium in the transducing steps.…”

“…a continuous increase in the rate of 02 evolution throughout the light portion of the light/dark cycle (12). When cells were followed for four consecutive days, the normal rhythm in 02 evolution was not observed ( 12), which argues against a phase delay or a transient inhibition in the expression ofthe rhythm. In addition, a naturally occurring population of Euglena gracilis was isolated in which the photosynthetic reactions were not controlled by or coupled to the biological clock but were, instead, influenced only by environmental parameters ( 13).…”

“…The previously reported observation that Ca2`and calmodulin were involved in coupling the photosynthetic light reactions to the biological clock (12) suggested that the inositol trisphosphate signaling system might also be involved in these steps. IP3 is hydrolyzed from the plasma membrane lipid phosphatidylinositol (2,3).…”

“…2A). TMB-8, by altering the level of intracellular Ca2+, may prevent the activation of calmodulin and thus uncouple the photosynthetic reactions from the clock (12 …”