Gary G. Coté

Amplitude Model for the Effects of Mutations and Temperature on Period and Phase Resetting of the Neurospora Circadian Oscillator

This paper analyzes published and unpublished data on phase resetting of the circadian oscillator in the fungus Neurospora crassa and demonstrates a correlation between period and resetting behavior in several mutants with altered periods: As the period increases, the apparent sensitivity to resetting by light and by cycloheximide decreases. Sensitivity to resetting by temperature pulses may also decrease. We suggest that these mutations affect the amplitude of the oscillator and that a change in amplitude is responsible for the observed changes in both period and resetting by several stimuli. As a secondary hypothesis, we propose that temperature compensation of period in Neurospora can be explained by changes in amplitude: As temperature increases, the compensation mechanism may increase the amplitude of the oscillator to maintain a constant period. A number of testable predictions arising from these two hypotheses are discussed. To demonstrate these hypotheses, a mathematical model of a time-delay oscillator is presented in which both period and amplitude can be increased by a change in a single parameter. The model exhibits the predicted resetting behavior: With a standard perturbation, a smaller amplitude produces type 0 resetting and a larger amplitude produces type 1 resetting. Correlations between period, amplitude, and resetting can also be demonstrated in other types of oscillators. Examples of correlated changes in period and resetting behavior in Drosophila and hamsters raise the possibility that amplitude changes are a general phenomenon in circadian oscillators.

Potassium Channels in Samanea saman Protoplasts Controlled by Phytochrome and the Biological Clock.

Leaflet movement in legumes depends on rhythmic, light-regulated ion fluxes in opposing regions of the leaf-moving organ. In flexor and extensor protoplasts from Samanea saman Merrill, opening and closing of K+ channels were rhythmic in constant darkness. When channels were open in flexor protoplasts they were closed in extensor protoplasts, and vice versa. The rhythms were shifted by a delay in the onset of constant darkness, a response typical of endogenous circadian rhythms. During the light period, the channels in flexor protoplasts were sensitive to red light that was followed by premature darkness; phytochrome was implicated as the photoreceptor.

Inositol 1,4,5-trisphosphate may mediate closure of K+ channels by light and darkness in Samanea saman motor cells.

Leaflet movements of Samanea saman (Jacq.) Merr. depend in part upon circadian-rhythmic, light-regulated K+ fluxes across the plasma membranes of extensor and flexor cells in opposing regions of the leaf-moving organ, the pulvinus. We previously showed that blue light appears to close open K+ channels in flexor protoplasts during the dark period (subjective night) (Kim et al., 1992, Plant Physiol 99: 1532–1539). In contrast, transfer to darkness apparently closes open K+ channels in extensor protoplasts during the light period (subjective day) (Kim et al., 1993, Science 260: 960–962). We now report that both these channel-closing stimuli increase inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in the appropriate protoplasts. If extensor cells are given a pulse of red light followed by transfer to darkness, channels still apparently close (Kim et al. 1993) but changes in Ins(1,4,5)P3 levels are complex with an initial decrease under red light followed by accumulation. Neomycin, an inhibitor of polyphosphoinositide hydrolysis, inhibits both blue-light-induced Ins(1,4,5)P3 production and K+-channel closure in flexor protoplasts and both dark-induced Ins(1,4,5)P3 production and K+ channel closure in extensor protoplasts. The G-protein activator, mastoparan, mimics blue light and darkness in that it both increases Ins(1,4,5)P3 levels and closes K+ channels in the appropriate cell type at the appropriate time. These results indicate that phospholipase C-catalyzed hydrolysis of phosphoinositides, possibly activated by a G protein, is an early step in the signal-transduction pathway by which blue light and darkness close K+ channels in S. saman pulvinar cells.

Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman

An improved protocol for the separation of inositol phosphates by high performance liquid chromatography was used to resolve inositol phosphates from pulvini (motor organs) of the legume, Samanea saman. The pulvini contained inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers which co-migrated with those of mammalian red blood cells, and one or more other inositol metabolites which, to our knowledge, have not been previously noted in preparations of inositol phosphates. The finding of inositol phosphates in Samanea which comigrate with mammalian inositol phosphates supports the possibility that the phosphatidylinositol cycle may function in signal transduction in plants as well as in animals.

Phosphoinositide Turnover and Its Role in Plant Signal Transduction

Plants often seem to be passive, background scenery of our world. Because they do not flee, attack, or vocalize, we often think of them as unresponsive. In reality, plants actively monitor their environment and respond to it. We often fail to recognize these responses because they are usually subtle: slow movements or developmental or biochemical changes. When a plant does respond rapidly and dramatically—a Venus’s flytrap snares its prey, or a disturbed Mimosa pudica folds its leaves—it is a powerful reminder that plants are not scenery but active players.

Membrane Lipids and Circadian Rhythms in Neurospora crassa

The possibility that membranes are involved in circadian rhythmicity in the ascomycete fungus Neurospora crassa is discussed. Neurospora is an ideal organism for testing this possibility; knowledge of its biochemistry and genetics is substantial, and mutants affecting circadian rhythms and/or lipid biochemistry are available.