H+ -pumping rhodopsin from the marine alga Acetabularia.

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.

Temporal morphology and cap formation in Acetabularia--I. Light-dark cycle perturbations.

Cap formation, a major developmental process in the alga Acetabularia, is influenced by a single perturbation of the entraining light-dark schedule and thus, presumably, of the circadian rhythms. This perturbation is brought about several weeks before cap formation, the most conspicuous expression of morphogenesis in Acetabularia. The effect is more pronounced on cap formation than on growth. It varies in importance with the circadian time at which the perturbation was brought about. The effect is dependent on the developmental state of the alga: transfer carried out during the logarithmic phase of growth produces a delay whose importance decreases with time. When carried out during the phase of slow terminal growth, the transfer induces a transitory acceleration of cap formation. When the algae approach their final length, no effect is elicited. Photoperiodism seems to be involved.

Temporal morphology and cap formation in Acetabularia--II. Effects of morphactin and auxin.

In order to support the hypothesis that circadian rhythms are implicated in cap formation, experiments were undertaken on the possible time-dependency of the effects of (a) a competitive inhibitor of auxins, morphactin and (b) of auxin (IAA). It was found that: (i) the inhibitory effect of morphactin varies dramatically with the time at which the several weeks' treatment was first begun; (ii) the maximum inhibition varies with development and decreases with time; (iii) IAA accelerates cap formation when the algae are submitted to IAA during the exponential growth phase; the effect is time dependent and decreases with time; (iv) IAA first applied on smaller algae has a transient inhibitory effect which is time dependent; (v) anucleate fragments also respond differentially to an IAA treatment begun at several times in the 24-hr cycle, most clearly when newly formed mRNA have been accumulated and (vi) the effect of iAA is not cumulative with that of a LD shift; that of morphactin is not, or only slightly, improved by a LD shift.