Kinetic analysis of a signal-transduction pathway by time-resolved somatic complementation of mutants.

Sensory control of sporulation in Physarum polycephalum plasmodia is mediated by a branched signal-transduction pathway that integrates blue light, far-red light, heat shock and the starvation state. Mutants defective in the pathway were isolated and three phenotypes obtained: blue-blind, general-blind and light-independent sporulating. When plasmodia of the blue-blind mutant Blu1 were exposed to a pulse of blue light and subsequently fused to non-induced wild-type plasmodia, the resulting heterokaryons sporulated, indicating a functional blue- light photoreceptor in the mutant. When the general-blind mutant Nos1 was fused to a wild-type plasmodium which had been induced by light, sporulation of the heterokaryon was blocked. However, the dominant inhibition of sporulation by Nos1 was gradually lost with increasing time between induction by light and time of fusion, suggesting that Nos1 can be bypassed by the time-dependent formation of a downstream signal-transduction intermediate. Phenotype expression in constitutively sporulating (Cos) mutants depended on starvation. The Cos2 product was titrated by fusing mutant plasmodia of different sizes to wild-type plasmodia of constant size and analysing the sporulation probability of the resulting heterokaryon. The titration curve indicates that a small change in the amount of Cos2 product can cause sporulation. We conclude that somatic complementation analysis allows the time-resolved evaluation of the regulatory function of mutations in a signal-transduction pathway without prior cloning of the gene. This shortcut allows us to characterize many mutants quickly and to select those for molecular analysis that display a well-defined regulatory function.

Functional mapping of the branched signal transduction pathway that controls sporulation in Physarum polycephalum.

Sporulation of starving plasmodia of Physarum polycephalum was found to be induced by far-red light, blue light or heat shock, each of which is perceived by a different input receptor system. The branched signal transduction pathway was mapped and the time-dependent formation of some of its components analyzed.

A photoreceptor with characteristics of phytochrome triggers sporulation in the true slime mould Physarum polycephalum.

Phytochrome is a ubiquitous photoreceptor in plants that controls a variety of responses to light, including gene expression, differential cell growth and intracellular movement of organelles. All phytochromes analysed so far are reversibly interconverted by light between an inactive and an active conformation, each of which has a different and characteristic absorbance spectrum. Based on photophysiological measurements we provide evidence, that a photoreceptor with these unique properties of phytochrome triggers sporulation in the true slime mould Physarum polycephalum.

Time-resolved detection of three intracellular signals controlling photomorphogenesis in Physarum polycephalum.

Incompetent plasmodia of Physarum polycephalum exposed to a light pulse sporulated after reaching the competent stage. Fusion of irradiated plasmodia with dark-incubated plasmodia and analysis of sporulation indicated the presence of a morphogenetic signal. It is concluded that a logic AND gate integrates the photoreceptor signal and the competence signal and controls the formation of the morphogenetic signal.

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.