Topology of an intracellular transduction chain (phototropism of Phycomyces): 1. Joint review of functional, temporal, and spatial aspects.

Two light-induced growth reactions in a unicellular cylindrical sporangiophore of Phycomyces blakesleeanus-vertical growth acceleration under symmetrical irradiation (photomecism) and directional growth under unilateral irradiation (phototropism)-share common input light perception as well as common output growth mechanism but have strongly divergent dynamics and other distinctive features. This divergence culminates in the phototropic paradoxes the main of which states that photomecism shows total adaptation, while phototropism does not adapt. The basis for this contradiction is that the phototropic transduction chain, unlike that of photomecism, faces a spatially non-uniform stimulus and processes a series of spatial patterns (light and absorption profiles, adaptation profile, etc.). The only way to resolve the paradoxes and correlate features of both responses within a single transduction chain is to assume non-local signal transduction, e.g. a cross-talk between different azimuthal locations within the cylindrical cell. On the other hand, to establish the presence of an appropriate cross-talk is equivalent of gaining insight into the topology of the transduction chain. This series of two papers contains a review reconsidering the entire field from this viewpoint (Paper 1) and a mathematical model of pattern transduction which unifies features of phototropism and resolves the paradoxes (Paper 2). At the same time, this is the first "proof of concept" for the "activity/pooling (a/p) networks"-a specific mathematical apparatus designed to analyse systemic properties and control in metabolic pathways.

Mutants of phycomyces with decreased gallic acid content.

Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycete Phycomyces blakesleeanus, much more so than in the basal mycelium or the culture medium. The actual concentrations vary with illumination, age of the culture, and composition of the medium. We devised a simple screening procedure to isolate hba mutants whose sporangiophores contained less gallic acid than the wild type. The most useful mutant had very low concentrations of hydroxybenzoic acids in the sporangiophores, but about the same as the wild type in the basal mycelium and the medium. The mutant was only slightly different from the wild type in growth and morphology. Mutant and wild-type sporangiophores grew away from ultraviolet C sources (260 nm) equally well. Contrary to previous conjectures, ultraviolet tropism does not depend on the ultraviolet absorption of gallic acid or other free hydroxybenzoic acids in the sporangiophore. Against expectations, phenols did not impair DNA extraction: sporangiophores produced better DNA preparations than basal mycelia and the hba mutant only slightly better than the wild type.

General theory of three-dimensional radiance measurements with optical microprobes.

Measurements of the radiance distribution and fluence rate within turbid samples with fiber-optic radiance microprobes contain a large variable instrumental error caused by the nonuniform directional sensitivity of the microprobes. A general theory of three-dimensional radiance measurements is presented that provides correction for this error by using the independently obtained function of the angular sensitivity of the microprobes.

Theory of equidistant three-dimensional radiance measurements with optical microprobes.

Fiber-optic radiance microprobes, increasingly applied for measurements of internal light fields in living tissues, provide three-dimensional radiance distribution solids and radiant energy fluence rates at different depths of turbid samples. These data are, however, distorted because of an inherent feature of optical fibers: nonuniform angular sensitivity. Because of this property a radiance microprobe during a single measurement partly underestimates light from the envisaged direction and partly senses light from other directions. A theory of three-dimensional equidistant radiance measurements has been developed that provides correction for this instrumental error using the independently obtained function of the angular sensitivity of the microprobe. For the first time, as far as we know, the measurements performed with different radiance microprobes are comparable. An example of application is presented. The limitations of this theory and the prospects for this approach are discussed.

Specific tropism caused by ultraviolet C radiation in Phycomyces.

The giant sporangiophores of Phycomyces blakesleeanus turn towards blue and away from ultraviolet C sources (wavelength under 310 nm). We have isolated fifteen mutants with normal blue tropism but defective ultraviolet tropism. Wild-type sporangiophores described a double turn when exposed successively to blue and ultraviolet beams coming from the same side; under certain conditions, the mutants turned only to the blue. The new uvi mutations modified the behaviour in heterokaryosis and were lethal in homokaryosis, i.e., they affected essential cellular components. The responses of the wild type and one of the mutants were registered and evaluated with a computer-aided device. The mutant behaved normally under blue light, but took longer than the wild type to turn away from the ultraviolet source. With very weak ultraviolet stimuli (10(-8) and l0(-9) W m-2), the wild type turned towards the source, but the mutant did not respond. Calculations of absorbed-energy distributions in the sporangiophore showed that Phycomyces responds differently to similar spatial distributions of blue and ultraviolet radiations. Wild-type and mutant sporangiophores had the same high ultraviolet absorption due to gallic acid. We conclude that ultraviolet tropism is not just a modification of blue phototropism due to the high ultraviolet absorption of the sporangiophores. Phycomyces has a separate sensory system responsive to ultraviolet radiation, but not to blue light.

Estimation of optical parameters in a living tissue by solving the inverse problem of the multiflux radiative transfer.

Calculations of radiative transfer require knowledge of the absorption and scattering coefficients and the asymmetry factor of scattering in the medium. A method is presented for estimating these coefficients in living plant leaves from fiber-optic measurements. We consider the plant leaf as consisting of two layers of different refractive indices and with reflecting surfaces. Light intensities at the boundaries of these layers in several irradiated plant leaves have been measured using a thin (70-microm) glass fiber connected to a photomultiplier. The diffuse reflection and transmission were measured with an integrating sphere. From these values we derive an estimation of the scattering and absorption coefficients and the asymmetry factor of scattering applying an inversion of the multiflux theory of light propagation in turbid media. In addition, we compare these coefficients with those obtained by using the Kubelka-Munk theory.

What do we know about the non-uniform perception of a phototropic stimulus in Phycomyces?

For a comprehensive study of phototropism in sporangiophores of the fungus Phycomyces, quantitative treatment of spatial aspects is necessary. The first step in quantifying spatial factors of phototropic signal processing is the elucidation of the non-uniform light profile, predominantly caused by a lens effect in the cylindrical body of the sporangiophore. Herein we compare recently presented theoretical and experimental studies of light profiles. Errors and ambiguities arising from instrumental limitations and arbitrary assumptions are revealed. On the other hand, by combining theoretical and experimental results we have been able to select out the reliable information, which can now be applied in phototropic studies.

A Novel Effect in Phycomyces Phototropism : Positive Bending and Compensation Spectrum in Far UV.

A novel effect-positive phototropic bending under far UV irradiation (between 260 and 305 nanometers) at low intensities-is reported. Natural compensation points (intensities which cause no bending under unilateral irradiation) have been determined for different wavelengths. The curve connecting these points, the compensation spectrum, divides the intensity-wavelength plane into areas of negative and positive tropism. It is further shown that a highly asymmetrical pattern of light stimulus within the sporangiophore underlies the symmetrical growth response at each compensation point. This suggests that some unknown additional factor is involved in perceiving a UV stimulus at the level of the photoreceptor. It is also demonstrated here that positive tropism in the UV range is due to a lens effect. We conclude that the hypothesis of optical attenuation of the stimulus (considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism heretofore neglected by others.

Geometrical optics approach to the intensity distribution in finite cylindrical media.

When irradiated unilaterally, certain cylindrical fungi act like a cylindrical lens. This behavior is the basis of phototropism (directed growth caused by asymmetrical illumination). A quantitative treatment of phototropism implies the description of light distribution at the inner boundary of finite nonscattering and weakly scattering homogeneous and radially stratified cylindrical media. This problem is solved using geometrical optics. First parameter representations for caustic and wavefronts within a homogeneous cylinder are derived. Then, the lens properties of the system are expressed by expansion coefficients. Geometrical optics fails at the caustic; there the field distribution is calculated with Airy functions. On the basis of intensity distributions, formulas for the absorption of light by oriented photoreceptors located at the wall of the system are derived. The theory is then applied to a special biological object.

Photon flux gradients in layered turbid media: application to biological tissues.

Estimation of photon flux gradients in biological tissues is necessary in many photobiological studies. A straightforward application of two-flux theories of light propagation in intensely scattering media to biological tissues is in many cases not adequate for two reasons: First biological tissues are layered objects with different absorption and scattering in different layers where there is no possibility of measuring photon fluxes within the object. Second, the multiple reflections at the boundaries make significant contributions to the photon fluxes. This paper presents a procedure for estimating, on the basis of measured transmission and remission, phenomenological absorption and scattering coefficients, the thickness of the layers, and the photon flux gradients in a two-layered tissue. The method, which is based on a combined treatment of the Kubelka-Munk equations for both layers, accounts for multiple reflections and does not require any measurements within the object. An example of application to a green leaf is given.

Correcting remission and transmission spectra of plant tissue measured in glass cuvettes: a technique.

A method for measuring transmittance and reflectance of plant material in glass cuvettes using a single-beam spectrophotometer with an integrating sphere attachment is described. This method requires extensive processing of the originally measured values as these are distorted by multiple reflections of the diffusely transmitted or reflected light. Most of the theories on light interaction with scattering materials require data processing to meet the theory's prerequisites. Therefore, the complicated evaluation does not restrict the applicability of the method. Special attention is given to the 60 degree/diffuse incidence requirement of the Kubelka-Munk theory. It is argued that the 60 degree/diffuse requirement is not essential for thick scattering layers. It is further stressed that a better knowledge of the optical properties should be of great help in many fields of biology.

A comparative study of the responsivity of Sinapis alba L. seedlings to pulsed and continuous irradiation.

Anthocyanin formation in 36h dark grown Sinapis alba L. seedlings and inhibition of hypocotyl elongation in 36h and 54h dark grown and 54h and 7 day light grown seedlings in response to continuous red light could be substituted for by hourly 5 min light pulses where the total fluence over the irradiation period is the same. These pulses are partially (36h) or almost totally (54h and 7 day) reversible by subsequent far-red (RG 9) light pulses. In contrast to 654 nm light, hourly light pulses with 552 nm, 449 nm and 715 nm can at best only partially substitute for continuous irradiation. These data are discussed with respect to the commonly used models for the phytochrome high irradiance response.