Light and dark adaptation in Phycomyces phototropism

Light and dark adaptation of the phototropism of Phycomyces sporangiophores were analyzed in the intensity range of 10(-7)-6 W X m- 2. The experiments were designed to test the validity of the Delbruck- Reichardt model of adaptation (Delbruck, M., and W. Reichardt, 1956, Cellular Mechanisms in Differentiation and Growth, 3-44), and the kinetics were measured by the phototropic delay method. We found that their model describes adequately only changes of the adaptation level after small, relatively short intensity changes. For dark adaptation, we found a biphasic decay with two time constants of b1 = 1-2 min and b2 = 6.5-10 min. The model fails for light adaptation, in which the level of adaptation can overshoot the actual intensity level before it relaxes to the new intensity. The light adaptation kinetics depend critically on the height of the applied pulse as well as the intensity range. Both these features are incompatible with the Delbruck-Reichardt model and indicate that light and dark adaptation are regulated by different mechanisms. The comparison of the dark adaptation kinetics with the time course of the dark growth response shows that Phycomyces has two adaptation mechanisms: an input adaptation, which operates for the range adjustment, and an output adaptation, which directly modulates the growth response. The analysis of four different types of behavioral mutants permitted a partial genetic dissection of the adaptation mechanism. The hypertropic strain L82 and mutants with defects in the madA gene have qualitatively the same adaptation behavior as the wild type; however, the adaptation constants are altered in these strains. Mutation of the madB gene leads to loss of the fast component of the dark adaptation kinetics and to overshooting of the light adaptation under conditions where the wild type does not overshoot. Another mutant with a defect in the madC gene shows abnormal behavior after steps up in light intensity. Since the madB and madC mutants have been associated with the receptor pigment, we infer that at least part of the adaptation process is mediated by the receptor pigment.     

Light and dark adaptation in Phycomyces light-growth response

Sporangiophores of the fungus Phycomyces exhibit adaptation to light stimuli over a dynamic range of 10(10). This range applies to both phototropism and the closely related light-growth response; in the latter response, the elongation rate is modulated transiently by changes in the light intensity. We have performed light- and dark- adaptation experiments on growing sporangiophores using an automated tracking machine that allows a continuous measurement of growth velocity under controlled conditions. The results are examined in terms of the adaptation model of Delbruck and Reichardt (1956, Cellular Mechanisms in Differentiation and Growth, 3-44). The "level of adaptation," A, was inferred from responses to test pulses of light by means of a series of intensity-response curves. For dark adaptation to steps down in the normal intensity range (10(-6)-10(-2) W/m2), A decays exponentially with a time constant b = 6.1 +/- 0.3 min. This result is in agreement with the model. Higher-order kinetics are indicated, however, for dark adaptation in the high-intensity range (10(-2)-1 W/m2). Adaptation in this range is compared with predictions of a model relating changes in A to the inactivation and recovery of a receptor pigment. In response to steps up in intensity in the normal range, A was found to increase rapidly, overshoot the applied intensity level, and then relax to that level within 40 min. These results are incompatible with the Delbruck-Reichardt model or any simple generalizations of it. The asymmetry and overshoot are similar to adaptation phenomena observed in systems as diverse as bacterial chemotaxis and human vision. It appears likely that light and dark adaptation in Phycomyces are mediated by altogether different processes.     

System analysis of Phycomyces light-growth response. Wavelength and temperature dependence.

The light-growth response of the Phycomyces sporangiophore was studied further with the sum-of-sinusoids method of nonlinear system identification. The first- and second-order frequency kernels, which represent the input-output relation of the system, were determined at 12 wavelengths (383-529 nm) and 4 temperatures (17 degrees, 20 degrees, 23 degrees, and 26 degrees C). The parametric model of the light-growth response system, introduced in the preceding paper, consists of nonlinear and linear dynamic subsystems in cascade. The model parameters were analyzed as functions of wavelength and temperature. At longer wavelengths, the system becomes more nonlinear. The latency and the bandwidth (cutoff frequency) of the system also vary significantly with wavelength. In addition, the latency decreases progressively with temperature (Q10 = 1.6). At low temperature (17 degrees C), the bandwidth is reduced. The results indicate that about half of the latency is due to physical processes such as diffusion, and the other half to enzymatic reactions. The dynamics of the nonlinear subsystem also vary with wavelength. The dependence of various model components on wavelength supports the hypothesis that the light-growth response, as well as phototropism, are mediated by multiple interacting photoreceptors.     

Interaction between gravitropism and phototropism in sporangiophores of Phycomyces blakesleeanus

The interaction between gravitropism and phototropism was analyzed for sporangiophores of Phycomyces blakesleeanus. Fluence rate-response curves for phototropism were generated under three different conditions: (a) for stationary sporangiophores, which reached photogravitropic equilibrium; (b) for sporangiophores, which were clinostated head-over during phototropic stimulation; and (c) for sporangiophores, which were subjected to centrifugal accelerations of 2.3g to 8.4g. For blue light (454 nm), clinostating caused an increase of the slope of the fluence rate-response curves and an increase of the maximal bending angles at saturating fluence rates. The absolute threshold remained, however, practically unaffected. In contrast to the results obtained with blue light, no increase of the slope of the fluence rate-response curves was obtained with near-ultraviolet light at 369 nm. Bilateral irradiation with near-ultraviolet or blue light enhanced gravitropism, whereas symmetric gravitropic stimulation caused a partial suppression of phototropism. Gravitropism and phototropism appear to be tightly linked by a tonic feedback loop that allows the respective transduction chains a mutual influence over each other. The use of tropism mutants allowed conclusions to be drawn about the tonic feedback loop with the gravitropic and phototropic transduction chains. The results from clinostating mutants that lack octahedral crystals (implicated as statoliths) showed that these crystals are not involved in the tonic feedback loop. At elevated centrifugal accelerations, the fluence-rate-response curves for photogravitropic equilibrium were displaced to higher fluence rates and the slope decreased. The results indicate that light transduction possesses a logarithmic transducer, whereas gravi-transduction uses a linear one.     

Negative phototropism in the piloboloid mutants of Phycomyces blakesleeanus Bgff.

The sporangiophore (spph) of a piloboloid mutant, genotype pil, of Phycomyces ceases elongation and expands radially in the growth zone shortly after reaching the developmental stage IV b. The pil spph is always negatively phototropic to unilateral visible light when its diameter exceeds 210 m. Photoinduction of spph initiation, light-growth response, threshold of light energy fluence rate for the negative phototropism, avoidance and gravitropism in the pil mutant are all normal. In liquid paraffin, the pil spph shows negative phototropism as does the wild-type spph. Genetic analyses indicate that the negative phototropism of the pil mutant is governed by the phenotypic characteristics of pil but not by specific gene(s) responsible for negative phototropism. These facts imply that the reverse phototropism of the pil mutant results from a loss of the convergent lens effect of the cell because of the increase in cell diameter.Abbreviations spph(s) sporangiophore(s) - wt(s) wild type(s)     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Wavelength dependence of bilirubin photoreactivity

The rates and products of the self-sensitized photoreactions of bilirubin IXα vary with excitation wavelength, solvent and the presence or absence of oxygen. Radical or Type I photooxidation reactions of bilirubin are implicated in degassed chloroform or methanol. Quantum yields for the disappearance of bilirubin vary from 10^?2 to 10^?4 with excitation wavelengths of 440, 340 and 280 nm and with the higher quantum yield generally appearing in chloroform solvent and/or excitation at 280 nm. Bilirubin is stable in degassed chloroform to irradiation at 440 nm.     

Wavelength dependence of visual acuity in goldfish

Visual acuity was measured in a two-choice training experiment with food reward. Four goldfish were trained to select a homogeneously illuminated testfield when a high-contrast grating (transparancy) was shown for comparison at the second testfield. Measurements were performed for white and monochromatic testfield illuminations in the light adapted state. Fourteen wavelengths between 404 nm and 683 nm were tested. For each wavelength (and white light) the testfield intensity was determined for which spatial resolution was highest. Between 446 nm and 683 nm maximal values of 2.0 cycles/deg (corresponding to a visual acuity of 15' of arc) were found. At 404 nm and in the ultraviolet resolution was lower (0.6 and ~0.25–0.35 cycles/deg, respectively). Cone and small ganglion cell densities may equally account for visual acuity. The action spectrum of maximal visual acuity is very similar to the spectral sensitivity function representing recognition of "colour". Measurements under reduced room illumination and after treatment with Ethambutol further indicate that the detection of high contrast gratings is processed by the same "channel" as colour vision. A similar separate and parallel processing of "colour" and "form" on the one hand, and "brightness" and "motion" on the other hand was found in humans.     

Photogravitropic equilibrium in Avena coleoptiles: fluence rate-response relationships and dependence on dark adaptation and clinostating

Phototropism of Avena coleoptiles was measured in response to blue-light irradiation lasting between 2 and 24 h. During this time the coleoptiles established a bending angle of photogravitropic equilibrium that was dependent on the time of irradiation and also on the pretreatment in light or darkness prior to stimulation. The absolute threshold for the photogravitropic equilibrium in response to blue light was 10(-8) micromol m(-2) s(-1). Photon fluence rate-response curves, which were generated after several hours of dark adaptation, had a characteristic shape with a prominent optimum in the middle of the dynamic range. Curves which were generated without prior dark adaptation displayed no such optimum. Clinostating dark-adapted coleoptiles caused an increase of sensitivity and responsiveness during a 2-h period of unilateral irradiation. The advantages and the drawbacks of long-term irradiation experiments for the investigation of phototropism and the generation of action spectra are discussed.     

Growth-rate adaptation of Phycomyces sporangiophores to partial depletion of oxygen

The growth rate of Phycomyces blakesleeanus sporangiophores was found to be very sensitive to sudden changes in the oxygen concentration. A change from 20% to 15% oxygen elicits a transient decrease in the growth rate which returns to normal 10 min after altering the concentration. After a step change to 10% oxygen, the growth rate shows two minima at 6–8 and 30–35 min and it reaches about 80% of its original value 50 min after this change. A threshold curve for this negative growth response shows that sporangiophores begin to sense a decrease in the oxygen concentration from 20% to 17%. Seven phototropically abnormal mutants with defects in the genes madA to madG were tested for the oxygen response. Two strains, C149madD120 and C316madF48, were found to have recoveries different from those of the wild type after step changes from 20% to 10% oxygen.     

Light and dark adaptation of halorhodopsin

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.     

Threshold and adaptation in Phycomyces. Their interrelation and regulation by light

The absolute light sensitivity of Phycomyces sporangiophores was determined by analyzing the intensity dependence of the phototropic bending rate and of the light growth and dark growth responses to step changes of the intensity. We found that the different methods give approximately the same results for the wild-type strain, as well as for several behavioral mutants with defects in the genes madA, madB, and madC. A crucial factor in the determination of thresholds is the light intensity at which the strains grow during the 4 d after inoculation and prior to the experiment. When the wild-type strain grows in the dark, its threshold for the bending rate is 10(-9) W X m-2, compared with 2 X 10(-7) W X m-2 when it is grown under continuous illumination. Further, the maximal bending rate is twice as high in dark-grown strains. This phenomenon is further complicated by the fact that the diameter and growth rate of the sporangiophores also depend on the illumination conditions prior to the experiment: light-grown sporangiophores have an increased diameter and an increased growth rate compared with dark-grown ones. Some of the behavioral mutants, however, are indifferent to this form of light control. Another factor that is controlled by the growth conditions is adaptation: the kinetics of dark adaptation are slower in light-grown sporangiophores than in dark-grown ones. We found empirically a positive correlation between the slower dark adaptation constant and the threshold of the bending rate, which shows that the two underlying phenomena are functionally related.