Subliminal light control of dark adaptation kinetics in Phycomyces phototropism

The dark adaptation kinetics of Phycomyces phototropism depend critically on the experimental protocol. When sporangiophores that had been light-adapted to a fluence rate of 1 W m-2 at 447 nm were exposed to dim unilateral light, the adaptation kinetics showed exponential decay (6 min time constant). However, when light-adapted sporangiophores were kept for variable intervals in darkness (i.e. in presence of traditional red safelight) and then exposed to dim unilateral test light, the decay kinetics of adaptation were biexponential with a rapid decay during the first minute (1 min time constant), followed by a slow recovery (11 min time constant). Thus, the dim subliminal light given after the sporangiophores had been adapted to 1 W m-2, was actually perceived, and exerted control over the dark-adaptation process. The observed acceleration of dark-adaptation kinetics constitutes a novel light effect of the sporangiophore. At wavelength 383 nm this effect was not observed. Because a beta-carotene lacking mutant, L91 (genotype carB), was unmodified in dark-adaptation kinetics measured in the presence or absence of subliminal light, it appears that beta-carotene is not involved in the photocontrol of adaptation.     

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

EFFECT OF CALCIUM ON DARK ADAPTATION IN Phycornyces PHOTOTROPISM

Adaptation processes enable phototropism of Phycomyces to operate over a 10-decade range of blue-light intensity (1 nW m-2-10 W m-2). To investigate the influence of calcium on dark adaptation, the phototropic latency method was employed with the modification that sporangiophores were temporarily immersed in solutions containing CaCl2 or LaCl3. Following such treatment, the time course of bending was found to have two components with distinct latencies and bending rates. After immersion in darkness for 30 min in LaCl3 solution or 1 h in a solution of CaCl2, MgCl2, or the calcium chelator EGTA, each sporangiophore was adapted to a blue light beam (1 W m-2) for 45 min by rotation around its vertical axis. Cessation of rotation defined the onset of the phototropic stimulus, at which time the intensity was reduced by as much as 10(3)-fold. For a 10(2)-fold reduction (to 10(-2) W m-2), immersion in CaCl2 (10-100 microM) reduces the latency 13 min for the early bending component and 18 min for the late component, whereas treatment with the calcium-channel blocker lanthanum (0.1-11 microM LaCl3) increases the latency 12 min for the early component and 13 min for the late component. EGTA (10 microM) also had an inhibitory effect, increasing the latency of the first and the second components by 7 and 10 min, respectively. In experiments performed similarly, but without the light adaptation treatment after immersion, no differences between calcium-treated and control sporangiophores were found. The bending rates of both components show only a weak dependence on calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACTION SPECTRUM FOR SUBLIMINAL LIGHT CONTROL OF ADAPTATION IN Phycomyces PHOTOTROPISM

Abstract -Adaptation processes enable phototropism and other blue light responses of Phycomyces to operate over a 10-decade range of Ruencc rate. Phototropic latency, used routinely to monitor the kinetics of sensitivity recovery after a step down in fluence rate, can be shortened by application of dim light for 35 min during the early part of the latency period. This light is termed subliminal , because it does not elicit phototropism under these experimental conditions; rather, it exerts its influence on the underlying adaptation kinetics. Fluence rate-response data for this latency reduction, obtained at 17 wavelengths of subliminal light from 347 to 742 nm, showed a variety of shapes that could be fit by zero, one, or two sigmoidal components, plus a constant term. At most wavelengths, the fluence-rate threshold for latency reduction by subliminal light tended to be well below the absolute threshold for phototropism, indicating that this effect is highly sensitive. An action spectrum for the sensitivity of the subliminal light effect, derived from the fluence rate-response curves, shows major peaks around 400 and 500 nm and a broad band from 570 to 670 nm, followed by a steep absorption edge. The sensitivity in the near ultraviolet region is relatively very low. The magnitude of the latency reduction also depends strongly on wavelength with a maximum at about 450 nm. The Huence-rate response data and the action spectrum–which is markedly different from that for phototropism and other blue-light responses of Phycornyces – indicate the participation of multiple pigments, or pigment states, in the photocontrol of adaptation.     

PHOTOTROPISM OF THE Phycomyces SPORANGIOPHORE: A COMPARISON WITH HIGHER PLANTS

Abstract— The basic phenomena of Phycomyces phototropism are described and compared with those of Avena and Zea coleoptiles and the seedlings of Arabidopsis, Cucumis, Helianthus, Raphanus and Sesamum. Symmetric redistribution of growth rate during curvature, a feature characteristic of Phycomyces phototropism, can in some cases also be found in plants. Common features are: complex fiuence-response curves, log-linear fluence dependence of the phototropic latency, relatively slow bi-exponential dark-adaptation and fast light-adaptation kinetics, action spectra indicating the ubiquitous flavin-like photoreceptor(s) as well as other non-flavin receptors, and spacial memory as indicated by phototropic reversal experiments. The problem how phototropic curvature is maintained continuously while the light-growth response adapts, remains unexplained ("phototropism paradox"). For Phycomyces it is shown that some of the most basic information processes, which include contrast recognition, wavelength sensitivity and sensitivity recovery (sensor adaptation) are mediated by the photoreceptor systems.     

Photoreception and Phototropism in Phycomyces: Antagonistic Interactions between Far-UV, Blue, and Red Light

Abstract— Phototropism of the sporangiophore of the fungus Phycomyces is mediated by UV and blue light. Classical phototropism action spectra with maxima near 280, 370 and 450 nm indicate a flavin-like photoreceptor. Blue light mediates positive phototropism while far-UV light mediates negative phototropism. To better understand the mode of interaction of far-UV with blue light we performed phototropism experiments in which sporangio-phores were placed for 4 h between sources of 280 and 454 nm light coming from opposite directions. The fluence rates of the far-UV were chosen such that unilateral light alone elicited 90° of negative bending. For blue light, moderate fluence rates were applied that elicited about 40° bending. Under conditions of bilateral irradiation the blue light substantially reduced the far-UV elicited phototropism. In the presence of tonic red light the antagonism between far-UV and blue light was greatly reduced. Red light, which by itself is phototropically ineffective, also reduced phototropic bending elicited by either far-UV or blue light. These observations are taken as indications for the existence of a red light-absorbing intermediate of the blue-light receptor. Because the far-UV/ blue-light antagonism disappeared almost completely in the presence of tonic red light, the antagonism may occur at the level of this receptor intermediate.     

Analysis of multiple photoreceptor pigments for phototropism in a mutant of Arabidopsis thaliana

The shape of the fluence-response relationship for the phototropic response of the JK224 strain of Arabidopsis thaliana depends on the fluence rate and wavelength of the actinic light. At low fluence rate (0.1 micromole m-2 s-1), the response to 450-nm light is characterized by a single maximum at about 9 micromoles m-2. At higher fluence rate (0.4 micromole m-2 s-1), the response shows two maxima, at 4.5 and 9 micromoles m-2. The response to 510-nm light shows a single maximum at 4.5 micromoles m-2. Unilateral preirradiation with high fluence rate (25 micromoles m-2 s-1) 510-nm light eliminates the maximum at 4.5 micromoles m-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 micromoles m-2 is unaffected. Based on these results, it is concluded that a single photoreceptor pigment has been altered in the JK224 strain of Arabidopsis thaliana.     

ARE THERE SEVERAL PHOTORECEPTORS INVOLVED IN PHOTOTROPISM OF Phycomyces blakesleeunus? KINETIC STUDIES OF DICHROMATIC IRRADIATION*

Abstract— Photogeotropic equilibrium angles were measured for Phycomyces blakesleeanus wild type firstly by means of dichromatic fluence rate response curves using simultaneous irradiation with near threshold 450 nm reference light (constant at 1.2 × 10^?8 W m^?2) and variable fluence rates of test light (498–630 nm) from the same side. These curves showed minima for test light fluence rates that were close to the photogeotropic threshold for these wavelengths. Secondly, the time course of this inhibitory effect was studied with both the inductive reference 450 nm light (2 × 10^?-⁷ W m^?2) and the test light (606 or 450 nm) given as light pulses of 2 s duration (2 s light/48 s dark periods for 6 h). The dark period between the onset of the inductive reference light and test light pulses was varied between 0 and 48 s. No inhibitory effects were observed for simultaneous pulses; however, inhibitory effects were demonstrated for delay times of 2 s and 20 s for 606 nm as well as 450 nm test light. If the test light pulses were given immediately before the inductive reference light, only 606 nm test light was effective in producing a significant inhibitory effect. The results are discussed with regard to a multichromophoric photoreceptor system and to the wavelength dependence of the effects observed. The data and conclusions favor a photoreceptor system with at least two separate chromophoric absorptions of the blue light receptor type, one acting positively, the other acting inhibitorily, and at least one other photoreceptor of presumably minor influence.     

STUDIES ON PHOTOTROPISM OF YOUNG SPORANGIOPHORES OF PILOBOLUS KLEINII*

Abstract— Young sporangiophores of the fungus, Pilobolus kleinii, respond to unilateral illumination by bending or by growing toward light of wavelengths between 312 and 530 mμ, with peaks of sensitivity near 360 and 450 mμ. Young sporangiophores exhibit a negative phototropic response to wavelengths shorter than 300 mμ, with a strong negative response at 280 mμ. Since the action spectrum did not correspond to the absorption spectrum of the pigmented zone as measured in vivo, and since colorless sporangiophores formed on media containing diphenylamine were capable of phototropic response, it is unlikely that the conspicuous orange-yellow pigment in young sporangiophores is the photoreceptor for phototropism. The results of probing with small beams of light and the behavior of sporangiophores submerged in mineral oil, together with measurements of the refractive index of the tip and base indicate that the photosensitive region is located in the tip of the young sporangiophore.     

Separate Sensory Pathways for Photomorphogenesis in Phycomyces

The zygomycete Phycomyces blakesleeanus develops two types of sporangiophores of very different size: macrophores and microphores. Blue light inhibits the development of microphores and stimulates the development of macrophores. These responses are called photomicrophorogenesis and photomacrophorogenesis, respectively; phototropism designates the growth of the macrophores toward blue light and photocarotenogenesis, the increased accumulation of β-carotene under blue light. We have isolated three pim mutants that develop microphores under continuous illumination at fluence rates that inhibit them in the wild type. The thresholds for photomicrophorogenesis in the mutants were higher than in the wild type, but the thresholds for photomacrophorogenesis and phototropism did not change. At least one of the pim mutants had a much higher threshold for photocarotenogenesis than the wild type. A madJ mutant strain, already known for its very defective phototropism, was defective for photomicrophorogenesis, but normal for photomacrophorogenesis and photocarotenogenesis. The requirement for the pim and madJ gene functions separates the two photomorphogenetic responses. The results indicate that the four responses to blue light are based on a combinatorial use of sensory transducers.     

LIGHT-INDUCED CAROTENE SYNTHESIS IN MUTANTS OF PHYCOMYCES WITH ABNORMAL PHOTOTROPISM

Mutants of Phycomyces. abnormal in their phototropic responses (the mad mutants), have been tested for their responses in light-induced carotene synthesis (LICS). The amount of carotene synthesized at any given fluence is significantly lower in the madA, madB and madD mutants than in the wild type. The amount of carotene is not lower in other mad mutants ( madC, madE, madF and madG ). The double mutant mad A madB and the triple mutant mad A madB madC show stronger effects. The wild type strain, as well as those carrying a single mad mutation ( madA and madB ) or those carrying two or three mad mutations ( madA madB. madA madB madC ) show closely similar sensitivity to LICS. This contrasts with phototropism and photoinitiation of sporangiophores which are sensitive to extremely small signals in the wild type and in which the madA mutation decreases the sensitivity by nearly a factor of 10⁴ and madB mutation by a factor of 10⁵. It appears that LICS does not share the signal amplification mechanisms characteristic of the other two responses.     

Kinetic separation of phototropism from blue-light inhibition of stem elongation

These experiments tested the hypothesis that phototropic bending arises when a light gradient across the stem differentially inhibits cell elongation because of direct inhibition of cell elongation by light (the Blaauw hypothesis). Continuous irradiation of dark-grown cucumber seedlings (Cucumis sativus L.) with unilateral blue light inhibited hypocotyl elongation within 30 s, but did not induce phototropic curvature until 4.5 h after the start of irradiation. Marking experiments showed that curvature began simultaneously at the top and bottom of the growing region. In situ measurements of the light gradient across the stem with a glass fiber optic indicated that a 5- to 6-fold difference in fluence rate was established on the two sides of the stem. The light gradient established at the start of irradiation was the same as that after 6 h of irradiation. Changes in gravitropic responsiveness during this period were also ruled out. Calculations show that the light gradient should have caused curvature which would be detectable within 30 to 60 min and which would extrapolate to the start of irradiation--if the Blaauw hypothesis were correct. The long lag for phototropism in this case indicates that rapid inhibition of cell elongation by blue light does not cause the asymmetrical growth of phototropism. Rather, phototropism is superimposed upon this separate light growth response.     

Action spectra for photogravitropism of Phycomyces wild type and three behavioral mutants (L150, L152, and L154)

We have measured fluence rate-response curves and action spectra for photogravitropism in Phycomyces wild type and in three recently isolated mutants with elevated phototropic thresholds. The action spectra were determined from least-squares fits of a sigmoidal function to the fluence rate-response data for each wavelength. The action spectrum for wild type has maxima near 383, 413, 452, and 490 nm and minima near 397, 425, and 469 nm. This photogravitropism action spectrum is very similar to the Phycomyces phototropic balance action spectrum between 413 but has significantly higher effectiveness below 400 nm and above 490 nm. These differences may be caused by dichroic effects of the oriented receptor pigment and/or by multiple receptor pigments. The action spectra of the three mutants differ significantly from one another and from that of wild type. Relative to the wild type spectrum, all three mutants exhibit a suppression in effectiveness near 425 nm, which is near the transmission peak of the broadband blue filter used to isolate the mutants.     

Antagonistic blue- and red-light regulation of cab-gene expression during photosynthetic adaptation in Scenedesmus obliquus.

During adaptation of the photosynthetic apparatus of the green alga Scenedesmus obliquus to various light qualities, the accumulation of chlorophylls and pigment-protein complexes (with specific consideration of chlorophyll a/b-binding (Cab) proteins) and cab-gene expression were determined. The fluence rate dependences for chlorophyll accumulation and cab-gene expression were very different. Very low fluence rates of violet (404 nm), blue (461 nm) and red (650 nm) light below the photosynthetic threshold, i.e. between 10(-3) and 10(-1) mumol m-2 s-1, inhibited all of these reactions in cells grown under heterotrophic conditions. At elevated fluence rates (above 1 mumol m-2 s-1), red light retained its negative regulation, whereas blue light stimulated pigment accumulation. Under autotrophic conditions the pattern was more complex, because chlorophyll accumulation was unaffected by light below the photosynthetic threshold. However, the expression of cab-genes was inhibited by red light but stimulated by blue light. Cells adapted to fluence rates, which ensured photosynthetic energy supply (above 1 mumol m-2 s-1), showed an increase in chlorophyll accumulation, blue light being more effective than red light. The results confirm and extend our previous discovery of two antagonistically acting photoreceptors in Scenedesmus which mediate and coordinate the complex functional and structural changes associated with photosynthetic adaptation. One of these receptor pigments is a blue-light receptor with positive action; the other is a violet-red-light receptor which can operate far below the photosynthetic threshold and exerts a negative regulation.     

DIFFERENTIAL SPECTROPHOTOMETRY OF Phycomyces MUTANTS WITH ABNORMAL PHOTORESPONSES

Abstract— The unidentified receptor pigments responsible for phototropism and other blue light responses of Phycomyces are expected to contribute only a minuscule fraction of the in vivo absorption spectra, even for carotene-deficient ( car genotype) albino strains. The variable amount of residual carotenoids in strains with mutations in the carA gene precluded their use for the comparison of phototropism (mad) mutants with phototropically normal controls. We turned instead to carB albino strains, all of which have negligible carotene content. For various mad genes, we isolated carB mad strains by crosses and mutagenesis, and compared them with carB controls, in search of spectral differences associated with the mad photoreception defects. The spectra and their second derivatives show cytochrome bands, as well as a minor peak near 480 nm. This peak was present in carB strains, so it is probably not due to carotenoids. Strains L26 and L136 (both carB madE , but with different genetic backgrounds) showed modifications in the480–500 nm region. Sporangiophores that lacked the green material that usually interferes with in vivo spectroscopy were obtained by the addition of potassium iodide to the culture medium. Dark-grown carB sporangiophores have a more pronounced second-derivative peak at 480 nm than do light-grown sporangiophores. Growth in the light, though, did not significantly alter the spectra of sporangiophores of L131 (carB madB) or L130 (carB madC). The spectral differences may be useful in the biochemical search for the receptor pigments.     

PHOTOINITIATION OF SPORANGIOPHORES IN PHYCOMYCES MUTANTS DEFICIENT IN PHOTOTROPISM AND IN MUTANTS LACKING β-CAROTENE

Abstract—The formation of sporangiophores from mature Phycomyces mycelium is inhibited in a closed system. Irradiation of the mycelium with blue light reverses the inhibition of spordngiophore formation. Dose response curves for this reaction are established for wild type. β-caroteneless mutants and for mutants that are deficient in phototropism.
Phototropic-negative mutants. altered in genes madA and madB , have a raised threshold in this light reaction. whereas mutants deficient in genes madD to madG are unaffected. β-caroteneless mutants deficient in genes carA, carB , or carR have a threshold raised by a factor of 100–2000 depending on the amount of residual synthesis of β-carotene.     

LIGHT-INDUCED CHANGES IN THE VESICULAR APPARATUS OF Phycomyces SPORANGIOPHORES DURING THE PHOTOTROPIC RESPONSE

Abstract— We studied the ultrastructure of laterally illuminated stage I sporangiophores of Phycomyces blakesleeanus to determine whether the phototropic response had any structural correlate. Stimulated sporangiophores conserved the distinctive division into the four longitudinal zones described for state I, and showed no significant differences in the distribution of major organelles in the sides proximal and distal to the light source. On the other hand, apical vesicles were asymmetrically distributed, being present in lower numbers in the faster growing (distal) side of the sporangiophore. We suggest that this asymmetrical distribution reflects the differential utilization of vesicles necessary to sustain the corresponding growth rate of each side. Illumination therefore, must affect the rate of vesicle migration and utilization, more than their rate of synthesis.     

PHOTOTROPISM IN PHYCOMYCES MUTANTS LACKING β-CAROTENE

Abstract. β-carotene and riboflavin are considered as the major candidates for the photoreceptor for physiological responses to blue light in Phycomyces and a number of other organisms. Mutants of Phycomyces blocked in all six steps of the biosynthesis of β-carotene from phytoene contain no detectable β-carotene (less than 4 times 10^-5 of wild-type amount) but exhibit phototropic responses identical to wild-type. Moreover, wild-type Phycomyces , while abundant in trans -β-carotene, contains no detectable cis -β-carotene, sometimes proposed as a photoreceptor candidate on the basis of the close similarity of the cis -species absorption spectrum in the near UV region to many action spectra for blue-light responses. These results indicate that β-carotene cannot be the photoreceptor for phototropism in Phycomyces.     

ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces WILD TYPE AND THREE BEHAVIORAL MUTANTS

Photogeotropic equilibrium action spectra in the range from 301 to 740 nm were made for Phycomyces wild type and the three behavioral mutants C47 ( madA35 ), C109 ( madBlOl ) and LI (madCIIQ) , all of which have a raised phototropic threshold. In addition to two broad peaks at 365 and 455 nm, typical for flavins, the wild type action spectrum shows three novel peaks, which have not been observed previously. These peaks are located at 414, 491 and 650 nm. The 650 nm peak has a relative quantum efficiency of 3 × 10⁻⁸ compared to the peak at 414 nm. The wavelength dependent shapes of the fluence-response curves of the bending angle and the aiming error angle indicate more than one receptor pigment for phototropism. The shape of the action spectrum of C47 is basically unaltered in comparison to wild type. C109 and LI show substantial differences from the wild type. In the near UV two small peaks at 334 and 365 nm appear; the 414 and 491 nm peaks present in wild type and C47 are missing and two new peaks at 529 nm (not well resolved in C109) and 567 nm are found. None of the three mad mutations affects the 650 nm peak. A model of the sensory transduction chain is presented, which incorporates these and other known features.     

ACTION SPECTRUM FOR ENHANCEMENT OF PHOTOTROPISM BY Arabidopsis thaliana SEEDLINGS

Fluence-response relationships have been measured at wavelengths from 350 to 760 nm for the enhancement of phototropism in Arabidopsis thulium L. (Heynh) strain “Estland” by an irradiation at each of these wavelengths, given 2 h prior to a 450 nm inductive unilateral irradiation. Action spectra have been constructed from these fluence-response relationships based on: (i) the fluence required to obtain a curvature of 25° (corresponding to an enhancement of 15°), (ii) the fluence required to obtain 50% of the maximum enhancement and (iii) the fluence threshold for enhancement by a pre-irradiation. The action spectra exhibit two maxima, one at 669 nm and a second at 378 nm. The height of the maximum at 669 nm is approximately 4 times the height of the maximum at 378 nm. Based on the action spectra, it is concluded that the enhancement of phototropism in A. thaliana is mediated by phytochrome.