Photoreceptor Pigment for Blue Light in Neurospora crassa

Irradiating the mycelium of Neurospora crassa with moderate intensities of blue light causes a reversible photoreduction of a b-type cytochrome. The action spectrum for the photoreduction of cytochrome b is very similar to the absorption spectrum of flavin pigments. Prolonged irradiation of the mycelium with strong blue light irreversibly bleaches flavin-like pigments and as these pigments are bleached the photoresponse of cytochrome b is lost. We conclude from these and other data that a flavin is the photoreceptor pigment for the photoreduction of cytochrome b. The close similarity between the action spectrum for the photoreduction of cytochrome b and action spectra for a number of physiological photoresponses suggests that this photoreceptor pigment controls a wide variety of photobiological processes in a wide diversity of organisms.     

An action spectrum for photoinduced sporulation in the fungus Trichoderma viride

When a dark grown colony of Trichoderma viride is exposed towhite light for a short time, sporulation occurs only in thenarrow region of mycelia produced just prior to illumination.The action spectrum of this light effect was obtained for wavelengthsbelow 520 mµ using monochromatic irradiation of knownintensity having a band width of 10 mµ. The action spectrumshows 4 distinct peaks at 320, 380, 430 and 480 mµ. Thewavelengths at 320 and 380 mµ are most effective in photoinducedsporulation. The longer wavelengths, 430 and 480 mµ, areconsiderably less effective. The possibility that a carotenoid or a flavin compound may bea photoreceptor in this photoinduced sporulation is discussed. (Received January 4, 1969; )     

Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes

An action spectrum for anthocyanin formation in dark-grown broom sorghum (Sorghum bicolor Moench, cv Acme Broomcorn and cv Sekishokuzairai Fukuyama Broomcorn) seedlings was determined over the wavelength range from 260 to 735 nanometers. The action peaks were at 290, 650, 385, and 480 nanometers in descending order of height. The action of the 290-nanometer peak was not affected by subsequently given far red light, whereas those of the other three action peaks were nullified completely. The nullification of the 385-nanometer peak action by far red light was reversible. When an irradiation at these action peaks was followed by a phytochrome-saturating fluence of red light irradiation, the action of the 290-nanometer peak remained, whereas that of the 385-nanometer peak as well as those of the 650- and 480-nanometer peaks was masked by the action of the second irradiation. These findings suggested that the 290- and 385-nanometer action peaks involved different photoreceptors, the latter being phytochrome. The blue light-absorbing photoreceptor as reported to be a prerequisite for phytochrome action in milo sorghum was not found to exist in the broom sorghums.

The action spectrum deprived of the involvement of phytochrome was determined in the ultraviolet region by irradiating with far red light following monochromatic ultraviolet light. The spectrum had a single intense peak at 290 nanometers and no action at all at wavelengths longer than 350 nanometers.

     

Action spectrum for fruiting in the basidiomycete Schizophyllum commune

An action spectrum for fruit body formation was determined in the range 280–723 nm for a dikaryon of Schizophyllum commune Fr. Action maxima occurred at 280 and 340–360 nm (main peak), and there were minor peaks at 437 and 467 nm. The quantum effectiveness at 360 nm was ca seven-fold compared to that of 437 nm light. Wavelengths longer than 500 nm were ineffective. Light also induced formation of brown pigment in the area producing fruit bodies. Wavelengths ranging from 260 to 300 nm injured aerial hyphae at the border of the colony. The possibility that a flavin or a pteridine may be the photoreceptor is discussed.     

Light-controlled Stem Elongation in Pea Seedlings Grown under Varied Light Conditions

There appears to be an orderly transition from one photosensitive state to another in light-controlled stem elongation in the garden pea, Pisum sativum L. cv. Alaska. Stem elongation in dark-grown plants is known to be phytochrome-controlled. However, seedlings are insensitive to phytochrome after a red light pretreatment. An action spectrum for inhibition in these seedlings has peaks at 440 and 620 nm. Protochlorophyll is suggested as the photoreceptor. If these red pretreated seedlings are further exposed to white light, sensitivity to 440 to 620 nm light is lost. Promotion by blue-green light is the only photoresponse shown by these seedlings. Inhibition of completely white light-grown seedlings required simultaneous exposure to high intensity blue light and 600 nm light.     

An Action Spectrum for Light-induced Primordium Formation in a Basidiomycete, Favolus arcularius (Fr) Ames

The action spectrum for the initiation of fruiting (primordium formation) in Favolus arcularius was determined on the equal response basis. The detectable effect of light was observed in the region between 350 to 560 nanometers, showing six distinct peaks at 374, 398, 424, 446, 480, and 514 nanometers. The half maximum response is reached with 1.8 × 10⁸ ergs per cm² at the most effective wavelength, 398 nanometers. Since the inhibitors, diphenylamine and quinacrine, had no consistent effect on the primordium formation, it is suggested that the possible photoreceptor pigment(s) may be neither carotenoid nor flavinoid.     

The ultraviolet action spectrum for stomatal opening in broad bean

The ultraviolet action spectrum for stomatal opening was measured using epidermal peels from leaves of broad bean (Vicia faba). The spectrum was calculated from hyperbolic fluence response curves using 11 wavelengths ranging from 275 to 459 nm. The action spectrum exhibits a major peak at approximately 280 nm and a minor peak at approximately 360 nm. The response at 280 nm is about three times greater than the response at 459 nm. Under the conditions utilized (i.e. the absence of saturating red light), stomatal opening saturated at extremely low fluence rates: <0.2 μmol m⁻² s⁻¹ at 280 nm, and approximately 1.0 μmol m⁻² s⁻¹ at 459 nm. The threshold for blue-light-induced stomatal opening was approximately 0.02 μmol m⁻² s⁻¹. In light-mixing experiments, the addition of 280 nm light to saturating 650 nm (red) light caused additional stomatal opening, which is indicative of separate photoreceptors. In contrast, adding 280 nm of light to saturating 459 nm (blue) light did not increase stomatal opening, suggesting that they both excite the same receptor. The results with white light were similar to those with blue light. We infer that ultraviolet light acts via the blue light photoreceptor rather than through photosynthesis. The additional absorbance peak at 360 nm suggests that the chromophore is either a flavin or a cis-carotenoid, both of which exhibit peaks in this region. It is proposed that the chromophore can be excited either directly by blue light or by energy transferred from the protein portion of the protein-pigment complex after it absorbs 280 nm light.     

A Spectroscopic View of Some Recent Advances in the Study of Blue Light Photoreception*

The blue to UV-A region of the spectrum, spanning the region of about 320–520 nm, strongly influences the growth and development of plants and fungi. Photomorphogenesis in plants is, to a great extent, controlled by phytochrome, but there are unique contributions of the blue region, which cannot be duplicated by any amount of red light. Phototropism is, with few exceptions, a purely blue light response. In fungi, the blue region dominates the photocontrol of growth and development, though some red light effects have been reported. Many blue light action spectra fit the definition of cryptochrome, a pigment class defined by its UV-A and blue peaks. The action spectrum, if measured to sufficient resolution, displays several minor maxima or shoulders in the blue region which call to mind the vibrational levels of carotenoids and flavins. Recent molecular genetic studies, as well as photobiological work, have shown that some cryptochromes are related to the DNA repair enzyme photolyase, while others appear genetically and spectroscopically distinct. In this review, we have applied established criteria from photobiology, in particular, comparison of action spectra with absorption spectra, to these recent results. It is apparent that photolyase homologs such as CRY1 can explain the blue light portion of the action spectrum for hypocotyl elongation, assuming participation of the oxidized flavin. In fungi, the photoreceptor question remains open. Identification of the nph1 gene in Arabidopsis may soon lead to a photoreceptor for higher plant phototropism. Also, we present a possible solution to the most recent version of the long-standing flavin-carotenoid controversy, the zeaxanthin hypothesis for higher plant phototropism. In conclusion, there appear to be at least three classes of cryptochromes.     

Effect of light quality on the photoinduction of carotenoid synthesis in Verticillim agaricinum

The action spectrum of photoinduction of carotenoid biosynthesis in Verticillium agaricinum has a high peak in the near-UV region with very small peaks in the blue, yellow and red regions. This suggests that neither a flavoprotein nor a carotenoid could be the photoreceptor, but a new unknown pigment system. Sporulation however is not photoregulated and is unaffected by light in the near-UV region. So, mycochrome cannot be implicated in the sporulation of this fungus. Further, sporulation and carotenogenesis must be under separate regulatory control in this organism, but not necessarily so in other organisms.     

Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings

The action spectrum for the light-activated destruction of phytochrome in etiolated Avena seedlings has been determined. There are 2 broad maxima, one between 380 and 440 mμ, the other between 600 and 700 mμ. peaking at about 660 mμ. On an incident energy basis, the red region of the spectrum is more efficient than the blue by about one order of magnitude in activating phytochrome disappearance. Both the red absorbing as well as the far-red absorbing forms of phytochrome are destroyed after exposure of Avena seedling to either red or blue light.

From the action spectrum and photoreversibility of pigment loss, we conclude that phytochrome acts as a photoreceptor for the photoactivation of its metabolically-based destruction. We suggest that another pigment might also be associated with the disappearance of phytochrome in oat seedlings exposed to blue light.

     

MICROSPECTROPHOTOMETRY AND THE PHOTORECEPTOR OF PHYCOMYCES I

By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.     

Reversal of blue light-stimulated stomatal opening by green light

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.     

Modification of Blue Light Photoresponses by Riboflavin Analogs in Neurospora crassa

The effect of riboflavin analogs on blue light responses in a riboflavin mutant of Neurospora crassa was studied. The analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, acted as photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were ineffective as photoreceptors for the induction of carotenoid synthesis. These results provide addtional evidence implicating a flavin photoreceptor for at least two blue light responses of Neurospora.     

Über die Wirkung von Kaliumjodid auf die lichtstimulierte endogene Atmung von Algen

Summary The endogenous respiration of an achlorophyllous mutant of Chlorella vulgaris is enhanced by small amounts of blue light. The action spectrum for this effect shows two peaks at 460 and 375 m, which points to a flavin or a carotenoid in cis configuration as the likely photoreceptor responsible. Weber's (1950) observation that in vitro potassium iodide (KI) quenches the fluorescence of riboflavin was employed to distinguish between these two pigments. — KI in concentrations from 0.033 to 0.50 M lowers the oxygen uptake in blue light increasingly (Fig. 1, Table 1), but not specifically: KI inhibits the respiration of exogenous glucose even more (Fig. 2). Furthermore neither the inhibition of endogenous nor that of exogenous respiration is iodide-specific; a decrease in both of them takes place with KNO₃ of corresponding concentrations, too (Table 2). The somewhat smaller inhibition with KNO₃ compared to that with KI fits the known observation that iodide has a greater inhibiting effect on metabolic reactions of plant cells than nitrate (Hewitt and Nicholas, 1963). — Finally it was observed that irradiation with blue light (>366 to <550 m) of the iodide solutions used liberates some iodine (Fig. 3). Since the promoting effect of iodine on the isomerisation of carotenoids is well documented (Zechmeister, 1962), it might be impossible to determine whether a flavin or a cis-carotenoid participates in a blue light specific reaction by using an iodide solution, as has been done several times recently.

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Herrn Prof. Dr. R. Harder zum 80. Geburtstag gewidmet.     

Effect of far-red and green irradiation on the nyctinastic closure of albizzia leaflets

The nyctinastic closing of Albizzia julibrissin pinnules is delayed by exposure to far-red radiation at 710 and 730 nanometers, with the former more effective than the latter. Far-red radiation at 750 and 770 nanometers has no effect on the process. Red light at 660 nanometers, which by itself has no effect, delayed closure when given before or simultaneously with far-red radiation at 750 or 770 nanometers. Low doses of green light, on the other hand, prevented all far-red radiations from delaying closure when given together with one of them. Effectiveness peaks at 550 nanometers. Green light by itself has no effect on the closing process.

From these and previous results, it is concluded that phytochrome is one of two photoreceptors in the process, that the other photoreceptor is an unknown pigment, and that the unknown photoreceptor requires some prior effect of the far-red-absorbing form of phytochrome before its action. Predictions are made of some of the properties of the unidentified pigment.

     

Euglena: The Photoreceptor System for Phototaxis*

SYNOPSIS. The photoreceptor structures (eyespot-paraflagellar body-flagellum) for Euglena phototaxis were investigated by electron microscopy. The paraflagellar body—the photoreceptor—is a highly ordered crystalline lamellar structure. Optical diffraction of the electron micrographs and resulting filtered images of the paraflagellar body suggest that it is formed of rods in a helical arrangement. The action spectra for phototaxis, the in situ spectrum by microspectrophotometry of the paraflagellar body, and flavin analysis of the organism indicate that the photoreceptor molecule is a flavoprotein. The phototaxis action spectrum is similar to the spectrum for O₂ evolution and implies that similar molecules participate in the photo-processes. As a result, a photochemical scheme is suggested in which a photo-excited flavin and a cytochrome participate in the photoprocess. The photochemistry and photoreceptor structures for Euglena phototaxis are likened to a photoneuro sensory cell.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants

Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.

The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway.

     

Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor.     

Untersuchungen Über Die Lichtabhängige Carotinoidsynthese

Zusammenfassung In der vorliegenden Arbeit wurde als erster Schritt zur Identifizierung des für die lichtabhängige Carotinoidsynthese verantwortlichen Acceptors ein genaues Wirkungsspektrum der Lichtinduktion bei Fusarium aquaeductuum aufgenommen.Als Voraussetzung dafür wurde nach den Bedingungen für eine optimale Ausnützung der Lichtinduktion zur Farbstoffbildung gesucht. Es konnte gezeigt werden, daß Glucosefütterung eine wahrscheinlich durch Abnahme der Carotinoidvorstufen bedingte Begrenzung der Pigmentsynthese verhindert. Die Menge der nach der Lichtinduktion gebildeten Carotinoide ist über einen Bereich von zwei Zehnerpotenzen vom Logarithmus der eingestrahlten Lichtmenge abhängig; dabei ist im ganzen untersuchten Bereich die Produktenregel gültig.Die Farbstoffbildung kann nur durch kurzwellige Strahlung unterhalb 520 nm induziert werden. Das Wirkungsspektrum zeigt zwei Maxima bei 375/380 nm und 450/455 nm, eine Schulter bei 430-440nm, sowie ein kleineres Maximum oder eine deutlich ausgeprägte Schulter zwischen 470 und 480 nm.Auf Grund der Ähnlichkeit des ermittelten Wirkungsspektrums mit den Absorptionsspektren von Flavoproteiden wird angenommen, daß der Lichtacceptor für die lichtabhängige Carotinoidsynthese sehr wahrscheinlich ein Flavoproteid ist; Versuchsergebnisse anderer Autoren an verschiedenen Organismen sprechen ebenfalls für diese Annahme. Das vorliegende Wirkungsspektrum ist mit dem des Phototropismus von Phycomyces-Sporangienträgern und von Avena-Koleoptilen praktisch identisch; es wird deshalb die Möglichkeit erörtert, ob nicht für alle Entwicklungs- und Bewegungsvorgänge, die durch kurzwellige Strahlung induziert werden, der gleiche Lichtacceptor verantwortlich sein könnte.

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Light-dependent carotenoid synthesisI. Action spectrum of photoinduction in Fusarium aquaeductuum

Summary As a first step towards the identification of the photoreceptor responsible for the light-dependent carotenoid synthesis, this paper presents an accurate action spectrum of photoinduction in Fusarium aquaeductuum.As a prerequisite for the determination of the spectrum the optimal conditions for the light-dependent synthesis of pigments were studied. Addition of glucose after illumination enhances the amount of pigment produced in the following darkness, indicating that the limiting factor for pigment formation may be a deficiency of carotenoid precursors. The amount of pigments produced depends on the logarithm of the incident light over a 100 fold range. The reciprocity law holds true over a wide range of time and light intensity.Carotenoid synthesis is induced only by light with wavelength shorter than 520 nm. The action spectrum has maxima at 375/380 nm and 450/455 nm, one shoulder at 430/440 nm and a further shoulder (or possibly a third maximum) between 470 and 480 nm.From this action spectrum carotenoids can be ruled out as possible photoreceptors. The spectrum resembles the absorption spectra of certain flavoproteins. It is therefore concluded that a flavoprotein is the acting photoreceptor; data of other investigators with different organisms also support this conclusion. The action spectrum presented also resembles the spectra of phototropism in Phycomyces-sporangiophores and Avena-coleoptiles; therefore, the possibility is discussed that the same photoreceptor might be acting in all cases in which development and movements are mediated by light of short wavelength.

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Meinem verehrten Lehrer, Herrn Prof. Dr. E. Bünning, zum 60. Geburtstag in Dankbarkeit gewidmet.