ANALYSIS OF THE BINDING OF PHYTOCHROME TO PARTICULATE FRACTIONS

Abstract— The binding of phytochrome to receptor sites in a particulate fraction of maize coleoptiles has been studied as a function of the level of far-red-absorbing phytochrome (P_fr) offered in vivo and in vitro. Evidence is presented that the binding is cooperative. The degree of cooperativity expressed by the Hill coefficient of the binding function is the same (1–6) both in vivo and in vitro , whereas the Hill coefficient of the state function in vivo is significantly higher (2-1). The highest Hill coefficient (3–5) was found for the in vitro binding function in squash hooks.     

AN ACTION SPECTRUM FOR PHOTOTAXIS BY PSEUDOPLASMODIA OF Dictyostelium discoideum

Abstract— The sensitivity of phototactic orientation of pseudoplasmodia (slugs) of the cellular slime mold Dictyosrelium discoideum has been measured for white light and monochromatic light using computer aided directional statistics. The zero threshold for white light was found at about 10^-5 Ix. An action spectrum for positive phototaxis has been calculated from fluence rate-response curves; it shows two major maxima at about 420 and 440 nm and secondary peaks at 560 and 610 nm. This action spectrum is significantly different from the one for phototactic orientation in Dictyostelium amoebae.     

AN ACTION SPECTRUM OF PHOTOREACTIVATING ENZYME FROM SEA URCHIN EGGS

An action spectrum for photoreactivating enzyme activity from unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus was determined. The range of the spectrum is 313 to 500 nm, with a maximum at 365 nm. Comparison of this spectrum with that for photorecovery of developmental damage in the sea urchin embryo indicates the general similarity of the two processes.     

THE ACTION SPECTRUM OF AN IN VITRO DNA PHOTOREACTIVATION SYSTEM

Abstract— The wavelength-dependence of in vitro photoreactivation of transforming DNA by yeast extract has been determined. There is an intensity-dependent lag at the beginning of the biological reaction. There is a similar lag in the splitting of thymine dimers by the yeast extract in the light, a process known to account for most or all of the increase in transforming activity of photoreactivated DNA. The most efficient wavelengths for photoreactivation are around 3550 and 3850 Å. Although the action spectrum is not very similar to flavin absorption, riboflavin at very low concentration inhibits photoreactivation, as it also inhibits a number of flavoenzymes, suggesting that the photoreactivating enzyme might be a flavoprotein.     

PHYTOCHROME ACTION: A REAPPRAISAL

Stems of fully green plants show at least two types of response to light. In one, P_fr inhibits elongation. The second is a promotion of elongation which operates only in light; the effectiveness of red and far-red wavelengths indicates that this response is also mediated through phytochrome. Studies of the de-etiolation process also provide evidence for two modes of action of phytochrome; one is a P_fr-dependent reaction, and the second requires continuous light (or frequent short irradiations). It is proposed that, in addition to reactions which require P_fr and proceed in darkness, an important reaction of phytochrome in green plants occurs only in light. We have termed these two modes of action of phytochrome “static” and “dynamic”. The static mode operates after a brief exposure to light which establishes P_fr; the potential responses are largely reversible by far-red and exhibit reciprocity. The dynamic mode operates only in light and the responses do not show reciprocity. We consider that this mode operates through the transition from one bound form of phytochrome to another. The possible involvement of these two modes of action of phytochrome in photoperiodic mechanisms is discussed.     

RED/FAR-RED PHOTOREVERSIBILITY: NOT AN APPROPRIATE PHYTOCHROME ASSAY FOR RED-LIGHT PREIRRADIATED CORN COLEOPTILES

Abstract— Based on measurements with a single beam spectrophotometer, it has been found that subsequent red/far red irradiation cycles, which are usually given to monitor phytochrome content by dual wavelength spectroscopy, induce chlorophyll-related absorption changes in maize coleoptiles. Therefore, the difference signal, usually measured between 730 and 800 nm or 660 and 730 nm after saturating red and far red irradiations, does not represent solely the phytochrome content of preirradiated samples.     

ACTION SPECTRA OF PHYTOCHROME IN VIVO*

Abstract— A method is described to determine spectral properties of phytochrome in vivo. For photochrome in 7-day-old dark-grown Cucurbita pepo L. seedlings the mole fraction of the far-red-absorbing form (P_fr) present at photoequilibrium at 664 nm was found to be 0.76 ± 0.02 in vivo. Based on reflectance measurements, the photon fluence rate just below the surface of the cotyledons was calculated. Local rates of photoconversion for known local fluence rates were measured across cotyledons after non-saturating irradiations with wavelengths between 544 and 781 nm and in situ molar photoconversion coefficients were obtained. In contrast to purified oat phytochrome, the in situ molar photoconversion coefficients for P_fr show a strong shoulder between 660 and 700 nm. The maximum of P_fr absorption is at 726 nm. An isosbestic point of phytochrome is found at 686 nm. The mole fraction of P_fr present at photoequilibrium with 686 nm light is 0.58. The ratio of photoconversion quantum yields (that for P_r→ P_fr divided by that for P_fr→ P_r) is 1.38 ± 0.06.     

DIFFERENTIAL GENE ACTIVATION AS A MODE OF ACTION OF PHYTOCHROME 730

Abstract— Phytochrome-induced photomorphogenesis in the mustard seedling ( Sinapis alba L.) which can be regarded as being representative of the dicotyledonous seedlings has been analysed. In the present paper a number of arguments are presented, including data on RNA and protein synthesis and on the effects of actinomycin D and puromycin, which support the hypothesis that the 'positive' photoresponses of the seedling can be explained by a differential gene activation through P,₃₀. 'Positive' photoresponses are those which are characterized by an initiation or an increase of biosynthetic or growth processes (e.g. biosynthesis of anthocyanin; growth of cotyledons). The lag-phase of this type of photoresponse is rather long, 'Negative' photoresponses are those which are characterized by an inhibition of growth processes or other physiological processes like translocation. Here the lag-phase is short. Inhibition of hypocotyl lengthening is a typical response of this sort. The concept of differential gene repression through P ₇₃₀ may serve as a working hypothesis to approach the causal analysis of phytochrome-induced 'negative' photoresponses.     

AN INSTRUMENT FOR ACTION SPECTRUM STUDIES IN DERMATOLOGY

Abstract— An instrument designed for convenient determination of action spectra for cutaneous photo-responses in man and experimental animals is described. Light from 450 W Xe lamp is dispersed by a concave holographic grating. The spectrum from 244 to 616 nm is projected as a planar strip (2 times 17 cm) intercepted by a grid with 31 ports. The bandwidth at each port is 12 nm and the size of the port increases from about 4 × 4 mm to 6 × 8 mm from the low to high wavelength limits, respectively. Typical fluence rates in quanta m^-2 s^-1 are 4.0 times 10¹⁹ at 298 nm, 16 times 10¹⁹ at 394nm and 22 times 10¹⁹ at 538 nm. Responses due to delayed erythema in normal skin and to musk ambrette photoallergy and solar urticaria in patients skin have been elicited with this instrument.     

A TWOFOLD ACTION OF PHYTOCHROME IN CONTROLLING CHLOROPHYLL a ACCUMULATION

Abstract— A pretreatment with light prior to continuous illumination with high intensity white light eliminates the lag phase in chlorophyll a accumulation and increases the steady-state rate of chlorophyll a accumulation. In mustard seedlings ( Sinapis alba L.) the effect of a pretreatment can be fully attributed to phytochrome. The effect of phytochrome on chlorophyll a accumulation is twofold. It is possible to separate the effect on the lag phase from the effect on the steady-state rate of accumulation. While the effect on the lag phase is a relatively fast process (occurring within less than 3 h) the effect on the rate requires a considerable period of time (at least 12 h) to become manifest.     

ULTRAVIOLET INACTIVATION OF THE ABILITY OF E. COLI TO SUPPORT THE GROWTH OF PHAGE T7: AN ACTION SPECTRUM

Abstract— Ultraviolet (UV)-irradiated E. coli K-12 wild-type cells were sensitized by a post-irradiation treatment with 10^-2 M 2, 4-dinitrophenol (DNP). This effect was not seen in strains carrying a uvr mutation, suggesting that DN P interferes with the excision repair process. The polA strain was sensitized to the same extent as the wild-type strain, while the exrA strain was not affected by DNP treatment.
Recombination deficient strains ( recA, recB and recA recB ) were protected by DNP treatment after UV irradiation. This protection was abolished by the addition of a uvr mutation (i.e., in strains recA uvrB and recB uvrB ).
Alkaline sucrose gradient sedimentation studies showed that DNP treatment interfered with the rejoining of DNA single-strand breaks induced by the excision repair process. This interference was apparently specific for the exr gene-dependent branch of the uvr gene-dependent excision repair process, since the uvr and exr strains were not sensitized while the wild-type and polA strains were sensitized.     

MULTIPLICATIVE ACTION OF A UV-B PHOTORECEPTOR and PHYTOCHROME IN ANTHOCYANIN SYNTHESIS

Using 290-nm light, which excites only a UV-B photoreceptor, and 385- and 660-nm light, which activate only phytochrome, the fluence rate-response curves of monochromatic irradiations for anthocyanin synthesis in the first internodes of broom sorghum (Sorghum bicolor Moench, cv. Acme Broomcorn) were analyzed. Although the two photoreceptors absorbed light independently, they multiplicatively increased the action of each other. Accordingly, when the fluence rates of both wavelengths were changed together, the resulting slopes of the fluence rate-response curves of double-log plots were steep compared with the slopes obtained with the respective monochromatic irradiations. The slopes of fluence rate-response curves for monochromatic irradiations at 325 to 345 nm were steeper than those at other wavelengths. This difference was shown to be due to the multiplicative actions of both photoreceptors.     

AN ULTRAVIOLET RADIATION ACTION SPECTRUM FOR IMMEDIATE PIGMENT DARKENING

The wavelength dependence for immediate pigment darkening (IPD) was investigated by exposing the midback skin of volunteers to a series of incremental fluences of narrow waveband radiation isolated by band-pass filters in the310–400 nm region. The threshold IPD fluence for each waveband was determined by visual assessment of the skin responses immediately after each exposure. The action spectrum, constructed from the mean threshold fluences, was broad and extended from 320 nm to 400 nm with a peak at around 340 nm. No IPD could be evoked at 310 nm, even after erythemogenic fluences. The spectrum was similar in each of the three skin types investigated (III, IV, V). The broad nature of the action spectrum within the UVA region suggests that IPD may serve as an alternative endpoint for measuring photoprotection against these wavelengths.     

AN ACTION SPECTRUM FOR LIGHT AVOIDANCE BY PHYSARUM NUDUM PLASMODIA

Abstract— Plasmodia of the myxomycete Physarum nudum , grown on agar in darkness, avoided a lighted field. The percentage of plasmodia migrating out at the lighted field was found to be dependent on light intensity. An action spectrum for this response has two maxima at 452 and 375 nm.     

ANALYSIS OF PHOTOTRANSFORMATION INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF AVENA PHYTOCHROME BY A MULTICHANNEL TRANSIENT SPECTRUM ANALYZER

Phototransformation of the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form (Pfr) was followed with a custom-built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000-dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115-mJ, 600-ns half-width, 655-nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15-20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half-life of 3 s at 25°C and a Q₁₀ of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1-25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ-mol^-1, 75kJ.mol^-1, and -220 J.mol^-1 K^-1, respectively, and for conventionally purified phytochrome 25kJ.mol^-1, 75kJ.mol^-1and —170 J.mol^-1 K^-1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.     

ACTION SPECTRUM FOR ANTHRACENE-INDUCED PHOTOSENSITIZATION OF HUMAN SKIN

Abstract— The action spectrum for photosensitization by topically applied anthracene was determined in human volunteers. Spectral reactivity was demonstrated in the range between 320 and 380 nm, with peak activity at around 360 nm. Three distinct inflammatory responses viz. immediate transient erythema, delayed erythema, and wealing were evoked following exposure to effective wavelengths. The action spectra for these responses were similar but the threshold doses were different. Prior treatment with a mast cell degranulating agent (codeine) abolished anthracene-UVA induced wealing but did not influence the erythema response. These findings suggest that photosensitized damage to cutaneous mast cells may be partially responsible for some of the observed inflammatory responses, but other sites of photochemical injury are also involved.     

SYNERGISTIC LETHALITY OF PHAGE T7 BY NEAR-UV RADIATION AND HYDROGEN PEROXIDE: AN ACTION SPECTRUM

Abstract— Ozonation of valerophenone oxime o-methyl ether (4) produced a stereoisomeric mixture of crystalline dimeric valerophenone peroxides, 5a and 5b . N-n-butyl-N-methoxybenzamide (6) and N-methoxy-N-phenylvaleramide (7) along with valerophenone (1). Thermolysis of the higher melting peroxide 5a at 170–180°C, where a chemiluminescence was visible from added perylene. gave 1 and butyl benzoate (8) in addition to small amounts of the Norrish Type 11 products of 1. i.e. acetophenone (2) and cis- and trans- 2-methyl-1-phenylcyclobutanols. Biacetyl-sensitized photolysis of 5a in benzene yielded 2 in much higher yield in addition to 1, 8, and biphenyl. These results suggest that the triplet excited state of 1 is formed by the decomposition of 1 in low yield in thermolysis and in much higher yield in sensitized photolysis. although some of the Type II products may not arise from the triplet valerophenone.     

A PORPHYROPSIN-LIKE ACTION SPECTRUM FROM Xenopus MELANOPHORES

Light causes an aggregation of the melanosomes in the melanophores of isolated dark adapted Xenopus tadpole tails. Dose response curves for 6 wavelengths of light were measured, and using a standard response, an action spectrum was derived that can be fitted to a porphyropsin absorbance curve.     

AN ACTION SPECTRUM FOR ULTRAVIOLET-INDUCED SPORULATION IN THE FUNGUS STEMPHYLIUM SOLANI WEBER

Abstract— An action spectrum for conidial (spore) production in the fungus Stemphylium solani Weber is presented. The response is in the far-ultraviolet region (far u.v.). The spectrum shows a single peak at 280 nm, a shoulder at 260 nm, a trough at 250 nm, followed by a rise which is still rising at 230 nm, the shortest wavelength studied. The u.v. induced spore stimulation can be interrupted temporarily by irradiation in the visible region within 4 hr after induction.