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.     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

ANALYSIS OF MULTIPLE PHOTORECEPTOR PIGMENTS FOR PHOTOTROPISM IN A MUTANT OF Arabidopsis thaliana

Abstract
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 μmol m^-2s^-1), the response to 450-nm light is characterized by a single maximum at about 9 μmol m^-2. At higher fluence rate (0.4 μmol m^-2s^-1), the response shows two maxima, at 4.5 and 9 μmol m^-2. The response to 510-nm light shows a single maximum at 4.5 μmol m^-2. Unilateral preirradiation with high fluence rate (25 μmol m^-2s^-1) 510-nm light eliminates the maximum at 4.5 μmol m^-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 μmol 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.     

REC A +-DEPENDENT SYNERGISM BETWEEN 365 NM AND IONIZING RADIATION IN LOG-PHASE ESCHERICHIA COLI: A MODEL FOR OXYGEN-DEPENDENT NEAR-UV INACTIVATION BY DISRUPTION OF DNA REPAIR

Abstract —The oxygen dependence of 365 nm inactivation of colony-forming ability of Escherichia coli has been investigated in two series of DNA repair-deficient K12 mutants grown to mid-exponential phase. All strains except a uvr A rec A double mutant are more sensitive to inactivation under O₂ and show a lower threshold dose. The inactivation of photoreactivating enzyme in a crude cell extract and DNA repair disruption are both reduced when irradiation is carried out under nitrogen. The rec A gene-dependent synergism between 365 nm and ionising radiation is reversible if cells are incubated in full growth medium before ionising radiation treatment. In a wildtype strain, incubation for 2.5 h in full growth medium after 10⁶ J m^-2 365 nm radiation changes a sensitised response to a protection from ionising radiation. Protection is not seen at 1.5 times 10⁶ J m^-2. A tentative model for near UV lethality in logarithmic phase cells is suggested which proposes two classes of lesions. One requires oxygen for it's induction, is rapidly fixed as a lethal event as a result of repair disruption, and is primarily responsible for cell death after aerobic 365 nm irradiation. The other lesion, possibly pyrimidine dimers, may lead to cell death under anaerobic conditions.     

PHOTOLYSIS OF PHOSPHODIESTER BONDS IN PLASMID DNA BY HIGH INTENSITY UV LASER IRRADIATION

Abstract— The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 times 10^-5 until a maximum value of 4.5 times 10^-4 is attained at intensities of 10¹¹ W m^-2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m^-2). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed.     

ANTAGONISTIC EFFECTS OF RED AND FAR-RED LIGHTS ON THE STABILITY OF PHOTOSYSTEM II IN PEA LEAVES EXPOSED TO HEAT

Abstract— The presence of light during exposure of intact pea leaves to high temperature (40°C) protects Photosystem II (PSII) against inactivation, as indicated by the preservation of the maximal variable 685 nm chlorophyll fluorescence and the photosynthetic oxygen evolution. This photoprotection was observed (i) to be saturated at low fluence rates ( ca 10 W m^-2) and (ii) to be strongly dependent on the spectral characteristics of the light. It was specifically induced by red light (630–670 nm) whereas other wavelengths were much less protective. A strong antagonism between red and far-red lights was also observed, with PSII stabilization by red light being partially cancelled by additional far-red light.     

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.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

COMPARATIVE ANALYSES OF ACTION SPECTRA OF GLYCOLATE EXCRETION ("PHOTORESPIRATION") AND PHOTOSYNTHESIS IN THE BLUE-GREEN ALGA AN ACYSTIS NIDULANS

Abstract. The action spectra were determined by measuring photosynthetic H¹⁴CO^-₃-fixation and ¹⁴C-glycolate excretion to the medium during 15 min exposure to light at 15 different wavelengths in the visible region using interference filters and a 2500 W high pressure Xe lamp at a constant photon flux of about 1.51 × 10¹⁹ quanta m^-2.s^-1 at all wavelengths.
When plotted on relative scales the action spectrum of glycolate excretion lies below that of photosynthesis at all wavelengths shorter than 517 nm. As glycolate excretion had an exponential relationship to photosynthetic rates, different methods were used to analyze for a specific blue light effect which demonstrated that the relative amount of glycolate excretion was depressed by blue light compared with that by green and red. The greatest difference was observed around 460–480 nm. However, on statistical grounds it is not permitted to draw a difference spectrum which might indicate the absorption characteristics of pigment(s) involved.
A hypothesis is discussed assuming that some glycolate is consumed in an oxidation process for supply of electrons to Photosystem I when Photosystem II is poorly excited in the blue region of the spectrum, which was the case for Anacystis used in the present investigation.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

RESPONSE OF THE WILD-TYPE and HIGH LIGHT-TOLERANT MUTANT OF Anacystis nidulans AGAINST PHOTOOXIDATIVE DAMAGE: DIFFERENTIAL MECHANISM OF HIGH LIGHT TOLERANCE

Abstract— A high light-tolerant mutant of Anacystis was able to tolerate about three-fold higher light energy irradiance (30 W m^-2) than the wild type (10 W m^-2). The loss of sulfhydryl content and rate of lipid peroxidation in the wild-type cells is lower than in the mutant cells at high light irradiance. This phenomenon in the wild type is probably due to high light-induced severe photoinhibitory conditions resulting in a decreased rate of O₂ evolution. Results on the bleaching of the N, N '-dimethyl- p -nitrosoaniline at high light irradiance show a higher rate of bleaching in the wild-type than in the mutant cells. Further, results on the rate of N, N '-dimethyl- p -nitrosoani)ine bleaching in the presence of radical scavengers like sodium azide, histidine and sodium formate (10 m M , each) suggest that singlet oxygen is the predominant oxygen species produced in both the wild-type and mutant cells under high light. However, a similar quenching effect of formate in the mutant cells is indicative of increased formation of hydroxyl radicals. This observation is further corroborated by higher rate of lipid peroxidation. In addition to this, the superoxide dismutase activity is higher in the mutant (1.2 unit) than in the wild type. Taken together, these results suggest that the cells of the high light-tolerant mutant have an efficient intracellular mechanism to transform the free oxygen radicals.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

THE EFFECT OF CYTOKININS ON GROWTH and PIGMENT ACCUMULATION OF RADISH SEEDLINGS (RAPHANUS SATIVUS L.) GROWN IN THE DARK and AT DIFFERENT LIGHT QUANTA FLUENCE RATES*

Abstract— The dependency of cytokinin effects upon irradiance was studied with radish seedlings ( Raphanus sativus L. cv. Saxa Treib). Kinetin (6-furfurylamino-purine) or BAP (6-benzylamino-purine) were applied via the roots of plants growing either in continuous darkness or under high (90 Wm^-2) or low intensity white light (10Wm^-2). Apart from the different development of plants at low and high fluence rates, the following cytokinin effects were found:
(1) Both cytokinins acted in a similar manner on growth characteristics and pigment accumulation at high and low light conditions, BAP being in many cases more effective than kinetin.
(2) When compared with the control, the cytokinins suppressed hypocotyl and root lengthening in the dark and light-grown plants. In darkness they led to increased cotyledon areas, whereas in the light the leaf expansion was suppressed.
(3) In the etiolated and low light grown plants, the anthocyanin content of the hypocotyls was enhanced due to the action of cytokinins, whereas under high light the anthocyanin accumulation was decreased.
(4) In the cotyledons of etiolated plants, more phototransformable protochlorophyll(ide) and more carotenoids were formed when cytokinins were present. In green leaves the carotenoid content was diminished due to the action of cytokinins, particularly in plants grown in strong light. The chlorophyll a/b ratio was increased in the cytokinin-treated plants in most cases.
The results suggest a light dependency of the cytokinin effects. It is believed that the response of a plant towards exogenously applied cytokinins is similar to that with high intensity light.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

PHOTOBIOLOGICAL ACTIVITY OF MARMESIN (5-B-HYDROXYISOPROPYL-4–5 DIHYDROFUROCOUMARIN) IN CHINESE HAMSTER V79 CELLS

Abstract— Marmesin was isolated from the medicinal plant, Afraegle paniculata. Its cytotoxicity and mutagenicity in Chinese hamster V79 cells when sensitized to near ultraviolet (NUV) and long wavelength ultraviolet light or black light (BL) were assayed.
Marmesin was extremely cytotoxic in the dark. This cytotoxicity was photoenhanced in NUV and BL; the photoenhanced lethality being higher in NUV than in BL. The LD₅₀ of marmesin under NUV and BL photosensitization were 0.002 μ M and (0.012 μ M ), respectively. In the absence of NUV and BL, marmesin's LD₅₀ was 0.013 μ M . NUV and BL without marmesin were not significantly cytotoxic at the fluence rates of 0.29 W/m² and 4.2 W/m², respectively, for up to 20 min. In contrast to the observed high cytotoxicity of marmesin, its mutagenicity at the HGPRT locus (A_zG^r) was weak. The implication of this result in the high incidence of skin cancer in Nigeria in which A. paniculata is used as a medicinal plant is discussed.     

A PULSED LASER SYSTEM FOR THE STUDY OF MICRO-SECOND DELAYED FLUORESCENCE FROM PLANTS

Abstract—A new laser system is designed for the investigation of S delayed fluorescence. This is intended to overcome some undesirable features present in other current designs, such as low actinic light fluxes and long illumination times, or slow responses owing to long turn-off times of the stimulation source and the presence of fluorescence artifacts.
The system uses a pulsed argon-ion laser and bears little resemblance to either the modified Becquerel phosphoroscope used by Lavorel (1971) or to those systems requiring shutters. Cheapness of design together with the ability to adjust such parameters as pulse duration easily, make this design attractive to most laboratories in which short (10 μs) high intensity (10⁵ W m^-2) pulses with rapid (1.5 μs) turn-off times and high on-to-off contrast ratios (10⁶:1) are required.
Certain design criteria are strictly imposed in order to produce kinetic data that can be meaningfully analysed.