SIMULTANEOUS MEASUREMENT OF ACTION SPECTRA FOR PHOTOREACTIONS I AND II OF PHOTOSYNTHESIS

Abstract— We have devised a method of obtaining simultaneous action spectra for photoreactions I and II by analysis of direct and indirect effects involved in enhancement. The method requires previous determination of the neutral wavelength which gives maximum quantum yield by virtue of equal fractions of open reaction centers ( p and q ) for each photoreaction. A sufficient intensity of the neutral wavelength is used as a constant background. Upon addition of a weak modulated measuring light of intensity I_m and wavelength λ _m two amperometric signals are obtained for rate of oxygen evolution. A modulated signal (AC¯) isolates the direct effect of I_m and gives action of photoreaction II as AC/ I_m . An increment in total rate (ΔDC) also includes an indirect effect of I_m in perturbing reaction center conditions ( p and q ). From analysis of interaction of the two photoreactions, action for photoreaction I can be estimated as (2 ΔDC-AC)/ I_m . The method is applicable to whole cells, properly scales the two action spectra to each other, and removes contribution of the State 1-State 2 phenomena. Action spectra were obtained for Chlorella.     

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.     

ACTION SPECTRA FOR ULTRAVIOLET KILLING AND PHOTOREACTIVATION IN THE BLUE-GREEN ALGA AGMENELLUM QUADRUPLICATUM

Abstract— The action spectrum for photoreactivation has been determined in a coccoid blue-green alga, Agmenellum quadruplicatum. The spectrum is rather similar to that recorded for Streptomyces griseus conidia, with some suggestion of a little more structure. The action spectrum suggests possible carotenoid involvement; however, no other evidence for this could be found. The action spectrum for u.v. killing is also broad with some evidence of fine structure. The possible implication of tetrahydropteridines or c -phycocyanin as chromophores in the region 240–300 nm, along with DNA, is pointed out.     

EXCITATION AND FLUORESCENCE SPECTRA OF THE CHROMOPHORE ASSOCIATED WITH THE DNA-PHOTOREACTIVATING ENZYME FROM THE BLUE–GREEN ALGA ANACYSTIS NIDULANS

DNA-PHOTOREACTIVATING enzymes can be classified as deoxyribonucleate cyclobutane dipyrimidine photolyases*. Such an enzyme was recently purified 3760-fold from the blue-green alga Anacystis niduluns [8]. The absorption spectrum of the enzyme revealed a small peak at 418 nm that was attributed to an impurity. The enzyme has now been purified further, by affinity chromatography on far-ultraviolet (far-u.v.) irradiated DNA non-covalently bonded to cellulose, and its excitation and fluorescence spectra measured. These spectra reveal the presence of a non protein chromophore associated with the algal photolyase. The peak wavelengths in the excitation and absorption spectra in the visible region are almost identical and close to that observed in the in vitro photoreactivation action spectrum [8], observations supporting the view that this chromophore is involved as a cofactor in DNA photo reactivation.     

EXCITATION ENERGY TRANSFER IN THE LIGHT HARVESTING ANTENNA SYSTEM OF THE RED ALGA Porphyridium cruentum AND THE BLUE-GREEN ALGA Anacystis nidulans: ANALYSIS OF TIME-RESOLVED FLUORESCENCE SPECTRA

Abstract— Time-resolved fluorescence spectra of intact cells of red and blue-green algae Porphyridium cruentum and Anacystis nidulans were measured by means of a ps laser and a time-correlated photon counting system. Fluorescence spectra were observed successively from various pigments in the light harvesting system in the order of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and chlorophyll a (Chl a ). The spectrum changes with time in the range of0–400 ps in P. cruentum and of0–1000 ps in A. nidulans . The time-resolved spectra were analyzed into components to obtain the rise and decay curve of each fluorescence component. Overall time behaviors of the sequential fluorescence emissions from various pigments can be interpreted with a decay kinetics ofexp(–2 kt ^½). The rate constants of the energy transfer show that the energy transfer takes place much faster in the red alga P. cruentum than in the blue-green alga A. nidulans , particularly in the step PCAPC. Results also indicated that a special form of APC, far-emitting APC, exists in the pigment system of A. nidulans , but it does not mediate a main energy transfer from phycobilisome to Chl a.     

EVIDENCE FOR DARK REPAIR OF FAR ULTRAVIOLET LIGHT DAMAGE IN THE BLUE-GREEN ALGA, GLOEOCAPSA ALPICOLA

Abstract— The inactivating effect of far UV light on the unicellular blue-green alga Gloeocapsa alpicola could be totally reversed by exposure to blue light immediately after irradiation. However, if the irradiated cells were held in the dark before exposure to blue light, reversal became progressively less efficient and almost disappeared after 60–80 h holding. Caffeine and acriflavine inhibited loss of photoreversibility, suggesting an involvement of excision functions. Chloramphenicol and rifampicin slightly increased the rate of loss of photoreversibility, indicating that inducible functions play only a minor role. Split UV dose experiments indicated that light-dependent repair remained operational during dark liquid holding. These results provide preliminary evidence for dark repair in G. alpicola .     

THE ACTION SPECTRA FOR PHOTOREACTIVATION AND PHOTOREPAIR IN ICR 2A FROG CELLS

Abstract— Action spectra for photoreactivation (enhancement of colony forming ability) and photorepair (monomerization of pyrimidine dimers in DNA) were obtained for ICR 2A frog cells over the334–577 lira range. These spectra were very similar with peaks at 435 nm and little effectiveness at wavele.     

THE UV ACTION SPECTRA FOR THE CLONE-FORMING ABILITY OF CULTURED HUMAN MELANOCYTES AND KERATINOCYTES

Abstract Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone-forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22-1.92 for the UV-C and UV-R wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV-A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV-C and UV-B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV-A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV-A light.     

ACTION SPECTRA FOR THE IRIDOPHORE LIGHT RESPONSE IN THE NEON TETRA

Abstract The iridophores of some fishes such as the neon tetra ( Paracheirodon innesi ) are composed of alternating layers of guanine and cytoplasm. In the dark adapted state, the guanine layers are close together and reflect violet light. After illumination these layers separate and the iridophores reflect green light. The spacing change results from a direct action of light. Dose-response curves show that more than one light sensitive mechanism are involved, but that at threshold the action spectrum fits a 461 nm porphyropsin absorbance curve.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

ACTION SPECTRA FOR ACCUMULATION OF INORGANIC CARBON IN THE CYANOBACTERIUM, Anabaena variabilis

Abstract— Action spectra for accumulation of inorganic carbon were obtained for Anabaena variabilis , strainM–2, in the presence and absence of photosynthetic CO₂ fixation. The action spectrum for inorganic carbon accumulation in the presence of CO₂ fixation showed a peak around 684 nm, corresponding to chlorophyll a absorption in PS 1, while that for CO₂ fixation showed a peak around 630 nm, corresponding to phycocyanin absorption in PS 2. The action spectra obtained in the presence of iodoacetamide or diuron, which inhibit CO₂ fixation, showed two peaks, one at about 684 nm and the other at 630 nm, with the 630 nm peak height 80 to 90% of the 684 nm peak. These results indicate that inorganic carbon transport in A. variabilis can be driven with near equal efficiency by energy derived from absorption in photosystem 1 alone and with energy transferred to PS 1 after absorption by PS 2.     

ACTION SPECTRA FOR THE APPEARANCE OF DIFFERENCE ABSORPTION BANDS AT 480 AND 520 mμ IN ILLUMINATED CHLORELLA CELLS AND THEIR POSSIBLE SIGNIFICANCE TO A TWO-STEP MECHANISM OF PHOTOSYNTHESIS

Abstract— In the difference absorption spectrum of thin, actively growing* aerobic suspensions of Chlorella pyrenoidosa , both the 480 mμ (negative) and the 520 mμ (positive) bands are produced by light absorbed in chlorophyll b and chlorophyll a; the ratio of absorption changes caused by equal number of incident quanta of 650 mμ light and those of 680 mμ light, is about 1.2. Both effects are partially inhibited by DCMU. Upon replacing air with argon, the effects are increased several fold and become relatively insensitive to DCMU. The increase is stronger in the absorption region of chlorophyll a , than in that of chlorophyll b ; the ratio of the absorption changes, caused by equal numbers of 650 mμ and 680 mμ quanta decreases to about 0.8, for both effects. Variable (as regards the exact ratios of absorption changes), but parallel results for 480 and 520 mμ bands were obtained with cultures having low quantum yield of photosynthesis. This parallelism in the behavior of the 480 mμ and the 520 mμ band suggests that at least part of these two bands have a common origin. However, many observations suggest that both difference bands may have a multiple origin; as a working hypothesis, this origin is discussed in terms of three reactions: Reaction A—Photoreduction of chlorophyll a in system II; Reaction B—Photooxidation of chlorophyll b in system II; and Reaction C—Photooxidation (perhaps of a carotenoid) in system I.     

ELECTROCHROMIC BAND SHIFTS OF CAROTENOID IN A BLUE-GREEN ALGA

Abstract— The excitation with a short flash of cells of a blue-green alga, Synechococcus sp., induced, besides photooxidation of cytochrome c -553 and P-700, small absorption changes of complex kinetics in the wavelength region between 450 and 570 nm. The absorption changes were resolved into two kinetic components different in their sensitivity to gramicidin D.
The ionophore-sensitive component (Gs), which rose very rapidly on flash illumination and decayed with a half-time of 3 ms, has spectral features indicating a red shift of carotenoid absorption bands. Gs was sensitive to valonomycin but not to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation rate of Gs was markedly slowed down in the presence of tri- n -butyltin chloride. Phenazine methosulfate induced a secondary slow rise following the initial rapid rise. A similar slow rise appeared in the dark-starved cells but disappeared on the addition of methyl viologen. It is concluded from these results that Gs is an electrochromic band shift of carotenoid responding to the electric field formed by the primary charge separation of the photosystem I reaction center and its decay is related to the proton translocation through a proton channel of the membrane.
The ionophore-resistant component rose and decayed with the half-times of 0.2 and 2 ms, respectively. Its difference spectrum suggests a blue band shift of carotenoid. The ionophore-resistant component was also insensitive to DCMU. However, this component may be related in some way to flash-induced electron flow, because the photoresponse was altered by dibromothymoquinone, bathophenanthroline and 2- n -heptyl-hydroxyquinoline- N -oxide or the dark starvation of cells, which were all effective in inhibiting the cytochrome c -553 reduction.     

ON THE DISTRIBUTION OF EXCITATION ENERGY TO TWO PHOTOREACTIONS OF PHOTOSYNTHESIS

Abstract— From kinetic analysis of the Z-scheme we have derived expressions for fractions of open reaction centers and throughput quantum yield. The fraction of absorbed quanta available to photoreaction II has been treated by the wavelength dependent function, α. However, for purposes of analysis in terms of throughput electron flow it is necessary to introduce a related function, α', for actual processing of excitations by photoreaction II; α' differs from α if the individual quantum yields of the two photoreactions are not equal. 'Included in our findings are the following. As a close approximation quantum yield is maximum at α'= 0.5 and a symmetrical function of α' around the line α' =0.5. As previously noted, lowering of the apparent equilibrium constant between the photoreactions flattens dependence of quantum yield on α' near α' = 0.5 but also lowers the attainable quantum yield. For any given equilibrium constant the effective fractions of open reaction centers are equal at α' = 0.5 and mirror images of each other around the line α' = 0.5. Slow changes in quantum yield following changes in wavelength (the State 1-State 2 phenomenon) are explainable in terms of changes in α' which also have the effect of flattening dependence of quantum yield on wavelength. We have used these changes to estimate the neutral wavelength for α' = 0.5 as 681–682 nm in Chlorella .     

ACTION SPECTRA FOR LETHALITY IN RECOMBINATION-LESS STRAINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI*

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near-UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l -tryptophan toxic for recA mutants (now known to be H₂O₂) suggest that H₂O₂ photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near-UV radiation.     

STATE I-STATE II TRANSITIONS IN THE THERMOPHILIC BLUE-GREEN ALGA (CYANOBACTERIUM) Synechococcus lividus*

Time courses of state I-state II transitions were measured in the thermophilic blue-green alga (Cyanobacterium), Synechococcus lividus, that was grown at 55°C. The rate of the state I–II transition using light II illumination was the same as that in the dark, and the dark state was identified to be state II. Therefore, light regulation attained by state transitions is produced by the state II–I transition induced by system I light. The redox level of plastoquinone did not affect this dark state II. Arrhenius plots of the state transitions showed a break point around 43°C that corresponded to the phase transition temperature of this alga. Since both the state I–II and II–I transitions were very much temperature-independent, we could keep the alga in either state for a long time at a “low” temperature such as room temperature. Activities of both photosystems I and II in states I and II were also measured. After a state II–I transition, the system II activity increased about 16% and at the same time, svstem I activity decreased about 30%.     

ACTION SPECTRA FOR HEMATOPORPHYRIN DERIVATIVE AND PHOTOFRIN II WITH RESPECT TO SENSITIZATION OF HUMAN CELLS IN VITRO TO PHOTOINACTIVATION

Abstract— Exposure of Raji cells to haematoporphyrin derivative (HPD) and red light caused marked cytotoxicity. This was completely inhibited under anaerobic conditions. By using sodium dithionite in aqueous solutions, precise and graded oxygen concentrations could be achieved. Cytotoxicity was directly proportional to the oxygen concentration of the medium until a maximum was reached at a pO₂ of 90 mm Hg. Sodium dithionite did not affect the viability of test cells and did not alter the chromatographic profile of HPD. Dithionite did not interfere with the uptake of HPD by cells. Dependency of phototoxicity upon aerobic conditions suggests that the cytotoxic agent is derived from oxygen and is consistent with the hypothesis that singlet oxygen and/or oxygen-derived free radicals play an important role in photochemotherapy with HPD.     

ACTION SPECTRA FOR SUPEROXIDE GENERATION AND UV AND VISIBLE LIGHT PHOTOAVOIDANCE IN PLASMODIA OF Physarum polycephalum

Abstract— The initial photochemical process leading to photoavoidance by plasmodia of an albino strain of Physarum Plasmodium was studied. Superoxide (O), detected as superoxide dismutase (SOD)-inhibitable electron spin resonance (ESR) signal of a spin trap (tBN), was formed upon irradiation. The amount of O formed increased linearl) with log fluence rate above the threshold. The photoavoidance to radiation at wavelengths between200–800 nm also showed the similar linear relationship in log fluence rate-response curves. Thresholds for photoavoidance and O generation agreed with each other and the action spectra showed peaks at about 260, 370, and 460 nm. Thus, active oxygen generated by photosensitization seems to trigger the UV and blue light photoavoidance.