ACTION SPECTRA FOR PHOTOREACTIONS I AND II OF PHOTOSYNTHESIS IN THE BLUE-GREEN ALGA ANACYSTIS NIDULANS

Abstract— Action spectra for photoreactions I and II of photosynthesis were obtained for Anacystis nidulans and three of its variants which had altered chlorophyll/phycocyanin ratios. The spectra are properly scaled to each other. They provide information on contributions of phycocyanin and chlorophyll to initial absorption and final distribution of excitation energy to reaction centers I and II. In normally pigmented cells the light harvesting pigments for photoreaction I include about 40% of the phycocyanin and 84% of the chlorophyll. Both in normal cells and in cells with altered pigmentation excitation energy from phycocyanin is delivered to photoreaction II via a small number of chlorophylls. In response to alterations in chlorophyll/phycocyanin ratio Action I spectra showed large variations whereas Action II spectra were essentially invariant. The result is taken to mean that alteration in chlorophyll components in Anacystis is attended by a special restriction: there are only small changes in amount of chlorophyll accessible to photoreaction II in the face of large changes in amount committed to photoreaction I.     

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 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.     

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.     

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.     

ACTION SPECTRA FOR UV INDUCTION AND PHOTOREVERSAL OF A SWITCH IN THE DEVELOPMENTAL PROGRAM OF THE EGG OF AN INSECT (SMITTIA)*

Abstract— An action spectrum was established for the induction by ultraviolet (UV) radiation of the aberrant body segment pattern 'double abdomen' in the egg of the Chironomid midge Smittia. The action spectrum shows a peak at 285 nm wavelength, a shoulder at 265 nm, a slight increase from 245 to 240 nm, and a steep decline towards 300 nm. Corrections for wavelength-dependent shielding within the egg result in transformation of the shoulder at 265 nm into a minor peak. These results are compatible with the assumption that a nucleic acid-protein complex is involved in the initial photoreaction. This assumption is supported by the fact that the UV induction of the aberrant body segment pattern 'double abdomen' in the egg of Smittia is photoreversible. Wavelengths effective in this photoreversal range from 310 to 460 nm, with a peak at 440 nm. These spectral characteristics agree with action spectra for photoreactivation in other systems. Indirect photoreactivation does not occur in the egg of Smittia under the conditions of the experiments. Photoreversal with 440 nm radiation at high dose rate is temperature-dependent. The results support the assumption that the molecular basis for photoreversal in the egg of Smittia is similar to the predominant molecular mechanism of direct photoreactivation in other systems. Targets possibly involved in the UV induction of the 'double abdomen' are discussed.     

PHOTOTACTIC AND PHOTOKINETIC ACTION SPECTRA OF THE DIATOM NITZSCHIA COMMUNIS

Abstract— Three different types of photomotion reactions, photokinesis, photo-topotaxis and photo-phobotaxis, have been investigated in the diatom Nitzschia communis . In photokinesis the active light appears to be absorbed by the photosynthetic pigments. Apparently, the acceleration of movement by light is due to an additional ATP supply from photosynthetic phosphorylation, as it is the case in the blue-green algae of the genus Phormidium . On the other hand, no correlations exist between photo-phobotaxis and photosynthesis, because red light does not induce photo-phobotactic responses in N. communis . The phobotactic action spectrum resembles that one of photo-topotaxis. In the visible region only violet, blue and blue-green light is active in both the reactions. However, it is not yet clear whether or not the stimuli are mediated by the same photoreceptor, mainly because of the different sensitivity of both the reactions to u.v.     

AUTOMATIC RECORDING OF ACTION SPECTRA OF PHOTOBIOLOGICAL PROCESSES, SPECTROPHOTOMETRIC ANALYSES, FLUORESCENCE MEASUREMENTS AND RECORDING OF THE FIRST DERIVATIVE OF THE ABSORPTION CURVE IN ONE SIMPLE UNIT

Abstract— A compact, rugged and simply constructed instrument has been designed which measures action spectra of photosynthesis by delivering equal numbers of quanta between 400 and 720 nm to a sample placed upon the cathode of an oxygen sensor. The absorption spectrum is measured delivering equal numbers of quanta over the spectrum to a sample placed directly above a photoreceiver that has been adjusted in wavelength sensitivity to function as a quantum counter. All the details that are recorded by conventional high precision spectro-photometers for samples with relatively broad absorption bands (e.g. chlorophyll, carotenoids, phycobilins and living material such as algal suspensions) are resolved by the instrument. Corrected excitation spectra of fluorescence are obtained with the same instrument. Likewise the first derivative of the absorption curve may be scanned. Such recordings amplify details in the absorption spectrum, and they are particularly useful in analyses of small changes in slope. The functions described above may be operated separately or combined. The instrument has a total weight of about 10 kg, and the dimensions are ca . 60 × 40 × 30 cm. It is constructed for use in field laboratories and on board research vessels, and also for courses in biology where the principles behind photobiological analyses are illustrated. It is possible that a device of this type could be used for investigations of photosynthesis on other planets.     

PHOTODYNAMIC ACTION OF MEROCYANINE 540 IN ARTIFICIAL BILAYERS AND NATURAL MEMBRANES: ACTION SPECTRA AND QUANTUM YIELDS

The action spectra and quantum yields for singlet oxygen (1O2) generation by merocyanine 540 (MC540) in liposomes and isolated erythrocyte membranes were obtained using electron spin resonance techniques. Oxygen consumption was measured by spin label oximetry in the presence of histidine for fully-saturated dimyristoylphosphatidylcholine vesicles, mono-unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine vesicles and erythrocyte membranes. The quantum yield for the photogeneration of 1O2 by membrane-bound MC540 in aqueous buffer was determined to be 0.065 +/- 0.005, which is approx. 1/10 of the value determined for Rose Bengal under similar conditions. Using unilamellar liposomes and isolated erythrocyte membranes containing MC540 at different monomer/dimer ratios, we have observed that the action spectra of 1O2 generation closely overlap the absorption spectra of the monomeric dye in these systems. It is likely that factors which affect the monomer-dimer equilibrium of MC540 will influence the production of 1O2. These findings have important implications for the phototherapeutic efficacy of MC540.     

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.     

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 .     

ACTION SPECTRA OF CATION EFFECTS ON THE FLUORESCENCE POLARIZATION AND INTENSITY IN THYLAKOIDS AT ROOM TEMPERATURE

Abstract— The degree of polarization of chlorophyll- a (Chl- a ) fluorescence is known to monitor the extent of excitation migration and/or the orientation of the photosynthetic pigment molecules. We report here the effects of cations, at room temperature, on the degree of polarization of Chl- a fluorescence, and fluorescence intensity in thylakoids as a function of excitation wavelength. Observations of maxima at 650 and 675 nm in the cation-induced changes in the excitation spectrum for fluorescence at 730 and 762 nm, and, in the action spectra for the depolarization of fluorescence lead us to suggest that the regulation of the initial distribution of excitation to photosystem II involves the better coupling of Chl- b and- a in the light harvesting complex with Chl- a in the reaction center II complex.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

ACTION SPECTRA OF THE PHOTOPHOBIC RESPONSE OF BLUE AND RED FORMS OF Blepharisma japonicum

Abstract— When exposed, in the presence of molecular oxygen, to light intensities of the order of3–30 W m^-2, the ciliate Blepharisma japonicum changes its color from red to blue, because of the photooxidation of the photoreceptor pigment, blepharismin, to pxyblepharismin. Both red-and blue-pigmnentes cells show step-up photophobic responses. The action spectra f the light-dependent behaviour of the red and the blue form of Blepharisma have been determined; their structure is very similar to that the photosensing and phototransducing properties of blepharismin are maintained in its photooxidized form. oxyblepharismin.     

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 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 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.     

EFFECTS OF SHORT PULSES OF BLUE LIGHT ON THE ALKALINIZATION ASSOCIATED WITH THE UPTAKE OF NO3− AND CL− BY THE GREEN ALGA MONORAPHIDIUM BRAUNII AND RELATED ACTION SPECTRA

In Monoraphidium braunii, uptake of NO₃^?, NO₂^? and Cl^? is associated with proton transport and triggered by blue light (BL). Only 10 s after cells able to reduce NO₃^? to NH₄⁺ were irradiated with continuous, low-fluence BL in the presence of NO₃^?, an alkalinization of the medium began and only became interrupted by switching off the BL with a 60–90 s time lag. With 30 s BL pulses, the NO₃^?-dependent alkalinization lasted 3–5 min until it stopped. When the cells were exposed to continuous BL in the presence of Cl^?, the alkalinization also started within 10 s but lasted only 3 min. After that, the pH remained constant and decreased when the BL was switched off. With 30 s BL pulses, the Cl^?-dependent alkalinization lasted 3 min and then decreased to its initial value. The NO₃^?-dependent alkalinization shown by cells unable to reduce NO₃^? to NH₄⁺ was similar to that observed in the presence of Cl^?. These alkalinization rates fit the Bunsen-Roscoe reciprocity law. With 2 s pulses of high-fluence BL, the delay time of the NO₃ - or Cl^?-dependent alkalinizations was only 2 s, one of the fastest BL responses reported so far. The action spectra for Cl^? and NO₃^? uptakes proved to be very similar and matched the absorption spectra of flavins, including the 267 nm peak.