Delayed light production by blue-green algae,red algae,and purple bacteria

1. Blue-green algae, red algae, and purple bacteria all show the emission of delayed light. 2. The action spectra for the production of delayed light by three species of blue-green algae have one broad band with a peak at 620 mµ. 3. The action spectrum for production of delayed light by the red algae has one peak at 550 mµ with a shoulder from 600 to 660 mµ. 4. The emission spectra of the delayed light from both the blue-green and red algae were the same as from the green algae, Chlorella. 5. The action spectra for the production of delayed light by the different species of purple bacteria tested consisted of one or more bands not resolved between 800 and 900 mµ. 6. The emission spectrum of the delayed light from the purple bacteria was largely at wave lengths longer than 900 mµ.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.     

The identity of the fluorescent and delayed light emission spectra in Chlorella

1. The delayed light emission of Chlorella pyrenoidosa over the wave length range 400 to 950 mµ has been investigated. 2. Emission of delayed light is confined to the range 600 to 800 mµ. 3. To the precision with which the low light intensities involved can be measured with the apparatus in these experiments, the emission spectrum of the delayed light is the same as the spectrum of the fluorescent light. 4. Thus the delayed light must come from excited chlorophyll.     

Action spectra for photoreactivation of ultraviolet-irradiated Escherichia coli and Streptomyces griseus

Action spectra for photoreactivation (light-induced recovery from ultraviolet radiation injury) of Escherichia coli B/r and Streptomyces griseus ATCC 3326 were determined. The spectral region explored was 365 to 700 mµ. The action spectrum for S. griseus differed from that for E. coli, indicating that the chromophores absorbing reactivating energy in the two species were not the same. Reactivation of S. griseus occurred in the region 365 mµ (the shortest wave length studied) to about 500 mµ, with the most effective wave length lying near 436 mµ. This single sharp peak in the spectrum at 436 mµ suggested the Soret band typical of porphyrins. Reactivation of E. coli occurred in the region 365 to about 470 mµ, with the most active wave length lying near 375 mµ. The single, non-pronounced peak near 375 was probably not due to a Soret band, and the identification of the substance absorbing reactivating light in E. coli is uncertain. In neither species was the region 500 to 700 mµ active. The implications of these action spectra and their differences are discussed.     

The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll

1. The fluorescence spectra of the alga Porphyridium have been recorded as energy distribution curves for eleven different incident wave lengths of monochromatic incident light between wave lengths 405 and 546 mµ. 2. In these spectra chlorophyll fluorescence predominates when the incident light is in the blue part of the spectrum which is strongly absorbed by chlorophyll. 3. For blue-green and green light the spectrum excited in Porphyridium contains in addition to chlorophyll fluorescence, the fluorescence bands characteristic of phycoerythrin and of phycocyanin. 4. From these spectra the approximate curves for the fluorescence of the individual pigments phycoerythrin, phycocyanin, and chlorophyll in the living material have been derived and the relative intensity of each of them has been obtained for each of the eleven incident wave lengths. 5. The effectiveness spectrum for the excitation of the fluorescence of these three pigments in vivo has been plotted. 6. From comparisons of the effectiveness spectrum for the excitation of each of these pigments it appears that both phycocyanin and chlorophyll receive energy from light which is absorbed by phycoerythrin. 7. It is suggested that phycocyanin may be an intermediate in the resonance transfer of energy from phycoerythrin to chlorophyll. 8. Since phycoerythrin and phycocyanin transfer energy to chlorophyll, it appears probable that chlorophyll plays a specific chemical role in photosynthesis in addition to acting as a light absorber.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mµ rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mµ recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mµ) and longer (267 mµ) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mµ, and found to resemble the absorption of protein and lipide in the wave length region below 248 mµ. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mµ which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mµ) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mµ is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mµ is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mµ) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.     

THE EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

The Photoactivated Relaxation of Smooth Muscle of Rabbit Aorta

Smooth muscle of strips of rabbit aorta, placed in a state of active tonic contraction by addition of a stimulating drug, relaxes during exposure to light. The relaxation is reversible. The extent of relaxation produced by a standard exposure depends on the preexposure level of active contraction but not on the nature of the stimulating drug used to produce contraction. With strips brought to an intermediate level of contraction, the degree of relaxation (steady state levels) is a rectangular hyperbolic function of radiation intensity. The kinetics of the relaxation process during irradiation and the recovery process following irradiation are consistent with the hypothesis that the primary photoactivated material initiates a reaction or reactions leading to a product which inhibits some process involved in the production of active contraction. The photorelaxation does not require the presence of oxygen. It is potentiated by reducing the temperature of the aortic strip. The action spectrum of the photorelaxation shows relatively low effectiveness at wavelengths above 450 mµ. The effectiveness increases markedly and progressively as the wavelength is lowered below 450 mµ, reaching a peak at 310 mµ. A deep trough occurs at 280 mµ. However, both peak and trough probably result from internal filtering due to absorption by proteins in the aortic strip. It is surmised that if a correction could be made for this internal filtering, the action spectrum would rise continuously down to wavelengths at least as low as 250 mµ.     

Interplay between the Reactions to Light and to Gravity in Phycomyces

Sporangiophores of Phycomyces do not grow directly towards a horizontal beam of light, but equilibrate at an angle of about 30° above the horizontal. After describing several related observations, this paper suggests that the dioptric properties of an obliquely illuminated cylindrical lens, illustrated by a dummy cell, as well as a negative geotropic response, play major roles in determining the direction of growth. The shift of the equilibrium direction of growth towards the vertical, or a purely geotropic response, over a tenfold range of very low intensities (around 10⁶ quanta/cm² sec., or 10^-13watt/cm²) has been studied, and an action spectrum made, measuring the quantum fluxes producing a standard intermediate equilibrium direction of growth at different wavelengths. This may differ from the action spectra at higher intensities in lacking conspicuous maxima from 370 to 490 mµ. However, in the ultraviolet it parallels the other spectra, although without showing the much higher quantum efficiency of ultraviolet relative to visible light previously noted. Possible interpretations are discussed.     

PHOTOLYTIC LIPIDS FROM VISUAL PIGMENTS

A method is described for the preservation of iodopsin, the labile photopigment of daylight vision, by freeze drying in vacuo. The lipids released by the action of light on rhodopsin and iodopsin are found to be similar and to possess a labile absorption spectrum in chloroform, with a rising peak at about 390 mµ and a declining peak in the region of 470 mµ. After the change is complete the absorption spectrum resembles closely that of retinene.     

The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees; an electrophysiological study

The spectral sensitivities of the dorsal ocelli of cockroaches (Periplaneta americana, Blaberus craniifer) and worker honeybees (Apis mellifera) have been measured by electrophysiological methods. The relative numbers of quanta necessary to produce a constant size electrical response in the ocellus were measured at various wave lengths between 302 and 623 mµ. The wave form of the electrical response (ERG) of the dark-adapted roach ocellus depends on the intensity but not the wave length of the stimulating light. The roach ocellus appears to possess a single photoreceptor type, maximally sensitive about 500 mµ. The ERG's of bee ocelli are qualitatively different in the ultraviolet and visible regions of the spectrum. The bee ocellus has two types of photoreceptor, maximally sensitive at 490 mµ and at about 335 to 340 mµ. The spectral absorption of the ocellar cornea of Blaberus craniifer was measured. There is no significant absorption between 350 and 700 mµ.     

Respiratory Chain of Colorless Algae II. Cyanophyta

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad α-bands near 560 mμ. At −190° these bands split to give 4 peaks in the α-region for b and c-type cytochromes, but no α-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mμ. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mμ. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.     

Evidences from Action Spectra for a Specific Participation of Chlorophyll b in Photosynthesis

Rate of oxygen evolution in photosynthesis was measured as the current from a polarized platinum electrode covered by a thin layer of Chlorella. The arrangement gave a reproducibly measurable rate of photosynthesis proportional to light intensity at the low levels used and gave rapid response to changes in illumination. Two phenomena have been explored. The Emerson effect was observed as an enhancement of photosynthesis in long wavelength red light (700 mµ) when shorter wavelengths were added. Two light beams of wavelengths 653 and 700 mµ when presented together gave a photosynthetic rate about 25 per cent higher than the sum of the rates obtained separately. Large and reproducible transients in rate of oxygen evolution were observed accompanying change in illumination between two wavelengths adjusted in intensity to support equal steady rates of photosynthesis. The transients were found not to be specifically related to long wavelength red light. Both enhancement and the transients have identical action spectra which are interpreted as demonstrating a specific photochemical participation of chlorophyll b.     

Temperature and plant adaptation. I. Interaction of temperature and light in the synthesis of chlorophyll in corn

The effect of temperature and light intensity have been studied in relation to the greening of etiolated corn (Zea mays cv. Pioneer 309-B) seedlings. Chlorophyll accumulation is rapid at high temperature (28°) under all conditions of light intensity. At low temperature (16°), and particularly in combination with high light intensity (3000-4500 ft-c), the accumulation of both chlorophyll and carotene is inhibited.

Low pigment content at 16° is not directly due to a block in the pigment synthesizing mechanism, but rather to the photodestruction of chlorophyll prior to its stabilization in the membrane structure of the chloroplast lamellae. The parallel reduction in carotene content at high light intensity is probably a contributing factor, because of its role in protecting chlorophyll from photodestruction. The greater severity of photo-oxidation of chlorophyll at low temperature in corn when compared with wheat, appears to be due to a slower rate of protochlorophyllide synthesis and subsequent esterification. Thus in corn at 16° there is a prolongation of the photosensitive stage during chlorophyll synthesis. Photo-oxidation at 16° has also been shown to be a function of the incident light energy, with the photosynthetic pigments acting as receptors for their own destruction.

In comparison with the behavior of corn, wheat seedlings green rapidly at high light intensity at both 16° and 28°. This contrasting temperature response with respect to chlorophyll synthesis may underlie a fundamental difference in adaptation of these 2 species to growth in the temperate zones of the world.

     

The Nature of the Retinal Action Potential, and the Spectral Sensitivities of Ultraviolet and Green Receptor Systems of the Compound Eye of the Worker Honeybee

1. The retinal action potential consists principally of a sustained negative wave which persists for as long as the stimulus. Transitory negative on-effects and off-effects may also be present, particularly at long wave lengths (green, yellow, and red) and in the light-adapted eye. 2. Only the maintained component of the potential can be elicited under CO₂ anesthesia. The transient components are reversibly eliminated from the response at about the same time as the background noise of nerve and muscle spikes. It is suggested that the sustained component arises from the receptor cells, and the other components from second and higher order neurons. 3. The compound eye does not contain a homogeneous population of receptors. A green receptor system (maximum sensitivity at about 535 mµ) determines the response of the dark-adapted eye throughout most of the spectrum; during adaptation to yellow light, however, an ultraviolet receptor system is revealed, with maximum sensitivity at about 345 mµ. The anatomical bases of these receptor systems are unknown; however, they include both retinula cells and neurons in the optic ganglion. 4. There is no change in spectral sensitivity (Purkinje shift) in the first three logarithmic units above the threshold of the retinal action potential. 5. The relatively great effectiveness of near ultraviolet light in stimulating the positive phototaxis of the bee does not depend on excitation of the ultraviolet receptor of the ocellus.     

The ultraviolet light and photosensitized inactivation of tobacco mosaic virus

The quantum yield for the inactivation of tobacco mosaic virus has been determined at 253.7 mµ and found to be 4.3 x 10^–6. The possible significance of the observed one-hit process of inactivation has been discussed in terms of the kinetics and the rupture of model substances including nucleic acid. The ultraviolet light inactivation, which proceeds independent of oxygen, occurs without change in physicochemical properties, with the possible exception of an enhanced sensitivity to thermal denaturation. The photosensitized inactivation of virus by acriflavine has been found to proceed parallel with the destruction of the dye. The action was found to be dependent upon adsorbed dye, and the inactivation is enhanced by the presence of oxygen.     

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.

     

A STUDY OF THE BACTERICIDAL ACTION OF ULTRA VIOLET LIGHT : II. THE EFFECT OF VARIOUS ENVIRONMENTAL FACTORS AND CONDITIONS

1. Wide differences in the intensity of incident ultra violet energy are not accurately compensated by corresponding changes in the exposure time, so that the Bunsen-Roscoe reciprocity law does not hold, strictly, especially for bactericidal action on young, metabolically and genetically active bacteria. In the present series of experiments, however, the energies used at various wave lengths did not differ by so much as to cause a significant error in the reported reactions. 2. The longer wave length limit of a direct bactericidal action on S. aureus was found to be between 302 and 313 mµ. The shorter limit was not determined because the long exposures required vitiate quantitative results. Bactericidal action was observed at λ225 mµ. 3. The temperature coefficient of the bactericidal reaction approaches 1 and thus furnishes empirical evidence that the direct action of ultra violet light on bacteria is essentially physical or photochemical in character. 4. The hydrogen ion concentration of the environment has no appreciable effect upon the bactericidal reaction between the limits of pH 4.5 and 7.5. At pH 9 and 10 evidence of a slight but definite increase in bacterial susceptibility was noted, but this difference may have been due to a less favorable environment for subsequent recovery and multiplication of injured organisms. 5. Plane polarization of incident ultra violet radiation has no demonstrable effect upon its bactericidal action. In a third paper of this group the ratios of incident to absorbed ultra violet energy at various wave lengths and the significance of these relations in an analysis of the bactericidal reaction will be discussed.     

The interplay o light and heat in bleaching rhodopsin

Rhodopsin, the pigment of the retinal rods, can be bleached either by light or by high temperature. Earlier work had shown that when white light is used the bleaching rate does not depend on temperature, and so must be independent of the internal energy of the molecule. On the other hand thermal bleaching in the dark has a high temperature dependence from which one can calculate that the reaction has an apparent activation energy of 44 kg. cal. per mole. It has now been shown that the bleaching rate of rhodopsin becomes temperature-dependent in red light, indicating that light and heat cooperate in activating the molecule. Apparently thermal energy is needed for bleaching at long wave lengths where the quanta are not sufficiently energy-rich to bring about bleaching by themselves. The temperature dependence appears at 590 mµ. This is the longest wave length at which bleaching by light proceeds without thermal activation, and corresponds to a quantum energy of 48.5 kg. cal. per mole. This value of the minimum energy to bleach rhodopsin by light alone is in agreement with the activation energy of thermal bleaching in the dark. At wave lengths between 590 and 750 mµ, the longest wave length at which the bleaching rate was fast enough to study, the sum of the quantum energy and of the activation energy calculated from the temperature coefficients remains between 44 and 48.5 kg. cal. This result shows that in red light the energy deficit of the quanta can be made up by a contribution of thermal energy from the internal degrees of freedom of the rhodopsin molecule. The absorption spectrum of rhodopsin, which is not markedly temperature-dependent at shorter wave lengths, also becomes temperature-dependent in red light of wave lengths longer than about 570 to 590 mµ. The temperature dependence of the bleaching rate is at least partly accounted for by the temperature coefficient of absorption. There is some evidence that the temperature coefficient of bleaching is somewhat greater than the temperature coefficient of absorption at wave lengths longer than 590 mmicro;. This means that the thermal energy of the molecule is a more critical factor in bleaching than in absorption. It shows that some of the molecules which absorb energy-deficient quanta of red light are unable to supply the thermal component of the activation energy needed for bleaching, so bringing about a fall in the quantum efficiency. The experiments show that there is a gradual transition between the activation of rhodopsin by light and the activation by internal energy. It is suggested that energy can move freely between the prosthetic group and the protein moiety of the molecule. In this way a part of the large amount of energy in the internal degrees of freedom of rhodopsin could become available to assist in thermal activation. Assuming that the minimum energy required for bleaching is 48.5 kg. cal., an equation familiar in the study of unimolecular reaction has been used to estimate the number of internal degrees of freedom, n, involved in supplying the thermal component of the activation energy when rhodopsin is bleached in red light. It was found that n increases from 2 at 590 mµ to a minimum value of 15 at 750 mµ. One wonders what value n has at 1050 mµ, where vision still persists, and where rhodopsin molecules may supply some 16 kg. cal. of thermal energy per mole in order to make up for the energy deficit of the quanta.