Studies on the Second Emerson Effect in the Hill Reaction in Algal Cells

This paper shows that the “second Emerson effect”¹ exists not only in photosynthesis, but also in the quinone reduction (Hill reaction), in Chlorella pyrenoidosa and Anacystis nidulans. The peaks at 650 mμ, 600 mμ, 560 mμ, 520 mμ, and 480 mμ, observed in the action spectrum of this effect in the Hill reaction in Chorella, are attributable to chlorophyll b; the occurrence of an additional peak at 670 mμ, 620 mμ, and of two (or three) peaks in the blueviolet region suggests that (at least) one form of chlorophyll a contributes to it. In analogy to suggestions made previously in the interpretation of the Emerson effect in photosynthesis, these results are taken as indicating that excitation by light preferentially absorbed by one (or two) forms of chlorophyll a (Chl a 690 + 700), needs support by simultaneous absorption of light in another form of chlorophyll a (Chl a 670)—directly or via energy transfer from chlorophyll b—in order to produce the Hill reaction with its full quantum yield. In Anacystis, the participation of phycocyanin in the Emerson effect in the Hill reaction is revealed by the occurrence, in the action spectrum of this effect, of peaks at about 560 mμ, 610 mμ, and 640 mμ; a peak at 670 mμ, due to Chl a 670, also is present.     

The effect of caffeine and light on killing of the blue-green alga Anabaena doliolum by ultraviolet radiation

Summary Pretreatment of spores of the blue-green alga Anabaena doliolum with caffeine is antagonistic to UV lethality and posttreatment with caffeine is synergistic to UV lethality and mutagenicity. The results of photoreactivation experiments suggest that photoreactivation is independent of photosynthesis in blue-green algae.     

Inhibition of Photosynthesis in Certain Algae by Extreme Red Light

This paper shows that in Porphyridium cruentum and in Chlorella pyrenoidosa (but apparently not in Anacystis nidulans) “extreme red” light (> 720 mμ) can inhibit photosynthesis produced by “far red” light (up to 720 mμ). From the action spectrum of this phenomenon, it appears that an unknown pigment with an absorption band around 745 mμ must be responsible for it.     

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.     

Studies on electron-transport reactions of photosynthesis in plastome mutants of oenothera

Fluorescence characteristics and light-induced absorbance changes of 5 plastome mutants of Oenothera, all having a defect in photosynthesis, were investigated to localize the site of the block in their photosynthetic mechanism and to relate mutational changes in the plastome to specific biochemical events in photosynthesis. In 4 of the mutants examined photosystem 2 was largely, or completely, nonfunctional. Excitation of system 2 did not cause reduction of oxidized cytochrome f in these mutants. The system-2 dependent absorbance change at 518 mμ seen in normal leaves was absent in the mutants. Moreover, the mutants had a high initial fluorescence in the presence and in the absence of 3- (3,4-dichlorophenyl)-1,1-dimethylurea, which did not change during illumination, indicating that the reaction centers of system 2 were affected by the mutations. Photosystem 1 functioned normally.

A fifth mutant had an impaired photosystem 1. Even high intensity far-red light did not lead to an accumulation of oxidized cytochrome f as was seen in normal plants. Photosystem 2 was functioning, as evidenced by the fast reduction of the primary system-2 oxidant, and by the characteristics of the 518-mμ absorbance change.

Because 1 of the 2 photosystems is functional in all mutants, and because they all have the enzymes of the photosynthetic carbon cycle, it appears that the effect of the mutation is specific. The results suggest that the plastome controls reactions within the electron-transport chain of photosynthesis.

     

Spectral Studies of a Chlorophyll Pigment with Fluorescence Maximum at 698 mμ

A chlorophyll type pigment (F698) fluorescing maximally at 698 mμ at 77°K has been observed in preparations of chlorophyll. This fluorescence is quenched by small amounts of naturally occurring materials, including plastoquinone and the ubiquinones, and by nitrobenzene, probably by formation of a nonfluorescent complex. Fluorescence quenching does not occur in the presence of carotenes, xanthophylls, or reduced plastoquinone and ubiquinone. The fluorescence is sharply temperature dependent, with a steep rise in intensity occurring at 165°K. At 77°K the fluorescence yield is between 0.8 and 1.0. The red absorption maximum of the pigment is at 675 mμ at room temperature and at 688 mμ at 77°K. In vivo, a low temperature emission is also observed at 698 mμ, and this fluorescence is quenched by nitrobenzene. It is proposed that the pigment found in vitro is also the one responsible for emission at 698 mμ in vivo. A reaction of F698 with plastoquinone is suggested as the primary photochemical step in system II of photosynthesis.     

Photochemical and Nonphotochemical Reactions of Phytochrome in vivo

The nonphotochemical reactions of phytochrome in the coleoptiles of dark-grown corn seedlings were studied at 3 temperatures: 14°, 24°, and 34°. The data obtained show that the destruction of P_fr is the only measurable reaction occurring; reversion of P_fr to P_r was not found. The Q₁₀'s (2.7 and 3.5) and zero order kinetics found for the destruction reaction are consistent with the hypothesis that the reaction is enzyme-mediated.

In vivo action spectra for phytochrome transformation in the coleoptiles of darkgrown corn seedlings were obtained which agree qualitatively with those obtained by other workers for phytochrome-mediated physiological responses and in vitro action spectra. In vivo conversion of phytochrome by blue light, as determined from spectrophotometric measurements of phytochrome itself, is reported. Action peaks for P_r were found at 667 mμ and in the blue in the region of 400 mμ, with a broad shoulder from 590 mμ to 640 mμ. Action peaks for P_fr were found at 725 mμ and in the blue in the region of 400 mμ with a minor peak at 670 mμ, and a broad shoulder from 590 mμ to 640 mμ. The ratio of the quantum efficiencies of P_r at 667 mμ and P_fr at 725 mμ (Φ_r667/Φ_fr725) was estimated to be 1.0.

     

Absorption and Fluorescence of Water-Soluble Pigments Produced by Four Species of Pseudomonas

Pigments produced by four species of Pseudomonas during growth in three media were examined for visible and ultraviolet absorption. Ultraviolet fluorescence excitation and emission spectra were obtained. In all pigments, an absorption maximum occurs at 405 mμ. Ultraviolet excitation of fluorescence occurs primarily at 400 to 410 mμ, with smaller maxima at 460 mμ, or 525 mμ, depending on pH, bacterial species, or medium. Emission maxima, after excitation at 410 mμ, occur at 390 mμ, and 455 to 475 mμ. The differences in fluorescence spectra may be used for taxonomic classification of the Pseudomonas.     

Studies of Colloidal Chlorophyll in Aqueous Dioxane

The preparation and properties of a colloidal state of pure chlorophyll a in aqueous dioxane are described. The red absorption maximum is at 685± 1 mμ, depending on buffer concentration. The typical 672 mμ colloid (obtained by diluting an acetone solution with water) can be converted directly to the 685 mμ colloid by the addition of 1 M dioxane. The 672 → 685 mμ conversion is irreversible and is second order with respect to both 672 colloid and dioxane. It is shown that the formation of the 685 mμ colloid of chlorophyll a requires the Mg atom; no dioxane species is obtained with pheophytin or ethyl pheophorbide. Furthermore, of the transition metal salts of chlorophyll, Cu, Co, Ni, and Zn, only the Zn salt interacts with dioxane.     

Kinetics of Thymine Photodimerization in DNA

The kinetics of thymine photodimerization in E. coli DNA have been measured at various wavelengths of ultraviolet light. The initial quantum yield is not strongly dependent on wavelength. The ratio of thymine dimer to thymine in the photostationary state is much more dependent on wavelength. At the 235 mμ photosteady state 1.7 per cent of the thymine is present as dimer. This shifts to 6.5 per cent at 254 mμ and to 20 per cent of 275 mμ. While the change in position of the photosteady state with wavelength fails to fit a simple model, the data do indicate that not all thymines are capable of participation in dimer formation.     

Action Spectrum for the Appearance of the 520 mμ Difference Band in Illuminated Chlorella Cells

An action spectrum of the 520 mμ difference band in Chlorella is determined using dim illumination. Pigment (or pigments) absorbing most strongly at and above 680 mμ, probably the so-called “long-wave forms” of chlorophyll a appear to be the primary sensitizer of the 520 mμ effect.     

Structure, Growth, and Decomposition of Laminated Algal-Bacterial Mats in Alkaline Hot Springs

Laminated mats of unique character in siliceous alkaline hot springs of Yellowstone Park are formed predominantly by two organisms, a unicellular blue-green alga, Synechococcus lividus, and a filamentous, gliding, photosynthetic bacterium, Chloroflexus aurantiacus. The mats can be divided approximately into two major zones: an upper, aerobic zone in which sufficient light penetrates for net photosynthesis, and a lower, anaerobic zone, where photosynthesis does not occur and decomposition is the dominant process. Growth of the mat was followed by marking the mat surface with silicon carbide particles. The motile Chloroflexus migrates vertically at night, due to positive aerotaxis, responding to reduced O₂ levels induced by dark respiration. The growth rates of mats were estimated at about 50 μm/day. Observations of a single mat at Octopus Spring showed that despite the rapid growth rate, the thickness of the mat remained essentially constant, and silicon carbide layers placed on the surface gradually moved to the bottom of the mat, showing that decomposition was taking place. There was a rapid initial rate of decomposition, with an apparent half-time of about 1 month, followed by a slower period of decomposition with a half-time of about 12 months. Within a year, complete decomposition of a mat of about 2-cm thickness can occur. Also, the region in which decomposition occurs is strictly anaerobic, showing that complete decomposition of organic matter from these organisms can occur in the absence of O₂.     

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.

     

Light acclimation during and after leaf expansion in soybean

Soybean plants (Glycine max var. Ransom) were grown at light intensities of 850 and 250 μeinsteins m⁻² sec⁻¹ of photosynthetically active radiation. A group of plants was shifted from each environment into the other environment 24 hours before the beginning of the experiment. Net photosynthetic rates and stomatal conductances were measured at 2,000 and 100 μeinsteins m⁻² sec⁻¹ photosynthetically active radiation on the 1st, 2nd, and 5th days of the experiment to determine the time course of photosynthetic light adaptation. The following factors were also measured: dark respiration, leaf water potential, leaf thickness, internal surface area per external surface area, chlorophyll content, photosynthetic unit size and number, specific leaf weight, and activities of malate dehydrogenase, and glycolate oxidase. Comparisons were made with plants maintained in either 850 or 250 μeinsteins m⁻² sec⁻¹ environments. Changes in photosynthesis, stomatal conductance, leaf anatomy, leaf water potential, photosynthetic unit size, and glycolate oxidase activity occurred upon altering the light environment, and were complete within 1 day, whereas chlorophyll content, numbers of photosynthetic units, specific leaf weight, and malate dehydrogenase activity showed slower changes. Differences in photosynthetic rates at high light were largely accounted for by internal surface area differences with low environmental light associated with low internal area and low photosynthetic rate. An exception to this was the fact that plants grown at 250 μeinsteins m⁻² sec⁻¹ then switched to 850 μeinsteins m⁻² sec⁻¹ showed lower photosynthesis at high light than any other treatment. This was associated with higher glycolate oxidase and malate dehydrogenase activity. Photosynthesis at low light was higher in plants kept at or switched to the lower light environment. This increased rate was associated with larger photosynthetic unit size, and lower dark respiration and malate dehydrogenase activity. Both anatomical and physiological changes with environmental light occurred even after leaf expansion was complete and both were important in determining photosynthetic response to light.     

Action spectrum for an enhancement of endogenous respiration by light in chlorella

The oxygen consumption of a starved chlorophyll-free, yellow mutant of Chlorella vulgaris is enhanced by very small amounts of blue light (λ 450 mμ); a saturation level is reached at about 500 ergs cm⁻² sec⁻¹. At that intensity the respiration is about 3 times greater than in the dark. An action spectrum for the enhancement of respiration shows 2 peaks around λ 450 and 375 mμ. Flavins and cis-carotenoids are discussed as the pigments involved.     

Relationship between the Absorption and Emission Spectra and the "Red Drop" in the Action Spectra of Fluorescence In Vivo

The Stepanov equation, relating the intensity of emission, f_e($\bar{v}$), at a given frequency, and that of absorption, k($\bar{v}$), at the same frequency, is applied, in its modified form (see equation 3 in text) to suspensions of Chlorella, Porphyridium, and Anacystis and to chlorophyll solutions. This application can reveal whether the yield of fluorescence, Φ($\bar{v}$), is constant, or changes with frequency. In Chlorella (green alga) a sharp drop of Φ($\bar{v}$) is indicated towards the lower frequencies (longer waves), beginning around $\bar{v}$ = 1.48 × 10⁴cm^-1 (680 mμ); the Φ($\bar{v}$) function calculated from the Stepanov equation is in fair agreement with the directly determined action spectrum for the excitation of chlorophyll fluorescence in this organism. In Porphyridium (red alga) and Anacystis (blue-green alga) application of the Stepanov equation supports the conclusions, derived from direct measurements, of a much earlier “red drop” of the fluorescence excitation spectra. Direct measurements suggest that the drop in Porphyridium may begin at about 1.53 × 10⁴cm^-1 (654 mμ); in Anacystis, it may begin already above 1.57 × 10⁴cm^-1 (<637mμ). These results confirm the relation, postulated earlier by Duysens and others, between the action spectra of photosynthesis and of chlorophyll a fluorescence in algal cells. The relation of these findings to spectroscopic evidence, suggesting the existence of two main chlorophyll a components in vivo, in green as well as in red and blue-green algae, is discussed.     

Oxygen Concentration Inside a Functioning Photosynthetic Cell

The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 μM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth’s atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 μM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 μm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere.     

Photosynthetic Enhancement in the Diatom Phaeodactylum tricornutum

Enhancement phenomena in photosynthesis of the diatom Phaeodactylum tricornutum Lewin were studied by means of a Haxo oxygen electrode and 2 monochromatic light beams. It was necessary to correct for a minor non-linearity in rate oxygen evolution vs. intensity similar to that reported for Chlorella. Action spectra on complementary backgrounds and derived enhancement spectra were compared to in vivo absorption spectra in identifying the character of pigment systems 1 and 2. Fucoxanthin, chlorophyll c, and chlorophyll a-670 are clearly assignable to pigment system 2 which absorbs in excess at wavelengths 400 to 678 mμ. Chlorophyll a-1 (and a portion of the fucoxanthin) are assignable to pigment system 1 which absorbs in excess at wavelengths 678 to 750 mμ. Enhancement values were generally lower than those observed in Chlorella or Anacystis and lead to conclusion that diatom pigmentation provides an effective light harvesting apparatus.     

The Long Wave Length Forms of Chlorophyll a

When Euglena gracilis is cultured with light of low intensity (ca. 250 ft-c), an absorption band at 695 mμ is formed in an amount equal to about 20 per cent of the total chlorophyll absorption in this red region. An equally large proportion of C_a695 is observed in Ochromonas danica, irrespective of light intensity. Other algae tested appear to contain approximately 3 to 5 per cent of their chlorophyll as C_a695; this proportion does not increase as strikingly with lowering of the light intensity as it does in Euglena. C_a695 bleaches more readily than the other chlorophyll forms both reversibly, in whole cells, and irreversibly, in homogenates. Cells containing a large proportion of C_a695 have a fluorescence maximum at 708 mμ, as contrasted to the 687 mμ maximum in other algae. Occasionally, old cultures of Euglena contain cells with an absorption band at approximately 710 mμ. This absorption band is quite stable in aqueous extracts; when the pigment is transferred to ether an equivalent amount of pheophytin a is found to be present. Conditions leading to the formation of the 710 mμ absorption band are not yet known.     

Production of Fluorescence on Packaged Chicken

Development of fluorescence caused by pseudomonads proliferating on packaged chicken was determined by examination of the poultry under ultraviolet light and by measurements of absorption spectra. Asparagine broth inoculated with organisms from chicken showed absorption maxima at 270 mμ and 410 mμ; these peaks are characteristic of the fluorescent pigment, pyoverdine. Absorbance calculated as the ratio (A_270mμ + A_410mμ)/A_350mμ provided a convenient measure of amount of pigment produced; this ratio was related to numbers of fluorescing organisms recovered from chicken. Absorption peaks generally increased during the first few days the poultry was stored at 5 C and then declined during the latter part of the 7-day holding period.

Production of fluorescence was influenced by packaging materials. Fluorescence was not visible on poultry until counts of fluorescing bacteria were as great as 100,000 to 1,000,000 per cm². Growth of fluorescent pigment-producing pseudomonads on chicken was stimulated during storage after the poultry was dipped in solutions containing iron.