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.     

Four universal forms of chlorophyll a

We have matched the red absorption band measured at −196 C in a variety of chloroplast preparations with four major component curves representing forms of chlorophyll a having peaks at 661.6, 669.6, 677.1, and 683.7 nanometers. Chloroplast fractions enriched in one or the other of the two photochemical systems both contain these four major components, but system 1 preparations contain relatively more chlorophyll a 684. Chlorophyll a 677 and chlorophyll a 684 have greater bandwidths in system 1. Bands at longer wavelengths near 693 and 704 nanometers also often occur, but with far smaller heights than the above major bands. The longer wavelength bands are more common in system 1 than in system 2. In system 1 the half-widths of the four major bands in typical spectra average 11.3, 10.0, 10.3, and 10.8 nanometers while in system 2 they are 11.6, 9.8, 9.4, and 9.6 nanometers. Some spectra with sharper and some with wider bands were found, but the wavelengths were identical.     

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.     

Effect of method of preparation on the States of chlorophyll in euglena chloroplast fragments as determined by fluorescence spectroscopy

The state of chlorophyll in chloroplast fragments is affected by such factors as the ionic strength and pH of the suspending medium. With increasing ionic strength or at pH values other than neutrality, there is a decrease in the fluorescence yield of the form of chlorophyll with fluorescence maximum at 715 to 736 mμ (aggregate) and an increase in the yield of the form with fluorescence maximum at 685 mμ (monomer). (Positions of maxima cited are for 77°K.) These changes in yield are accompanied by modifications in absorption and fluorescence excitation spectra. It is also noted that these effects are similar to the ones brought about by pancreatic lipase, wheat germ lipase, pancreatic trypsin or urea. An interpretation is given which is consistent with the experimental data, namely, that the effects originate in conformational changes in the proteins to which the pigments are attached. These conformational changes give rise to an increase in the size of the aggregate and a decrease in the probability of energy transfer between the monomer and aggregates.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

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.     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : II. THE ABSORPTION CURVES OF PHOTOSYNTHIN FROM SEVERAL SPECIES OF BACTERIA

Absorption curves have been obtained in the spectral region of 450 to 900 mµ for the water soluble cell juice of four species of photosynthetic bacteria, Spirillum rubrum (strain S1), Rhodovibrio sp. (strain Gaffron), Phaeomonas sp. (strain Delft), and Streptococcus varians (strains C11 and orig.). These curves all show maxima at 790 and 590 mµ due to bacteriochlorophyll, whose highest band, however, occurs at 875, 855, or 840 mµ depending on the species. The bacteria that appear red rather than brown have a band at 550 mµ due to a carotinoid pigment. An absolute absorption curve of bacteriophaeophytin has maxima at 530 and 750 mµ. The extraction of cell juice by supersonic vibration does not change the position of the absorption bands or of the light absorbing capacity of the pigment.     

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.     

The arrangement of chloroplasts in cells influences the reabsorption of chlorophyll fluorescence emission. The effect of desiccation on the chlorophyll fluorescence spectra of Rhizomnium punctatum leaves

Chlorophyll fluorescence spectra measured with leaves are distorted by the effect of fluorescence reabsorption. A heterogeneous theoretical model simulating the effect of chloroplast arrangement in a cell on the distortion of chlorophyll fluorescence spectra due to reabsorption was formulated. Desiccation of leaves of the moss Rhizomnium punctatum was carried out as a simple model experiment. The parameters entering the model (maximal number of chloroplasts forming columns in a cell, chloroplast size and chlorophyll concentration in a chloroplast) were estimated by means of light microscopy and spectrophotometry. During the desiccation, a grouping of chloroplasts was observed by light microscopy and the chlorophyll fluorescence emission and excitation spectra of the leaves were measured at room temperature and at 77 K. The leaves were infiltrated with DCMU. The ratio F685/F735 of the main emission bands decreased by about 50% at room temperature and by about 30% at 77 K upon decreasing the leaf water content. No significant changes were found in the ratio E475/E436 of the bands of the leaf fluorescence excitation spectra at 77 K for both 685- and 735-nm emission wavelengths. The excitation spectra and mechanical dilution experiments indicated that no functional changes appeared upon desiccation at the level of energy transfer. Theoretical simulations were in a good agreement with the experimental dependencies. We were able to conclude that the grouping of chloroplasts in cells may enhance the effect of chlorophyll reabsorption and thereby cause a significant decrease of the F685/F735 ratio in the chlorophyll fluorescence spectrum.     

STUDIES ON THE CHLOROPHYLLS AND PHOTOSYNTHESIS OF THERMAL ALGAE FROM YELLOWSTONE NATIONAL PARK,CALIFORNIA, AND NEVADA

1. Myxophyceae normally growing at 65°C. evolved oxygen upon irradiation and showed evidence of retaining the power to carry on the process of photosynthesis at 20°C. This indicates that extra thermal energy is not essential for photosynthesis at least over a short period of time. 2. Chlorophyll a and b found in several species of Myxophyceae growing in waters ranging in temperature from 37–72°C. are essentially the same as found in plants growing all over the world. Certain standard chemical tests and spectroscopic examination of the chlorophylls were used as the criteria for these comparisons. The ratio of chlorophyll a to b often varied considerably but in general chlorophyll a showed an increase over the percentage found in most plants. 3. Green algae (Chlorella sp.?) were the only forms found at The Geysers, California. The temperature of the waters from which collections were made varied from 49–66°C. 4. Collections from Beowawe, Nevada were from waters ranging from 60–71°C. The algae belonged to the Myxophyceae and the species were like some of those found in Yellowstone National Park. 5. In some of the calcareous regions of Yellowstone National Park spectroscopic study of the chlorophylls revealed an unidentified absorption band at 548 mµ.     

Origin of Thylakoid Membranes in Chlamydomonas reinhardtii y-1 at 38 degrees C

The origin of thylakoid membranes was studied in Chlamydomonas reinhardtii y-1 cells during greening at 38°C. Previous studies showed that, when dark-grown cells are exposed to light under these conditions, the initial rates of accumulation of chlorophyll and the chlorophyll a/b-binding proteins in membranes are maximal (MA Maloney JK Hoober, DB Marks [1989] Plant Physiol 91: 1100-1106; JK Hoober MA Maloney, LR Asbury, DB Marks [1990] Plant Physiol 92: 419-426). As shown in this paper, photosystem II activity, which was nearly absent in dark-grown cells, also increased at a linear rate in parallel with chlorophyll. As compared with those made at 25°C, photosystem II units assembled during greening at 38°C were photochemically more efficient, as judged by saturation at a lower fluence of light and a negligible loss of excitation energy as fluorescence. Electron microscopy of cells in light for 5 or 15 minutes at 38°C showed that these initial, functional thylakoid membranes developed in association with the chloroplast envelope.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

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.     

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non‐photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light‐harvesting pigments. Copyright © 2015 John Wiley & Sons, Ltd.     

A Spectroscopic Analysis of a High Fluorescent Mutant of Chlamydomonas Reinhardi

Chloroplast fragments of a high fluorescent mutant of Chlamydomonas reinhardi, hfd 91, were compared against those of Acl⁺, a low chlorophyll variant of the wild type. The chloroplast fragments of the mutant which have a high invariant fluorescence yield lacked photochemical activities associated with photosystem II (PSII) but retained normal photosystem I (PSI) activities. The mutant fragments also lacked the low temperature (-196°C) light-induced absorbance changes due to the photoreduction of C-550 and the photooxidation of cytochrome (cyt) b-559 which are PSII-mediated reactions. A fourth-derivative analysis of the absolute spectra of the chloroplast fragments at different stages of reduction (obtained with ferricyanide, ascorbate, and dithionite) showed both the oxidized and reduced forms of C-550 and the reduced forms of cyt c-553, b-559, and b-564 in wild-type fragments. The mutant fragments lacked C-550 and an ascorbate-reducible cyt b-559 but contained cyt c-553, a dithionite-reducible cyt b-559, and cyt b-564.     

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 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.