Chlorophyll Yield Research Articles

ENERGY TRANSFER FROM CAROTENOIDS TO CHLOROPHYLL a IN BLACK LIPID MEMBRANES

Abstract— Black lipid membranes (BLM) were prepared from extracts of Chlorella and spinach chloroplasts. Excitation spectra of the 730 nm fluorescence of chlorophyll a in the BLM contained peaks identified as due to carotenoids and which therefore indicate sensitization of the chlorophyll fluorescence by them. The efficiency of this energy transfer was evaluated by comparison of the actual excitation spectra with those corresponding to 0 and 100 per cent transfer efficiency. Efficiencies were of the order of 40–50 per cent in BLM, but only 10 per cent in pigment solutions, when the mean distance between pigment molecules was 23 Å in both systems. The fluorescence quantum yield of chlorophyll a in such solutions was only 2 per cent of that found in BLM. Enhancement of energy transfer in BLM is considered to be mainly due to suppression of competing deactivation processes of excited carotenoid states, such as diffusional quenching by ground‐state molecules and internal conversion. Favorable orientation of pigment molecules in the BLM constitutes a further enhancement factor.

Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans. I. Relationship of slow fluorescence changes with structural changes

1. 1. Both normal and 3-(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned cells of the blue-green alga Anacystis nidulans show an extensive slow rise in the chlorophyll a fluorescence yield upon illumination (Papageorgiou and Govindjee, 1968). It was observed here that uncouplers of phosphorylation notably 5-chloro-3-( p-chlorophenyl)-4′-chlorosalicylanilide and 5-chloro-3- tert-butyl-2′-chloro-4′-nitrosalicylanilide suppress this slow rise in the fluorescence yield both in normal and poisoned samples. In comparison to these salicylanilides, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone seems to be less effective in this respect. 2. 2. Emission spectra measured with the salicylanilides in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells show that the uncouplers prevent a shift in the excitation transfer in favor of pigment system II, i.e. the transformation to the so-called “State 1”. In other words, in the presence of the uncoupler, the pigment systems remain arrested in “State 2”. According to this view, the slow fluorescence rise represents a shift from State 2 to State 1. 3. 3. Furthermore, the uncouplers suppress as well the light-induced structural alterations (as measured by a change in absorbance at 540 nm) in both normal and poisoned Anacystis cells. Fixation of cells with aldehydes, that immobilizes the structural alterations, also abolishes slow fluorescence yield changes. 4. 4. It was observed that the time course of the light-induced macroscopic structural alteration was slower as compared to slow fluorescence yield changes. It, therefore, seems that the microscopic conformational organization, that precedes the macroscopic volume changes, induces the slow changes in fluorescence yield by shifting the mode of excitation energy transfer. 5. 5. In conclusion, we suggest that an energy-dependent specific alteration of the organization between the two photosystems controls and regulates the mode of excitation energy transfer between the two photosystems, the efficiency of such a transfer in Anacystis is approximately 10%.

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane. 1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg 2+ causes an increase in φ f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ f. The PMS triggered decrease in φ f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg 2+ and Ca 2+ show that there is a relative increase of System II to System I fluorescence. 2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg 2+-induced increase of φ f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes. 3. In purified pigment System II particles, neither PMS nor Mg 2+ causes any change in φ f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg 2+-induced increase of φ f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion ( k h), whereas the Mg 2+ effect is due to a decrease in the rate of energy transfer ( k t), between the two photosystems. 4. From the relative ratio of φ f with DCMU and DCMU plus Mg 2+, we have calculated k t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10 8 s −1, and φ t (quantum yield of this transfer) to be 0.12.

Reaction kinetics of intermediates of the photosynthetic chain between the two photosystems

1. 1. Measurements are reported of light-induced changes in absorbance and in the yield of chlorophyll a fluorescence of intact algae. Most experiments were done with the red alga Porphyridium aerugineum. In alternating non-saturating illumination with light mainly absorbed by Photosystem 1 and 2, respectively, a periodical oxidation and reduction of the components of the photosynthetic chain, P700, cytochrome f, plastoquinone and Q, the primary electron acceptor of Photosystem 2, was observed. 2. 2. Lags observed in the oxidation and reduction kinetics agreed with the concept that the midpoint potentials of these compounds decrease in the order given above. The largest difference was observed between plastoquinone and cytochrome f; the kinetics of Q and plastoquinone were similar and suggested only a small potential difference between these compounds. 3. 3. Relative pool sizes of 15:1.8:1 electron equivalent were observed for plastoquinone, cytochrome f and P700. Quantum efficiencies suggested the same high rate of electron flow through plastoquinone and cytochrome f. The kinetics of these components indicated that the only large pool in the chain between the two light reactions is that of plastoquinone. 4. 4. Difference spectra obtained under conditions where all the above-mentioned components were alternately reduced and oxidized gave no evidence for cytochrome b participation in the chain between the two light reactions.

Absence of Photosystem 2 in heterocysts of the blue-green alga Anabaena

Heterocysts of the filamentous blue-green alga Anabaena cylindrica have a high concentration of the System 1 reaction center P700 and are able to photooxidize a cytochrome. They have a low yield of chlorophyll a fluorescence, do not show light-induced changes in fluorescence yield, show a very low intensity of delayed light emission and do not show Hill-reaction activity. It is concluded that heterocysts contain Photosystem 1 only.

Control of excitation transfer in photosynthesis V. Correlation of membrane structure to regulation of excitation transfer between two pigment systems in isolated spinach chloroplasts

1. Changes in fluorescence yield of chlorophyll a in isolated chloroplasts have been interpreted by means of regulation of excitation transfer between two pigment systems of photosynthesis 5–7. In order to investigate the relationship between the membrane structure of chloroplasts and the regulation of excitation transfer, changes of light scattering and chlorophyll a fluorescence of isolated spinach chloroplasts were measured upon addition of cations, Mg 2+ and Na +. The cations increased the intensities of both light scattering and fluorescence yield. The changes showed similar time courses and concentration dependences. These facts suggest that modification of membrane structure produced by the cations suppresses the excitation transfer between the two pigment systems. 2. In another case of structural change which is induced by light in the presence of N-methylphenazonium methosulfate, there was little correlation between light-scattering and fluorescence changes. 3. Changes in fluorescence yield induced by the addition of Mg 2+ were measured in disintegrated chloroplasts and fractionated particles. The effects of Mg 2+ on fluorescence were observed only in preparations of grana stacks, but not in preparations of stroma lamellae. These findings suggest that the excitation transfer is regulated between the two pigment systems located in the grana thylacoid membranes.

The Sites of Photoconversion of Protochlorophyllide to Chlorophyllide in Barley Seedlings

The photoreduction of protochlorophyllide a to chlorophyllide a in intact 6-day-old seedlings of etiolated barley (Hordeum vulgare) exhibits a small initial phase, followed by an induction period of about 1 hour before a rapid phase of additional chlorophyll formation begins. Cycloheximide, an inhibitor of protein synthesis, has no effect on the initial phase of conversion of preformed protochlorophyllide, but it either abolishes or severely inhibits the subsequent phase of rapid chlorophyll synthesis within 45 minutes of its application to the seedlings. An analysis of the biphasic inhibition process suggests that the lifetime of the enzyme controlling protochlorophyllide synthesis (probably delta-amino-levulinic acid synthetase) is not longer than 10 minutes.The rapid phase of chlorophyll formation can be effected by a series of brief (15 second) pulses of light spaced at least 5 minutes apart. When longer dark intervals are used, no increase is observed in the yield of chlorophyll per pulse. We interpret the findings to indicate that the photoconversion takes place at distinct enzymatic sites whose concentration does not increase during a period of 4 hours following the initial illumination. The sites can be used repeatedly with a turnover time determined by the removal of the product chlorophyllide and the synthesis and placement of a new protochlorophyllide molecule.

PH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H + status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.

On the enzymatic conversion of 14C-labeled chlorophyll a to 14C-labeled chlorophyll b

Through the addition of NADP + to homogenates of soybean leaves incubated in the dark at room temperature with uniformly 14C-labeled chlorophyll a, significant yields of 14C-labeled chlorophyll b (purified as pheophytin b) were obtained. The activity of the enzyme proposed as responsible for this conversion appears to be relatively unaffected by acetone, but is at least partially denatured by heating.

Low Temperature Reactions in Chloroplasts

Some photoreactions of photosynthetic materials (corn chloroplasts, spinach chloroplasts and subchloroplast particles rich in System II) are examined at 77°K. The fluorescence yield of chlorophyll is observed to decrease reversibly upon irradiation at 77°K. The presence of ferricyanide in the chloroplast preparations increases the rate of decline of fluorescence yield while the presence of benzylviologen and phenazine methosulfate inhibits the decline of fluorescence yield. Extracts of Ricinus communis leaf (castor bean) also inhibits the decline of fluorescence yield. Data is given showing the effect of linolenic acid, extracts of Ricinus leaf, ferricyanide, benzylviologen and phenazine methosulfate on the ratio of fluorescence intensities at 725 and 700 nm.

EFFECT OF AMMONIUM AND NITRATE CONCENTRATION ON CHLOROPHYLL INCREASES IN NATURAL TROPICAL PACIFIC PHYTOPLANKTON POPULATIONS1

Samples of nutrient‐poor water from the eastern tropical Pacific were enriched with a complete mixture of nonnitrogenous nutrients and with varying concentrations of nitrate and ammonium. They were then incubated in natural light on the deck of the ship and phytoplankton growth was measured daily by means of in vivo chlorophyll fluorescence. Growth rates were calculated from increases in fluorescence, and final yield of fluorescence as a function of nitrogen added was also calculated. In two out of four experiments plots of rates against concentration of nitrate or ammonium were hyperbolic and the concentrations giving a half‐maximal rate were about 1.5 µg‐atom/liter for ammonium and about 0.75 µg‐atom/liter for nitrate. In two other experiments a maximal growth rate was not achieved even at 10 µg‐atom/liter of added nitrogenous nutrient. The mean yield of chlorophyll for all experiments was 0.22 µg per µm of nitrogen added. Growth rates calculated from ammonium concentration at two of these experimental stations agreed well with growth rates calculated from 14C productivity and chlorophyll concentrations.

Fluorescence and phototransformation of protochlorophyll with etiolated bean leaves from −196 to +20°C

The fluorescence yield of convertible protochlorophyll(ide) decreases markedly between −196 and −90°C, while the decline of fluorescence yield of chlorophyll(ide) and non-convertible protochlorophyll is much less pronounced. A comparison of temperature dependence of fluorescence of etiolated leaves with fluorescence of photosynthetically active chloroplasts shows a similarity of the F656 protochlorophyll band in the former, with the F695 chlorophyll a band in the latter. A dark transformation after illumination can be measured only between −90 and −40°C, both with intact leaves and leaf homogenates.

Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg 2+, Ca 2+, Sr 2+, Ba 2+ and Mn 2+ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al 3+ showed similar but less significant effects on the fluorescence yields. Zn 2+ and Cd 2+ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM. In accordance with the results of our previous study concerning the effects of Mg 2+ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophyll a of pigment system II to that of pigment system I.

Control of excitation transfer in photosynthesis. II. Magnesium ion-dependent distribution of excitation energy between two pigment systems in spinach chloroplasts

1. 1. Effects of metal ions on the yields of chlorophyll a fluorescence were investigated in spinach chloroplasts. At room and liquid-nitrogen temperatures, addition of Mg 2+ increased the yields of two emissions of chlorophyll a, having peaks at 684 and 695 nm which were emitted from pigment system II and decreased the yield of another emission of chlorophyll a at about 735 nm which was emitted from pigment system I. Mg 2+ also changed the pattern of the fluorescence induction at room temperature to increase the variable component of fluorescence. 2. 2. Effects of Mg 2+ on the quantum yields of the two photoreactions were investigated in a region of weak light intensities. Mg 2+ accelerated the rate of photoreaction II measured by the Hill reaction with 2,6-dichlorophenolindophenol and depressed the rate of photoreaction I measured by the NADP + reduction in the presence of reduced 2,6-dichlorophenolindophenol and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. 3. 3. These facts indicate that Mg 2+ controls the distribution of excitation energy between the two pigment systems and that the control consists mainly in change in rate of excitation transfer from the bulk chlorophyll a molecules in pigment system II to those in pigment system I; namely, Mg 2+ suppresses the spillover of excitation energy from pigment system II to pigment system I.

Control of excitation transfer in photosynthesis. III. Light-induced decrease of chlorophyll a fluorescence related to photophosphorylation system in spinach chloroplasts

1. 1. A light-induced reversible decrease in fluorescence yield of chlorophyll a, designated as “fluorescence lowering”, was discovered in isolated spinach chloroplasts on illumination at high light intensities in the presence of N-methylphenazonium methosulfate and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The decrease in fluorescence yield amounted to 20–30%. 2. 2. The “fluorescence lowering” was markedly lessened by addition of uncouplers of photophosphorylation. The concentrations for 50% suppression of the light-induced effect were 2.0 μM atebrin, 2.5 μM carbonylcyanide m-chlorophenylhydrazone and 1 mM methylamine. These findings indicate the involvement of the photophosphorylation system in the “fluorescence lowering”. 3. 3. Kinetic analyses showed that the ‘fluorescence lowering’ commenced at about 0.2 sec and reached an half-level of the “fluorescence lowering” about 2 sec after onset of illumination. The half-decay time of the recovery after cessation of illumination was approx. 5 sec. 4. 4. The time response of the “fluorescence lowering” differed from that of the light-scattering change in the chloroplasts, thus indicating that the “fluorescence lowering” was not caused by the conformational change corresponding to the light-scattering change of the chloroplasts. 5. 5. In experiments at liquid-nitrogen temperature, preillumination before cooling decreased the yields of the three emissions of chlorophyll a at 684 nm, 695 nm and 735 nm.

Light-Induced Changes in the Fluorescence Yield of Chlorophyll a In Vivo : III. The Dip and the Peak in the Fluorescence Transient of Chlorella pyrenoidosa

The fluorescence transient of Chlorella pyrenoidosa, excited by saturating light absorbed mainly by system II, has a dip D between the peak I at 75 msec and the large peak P at 400 msec (the times depend on light intensity). This dip is observed in aerobic cells and in anaerobic cells where it is prominent. In anaerobic cells, the I-D decline is hastened almost equally by absorption of either 705 or 650 nm background light. In anaerobic cells, supplementary 700 and 710 nm light given during the transient slightly hastens and heightens P. Methyl viologen, an exogenous system I electron acceptor, eliminates P. Results suggest that system I action causes D, and that P is due to reduction of Q (fluorescence quencher) and intersystem intermediates caused by development of a block in oxidation of XH (X being the primary electron acceptor of light reaction I). Mathematical analysis suggests that if only two forms of Q participate beyond I, then system I action is required for D. If three forms participate, then the system Q --> QH --> Q' (see text) may explain D. The Malkin model (14), in its present form, does not allow D.

Light-Induced Changes in the Fluorescence Yield of Chlorophyll a In Vivo : I. Anacystis nidulans

he fluorescence yield of chlorophyll a in dark adapted Anacystis nidulans undergoes a slow change with continuous illumination. After the completion of the initial fast transient, the fluorescence yield rises from the level S to a plateau M within a minute, declining only after prolonged illumination. Both normal and 1,1-dimethyl-3(3'4'-dichloro)-phenylurea (DCMU)-poisoned Anacystis are capable of these changes. In normal Anacystis, the slow increase in the fluorescence yield (S --> M) requires light absorbed in system II while light absorbed in system I is ineffective. In DCMU-poisoned Anacystis, however, these changes are also promoted by light absorbed in system I. Addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), a photophosphorylation uncoupler acting near the photosynthetic electron transport chain, abolishes the rise from S to M in normal but has no effect in the DCMU-poisoned system. Phlorizin, a phosphorylase inhibitor, has very little effect. These results suggest that the light-induced variation in the fluorescence yield is related to the conformational changes which accompany photophosphorylation. The fluorescence yield of the auxiliary pigment phycocyanin remains constant throughout the interval of the light-induced changes in the fluorescence yield of chlorophyll a. Consequently, the fluorescence spectrum of the alga is variable on continuous illumination.

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP + reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin. 2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a 2). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a 2 from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a 1). The ratio of the fluorescence yields of chlorophyll a 1 and chlorophyll a 2 is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3. 3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP + reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP + reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively. 4. The constancy of the fluorescence yield of chlorophyll a 1 in the small fragments, under conditions at which P 700 is oxidized and NADP + is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a 1 is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a 1 to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment. 5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.

An analysis of the relations between fluorescence and photochemistry during photosynthesis

The dynamics of transfer and trapping of singlet excitation energy in photosynthetic systems have been analysed in terms of several models of the pigmented tissue. The formulations of Joliot and of Vredenberg and Duysens have been derived as special examples of a more general case. Published data on the relation between the yield of chlorophyll (or bacteriochlorophyll) fluorescence and the state of the energy-trapping molecules have been compared with theoretical predictions in an effort to decide what conclusions are afforded about the architecture of the photosynthetic tissue. It is concluded that in photosynthetic bacteria the migration of a singlet excitation quantum in the light harvesting bacteriochlorophyll aggregate extends over at least seven trapping molecules of the type P870, P985, etc.

Fluorescence of chlorophyll in photosynthetic systems. I. Analysis of "weak light effect" in isolated chloroplasts.

The fluorescence yield of chlorophyll in isolated spinach chloroplasts was studied in the steady state under various excitation light intensities, and a relationship between intensity and fluorescence was obtained. It was observed that the fluorescence yield in the strong light region was higher than that in the weak light region (“weak light effect”). Effects of electron carriers (ferricyanide, 2,6-dichlorophenolindophenol and menadione), photosynthetic inhibitors (3-(4′-chlorophenyl)-1,1-dimethylurea, 5-bromo-3-isopropyl-6-methyluracil and o -phenanthroline), heat treatment and temperature on the “weak light effect” were examined. To interpret the “weak light effect”, a back flow of electrons making a short circuit between the oxidized and reduced products of the oxygen-evolving photochemical reaction is inferred. The amount of the back flow is estimated to be 1·10 −5 –2·10 −5 equiv/mg chlorophyll·h.