Atusi Takamiya

Fluorescence of chlorophyll in photosynthetic systems III. Emission and action spectra of fluorescence—Three emission bands of chlorophyll a and the energy transfer between two pigment systems

The emission and action spectra of fluorescence of photosynthetic pigments, especially of chlorophyll a, were studied in spinach chloroplasts, Anacystis nidulans, Porphyra yeroensis, Porphyridium cruentum and other photosynthesis organisms at −196°, to elucidate the behavior of the pigments in fluorescence and energy transfer. At −196°, in most of the materials used, three emission bands of chlorophyll a were observed at 684 mμ, 695 mμ and 710–735 mμ (designated as F684, F695 and F-1). The emission and action spectra showed that F684 and F695 were excited by the light absorbed by phycobilins or chlorophyll b (pigment system II) and were excited less effectively, or not at all, by the light absorbed by chlorophyll a (pigment system I). F-1 was excited by both wavelenght regions. The light absorbed by pigment system I was more effective for F-1 than for F684 and F695, and the light absorbed by pigment system II was more effective for F684 and F695 than for F-1. A quantum yield of fluorescence as high as 0.75 was obtained in spinach chloroplasts at −196°. At this temperature, F-1 amounted to approx. three-quarters of the total quanta emitted, even upon excitation by 475-mμ light. It was concluded that two forms of chlorophyll a emitting F684 and F695 are contained in pigment system II, while F-1 is involved in pigment system I. Energy is transferred from pigment system II to I, at least at −196°, and presumbly also at physiological temperatures.

Fluorescence of chlorophyll in photosynthetic systems II. Induction of fluorescence in isolated spinach chloroplasts

Abstract Isolated spinach chloroplasts show a simpler time course of chlorophyll fluorescence in the induction period than that reported previously with living cells. The effects on the time course of many factors (addition of photosynthetic inhibitors, Hill oxidants, heat treatment, light intensity, pre-illumination and background light) were investigated. Inhibitors such as 3(4′-chlorophenyl)-1,1-dimethylurea, and o-phenanthroline caused the same remarkable effect on the time course, which was different from that caused by heat treatment of the chloroplasts. The back flow of electrons, which was considered in the analysis of steady-state fluorescence in our previous paper, is also taken into consideration in the present study. A scheme is proposed to account for the findings with respect to the fluorescence during the induction period. The quantity “work integral” is introduced to correlate the observed lowering of fluorescence during the induction period with the utilization of the absorbed light energy. The concentrations of the oxidation-reduction substances in the scheme were estimated from the “work integral” obtained under varied experimental conditions.

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

Abstract 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.

Distribution of plastocyanin in plants, with special reference to its localization in chloroplasts

Abstract 1. 1. Plastocyanin, a copper protein originally discovered in Chlorella ellipsoidea , was found to occur in green leaves of the following plants: spinach, parsley, carrot, turnip, Japanese scallion, and crown daisy. The copper protein was absent from the nonphotosynthetic tissues of the plants. It was not detected in the cells of the photosynthetic purple bacterium, Rhodopseudomonas palustris . 2. 2. Plastocyanin was found to be localized in the chloroplasts of green cells. Spinach chloroplasts contain plastocyanin at a ratio of about 300 chlorophyll molecules per atom of copper of plastocyanin. The chloroplast fragments also contain plastocyanin in concentrations comparable to that of the whole chloroplasts. The plastocyanin in chloroplasts or chloroplast fragments cannot be removed by simple washing with isotonic solution, but a considerable portion can be readily extracted by hypotonic treatment. From these findings, it was inferred that plastocyanin is associated with the chlorophyll-bearing subparticulate components of chloroplasts. 3. 3. The determination of copper has revealed that the copper of the isolated plastocyanin can account for about half the total copper present in the chloroplast.

Restoration of NADP photoreducing activity of sonicated chloroplasts by plastocyanin

Summary The effects of sonic treatment of Brassica chloroplasts on photochemical activities have been investigated. Sonic treatment caused a rapid and complete abolition of the Hill reaction with NADP as electron acceptor, whereas the reaction with ferricyanide or 2,6-dichlorophenolindophenol as electron acceptor was only slightly affected by the same treatment. The addition of a catalytic amount of plastocyanin to the sonicated chloroplasts induced a marked recovery of the lost activity with respect to NADP reduction, whereas the activities with ferricyanide and 2,6-dichlorophenolindophenol were not restored. The photoreduction of NADP with ascorbate and 2,6-dichlorophenolindophenol as the electron donor was also completely abolished by sonication of the chloroplasts and completely restored by addition of plastocyanin. These experimental results are discussed with respect to the role of plastocyanin as intermediary electron carrier in the photochemical electron transport in the chloroplasts.

Studies on the delayed light emission in spinach chloroplasts. I. Nature of two phases in development of the millisecond delayed light emission during intermittent illumination

Abstract The time course of changes in delayed light emission measured at 1.2 msec (average) after each excitation flash during intermittent illumination of spinach chloroplasts showed a complicated time-dependent change. In this time course, the existence of at least two components was found. One which developed slowly during illumination was selectively and thoroughly eliminated by addition of the uncouplers methylamine or a low concentration of carbonylcyanide 3-chlorophenylhydrazone (CCCP), or by uncoupling treatments of chloroplasts such as EDTA washing, sonic oscillation of chloroplasts and addition of a low concentration of Triton X-100. This component was found to be completely suppressed by addition of inhibitors of electron transport, 3-(4′-chlorophenyl)-1,1-dimethylurea (CMU), 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and o -phenanthroline. A suspending medium of pH 6.0 was optimal for the development of the component. The other component which developed rapidly at the onset of illumination was suppressed by the inhibitors CMU, DCMU, o -phenanthroline and high concentrations of CCCP. The highest yield of this component was observed at pH 4. The mechanism of the delayed light emission was discussed on the basis of the assumption that it is ultimately excited by a reverse reaction of the electron flow through Photoreaction II. It was concluded that the former, slowly developing component of delayed light emission is related to the amounts of accumulated highenergy intermediate or state of photophosphorylation, and the latter, fast-developing component to the amounts of reduced and oxidized products formed during illumination by Photoreaction II.

Inhibitory site of carbonyl cyanide m-chlorophenylhydrazone in the electron transfer system of the chloroplasts

Abstract The inhibitory effect of carbonyl cyanide m -chlorophenylhydrazone (CCCP) on the electron transport system of chloroplasts was investigated. At concentrations higher than 1 · 10 −7 M, CCCP acted as an inhibitor of the Hill reaction. Two different processes of CCCP inhibition were discernible; the one which was accomplished very rapidly and independently of light, and the other which developed slowly and only in light. Both processes of inhibition were not reversed by washing the inhibited chloroplasts. The rapid type of inhibition in the dark was observed not only in the Hill reaction in normal (untreated) chloroplasts, but also in ascorbate plus hydroquinonesupported NADP + photoreduction and manganese-supported 2,6-dichlorophenol-indophenol (DCIP) photoreduction in heated chloroplasts, whereas NADP + photoreduction supported by reduced DCIP was not inhibited. CCCP inactivated the Hill reaction by affecting the quantum efficiency of the reaction without influencing the rate constant of the dark reaction. There was no loss in the manganese content in chloroplasts caused by incubation with CCCP in the dark. The variable fluorescence in chloroplasts was decreased by CCCP at concentrations higher than 1 · 10 −7 M, with concomitant elimination of the induction phenomenon. However, on addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the CCCP-inhibited chloroplasts there occurred a recovery of the induction phase of fluorescence. The pool size of the electron acceptor of photosystem II decreased with increasing concentrations of CCCP, but the pool size of the primary electron acceptor determined in the presence of DCMU was not affected by the poison. It is postulated that the site of CCCP inhibition is located between the endogenous redox substance (Y 1 ) which receives electrons from artificial electron donors such as ascorbate, and the primary electron donor of photosystem II (Y 2 ).

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

Abstract 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.

Improvement in separation of system I and system II particles of photosynthesis obtained by digitonin treatment

By a combined use of digitonin treatment and subsequent centrifugation on a linear sucrose density gradient, the whole green material of the chloroplast lamellae was separated into System I and System II particle fractions, leaving no other fractions of intermediate properties at the final step of separation. Each of these particle fractions obtained had properties characteristic of System I or System II with respect to the molar ratio of chlorophyll achlorophyll b, the content of P700, the fluorescence emission spectrum at −196°;, photoreduction activities with ferricyanide and NADP+, and induction of fluorescence. About 40 and 50% of the total chlorophyll in the original chloroplasts were recovered in System I and System II particles, respectively. Only small amounts of total chlorophyll (less than 10%) were found as free chlorophyll detached from the lamellae through the digitonin treatment. These results support the view that the lamellae of chloroplasts are composed of about equal amounts of System I and System II particles on a chlorophyll basis.

Effects of various cations on separation of the two photochemical systems by digitonin treatment

Abstract The effects of various cations were investigated during separation of the two photochemical systems of photosynthesis by digitonin treatment of spinach chloroplasts. Satisfactory separation was obtained only when an appropriate concentration of cations was present in the treatment medium; optimum concentrations for divalent cations (Mg 2+ , Ca 2+ , Ba 2+ , Mn 2+ ) were 3–5 mM; for monovalent cations (Na + , K + , NH 4 + ), 50 mM. The modes of action of the salts ( e.g. MgCl 2 ) were investigated. The presence of the cations in the digitonin treatment mixture was an essential factor for separation of the two photochemical systems. Similar effects of cations were also observed when chloroplasts were disrupted in a French pressure cell. The effects of various salts and of light on the lamellar structure of the chloroplasts were investigated. There was no direct relationship between the electron microscopic changes of the chloroplast ( i.e. presence or absence of stacking of lamellae) and the separation of the two photochemical systems in the digitonin treatment.