MEMBRANE POTENTIAL AND MICROSECOND TO MILLISECOND DELAYED LIGHT EMISSION AFTER A SINGLE EXCITATION FLASH IN ISOLATED CHLOROPLASTS

Abstract— The effect of light-induced and salt-jump induced membrane potential on microsecond and millisecond delayed light emission from chloroplasts, following a single 10 ns flash, have been studied. Microsecond delayed light emission is shown to be independent of the membrane potential contrary to proposals that the activation energy for delayed light emission can be modulated by transmembrane electric fields. This result is discussed in terms of the possible origin of this short-lived emission. Millisecond delayed light after a single excitation flash is enhanced by membrane potential only if a proton gradient is present. By measuring changes in ms delayed light caused by simultaneous injection of KC1 and Na-benzoate (which creates a proton gradient) in the presence of valinomycin, the light-induced potential generated across the thylakoid membrane by a single excitation flash was calibrated and found to be 128 ± 10 mV in agreement with the recent measurements of Zickler and Witt (1976) based on voltage-dependent ionophores. It is concluded that the secondary charges that give rise to ms delayed light, after a single flash, do not fully span the membrane.     

ANALYSIS OF MICROSECOND FLUORESCENCE YIELD AND DELAYED LIGHT EMISSION CHANGES AFTER A SINGLE FLASH IN PEA CHLOROPLASTS: EFFECTS OF MONO- AND DIVALENT CATIONS

Abstract. New results are presented on the effects of mono- and divalent cations on concurrent changes in the microsecond yields and kinetics of chlorophyll a fluorescence and delayed light emission, and the light saturation curve for the latter at 100 μs, following a 10 ns flash at 337 nm. (1) The fluorescence yield increases exponentially from 3 to 30 μs (lifetime, τ, 6.4 ± 0.6/μs), and decays biphasically between 50 and 800μs. (2) The delayed light emission decays biphasically with two exponential phases: fast phase, T= 7–10μs, and slow phase, T= 33–40μs. (3) The light saturation curve for 100μs delayed light emission is satisfactorily represented by a one-hit Poisson saturation curve. (4) Addition of 5 mM NaCl to salt-depleted chloroplasts decreases (by as much as 40%) the yields of μs fluorescence and delayed light emission, and the subsequent addition of 5mM MgCl₂ increases the yields (≤2 × over samples with only NaCl). (5) The fluorescence yield rise and delayed light emission decay kinetics are independent of low concentrations of cations. The lifetime of the fast phase of fluorescence decay changes from ?90μs to ?160μs, when Na⁺ or Na⁺+ Mg²⁺ are added. Based on a detailed analysis presented in this paper, the following conclusions regarding the effects of low concentrations (few mM) of mono-and divalent cations in sucrose-washed chloroplasts at room temperature are made: (a) Na⁺ decreases (?6%) and Mg²⁺ increases (? 20% compared with the Na⁺ sample) the sensitization of photosystem II photochemistry: this effect is small, but significant. (b) Na⁺ increases and Mg²⁺ decreases the efficiency for radiationless transitions in singlet excited Chl a in the antenna and closed reaction center of PS II; this includes non-radiative energy transfer to PS I, intramolecular intersystem crossing and internal conversion. The ratio of the sum of the rate constants for radiationless transitions to that for fluorescence increases by ? 2-fold upon the addition of Na⁺, and is completely reversed by the addition of Mg²⁺. (c) The rate constant for the re-oxidation of Q^- decreases (about 50%) in the presence of Na⁺ or Na⁺+ Mg²⁺. These conclusions imply that cations produce multiple changes in the primary photoprocesses of PS II at physiological temperatures. It is proposed that these changes are mutually independent and can co-exist.     

EFFECT OF CHLORIDE AND BENZOATE ANIONS ON THE DELAYED LIGHT EMISSION IN DCMU-TREATED SPINACH CHLOROPLASTS

Abstract —Chloride anions, when added to DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]-treated spinach chloroplasts, change the rate of decay of the delayed light emission in the seconds region but do not change the shape or the temperature dependence of the decay. Benzoate anions, on the other hand, change both the rate and the shape of the decay of the delayed light emission. These results are consistent with a model in which the membrane potential and the structure of the reaction center affect the decay kinetics of the delayed light emission in the seconds region.     

THE EFFECT OF INHIBITORS AND UNCOUPLERS OF PHOTOSYNTHETIC PHOSPHORYLATION ON THE DELAYED LIGHT EMISSION OF CHLOROPLASTS*

Abstract— Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4-dichlorophenyl)-1, -1-dimethylurea or 1,10(ortho)-penanthroline) inhibited delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems.     

TIME COURSE OF MICROSECOND DELAYED LIGHT EMISSION FROM SCENEDESMUS OBUQUUS AT INTERMITTENT ILLUMINATION

Abstract— For dark adapted cells, the intensity of delayed light at the onset of a light period depends on the length of the preceding dark period. Under our experimental conditions, the exciting laser light seems to reduce the intermediate pool located between the two photosystems. If dark adapted cells are illuminated, mechanisms are established within about 20 s which appear to drain electrons from the intermediate pool.     

CORRELATION BETWEEN DELAYED LIGHT EMISSION AND FLUORESCENCE OF CHLOROPHYLL a IN SYSTEM II PARTICLES DERIVED FROM SPINACH CHLOROPLASTS

Abstract— The induction transient of delayed light of chlorophyll a, excited by repetitive flashes (0.5 ms in duration) and emitted 0.1 - 1.2 ms after the flashes, was measured in system II particles derived from spinach chloroplasts. An uncoupler, gramicidin S, was always added to the particles in order to eliminate the influence of the phosphorylation system on the delayed light and to isolate a direct relationship between the delayed light emission and the primary photochemical reaction, except for the experiments described in the next paragraph. The yield of delayed light emission from the system II particles was found to be about three–times higher than that of chloroplasts on a chlorophyll content basis. System I particles, on the other hand, emitted much weaker delayed light than chloroplasts. Upon intermittent illumination, induction of delayed light in system II particles showed a decrease from the initial rise level to the steady-state level. The initial rise level was the maximum. The fluorescence induction, on the other hand, exhibited an increase from the initial rise level to the maximum steady-state level. The induction of both delayed light emission and fluorescence arrived at their final steady-state levels after the same period of illumination. Induction of delayed light emission was measured under various conditions that changed the oxidation-reduction state of the primary electron acceptor, X, of photoreaction II: by adding an electron acceptor and an inhibitor of electron transport, and by changing the light intensity. The state of A'was monitored by measuring the fluorescence yield. The yield of delayed light emission excited by each flash was found to depend on the amount of oxidized form of X present before the flash. To examine the role of the primary electron donor Y of photoreaction II in delayed light emission, effects of electron donors of photoreaction II such as Mn²⁺, hydroquinone and p-phenylenediamine were investigated. These agents were found to markedly decrease the yield of delayed light emission without altering the pattern of its induction. They had little effect on the induction of fluorescence. These findings are interpreted by a mechanism in which transformation of the reaction center from the form (X^-Y⁺) into (X Y) produces a singlet excitation of chlorophyll a that is the source of millisecond delayed light emission. This reaction is probably non–physiological and must be very slow if compared to the transformation of (X^-Y⁺) into (X^-Y). Since the form (X^-Y⁺) is produced when the excitation is transferred to the reaction center in the form (XY), it is expected in this scheme that the yield of delayed light emission should depend on the amount of the form (X Y) present before the excitation flashes. Electron donors stimulate transformation of the reaction center from (X^-Y⁺) into (X^-Y). Since this reaction competes with the process of delayed light emission, electron donors are expected to suppress delayed light emission.     

FLASH INDUCED OSCILLATION OF THE LOW TEMPERATURE THERMOLUMINESCENCE BAND Zv IN CHLOROPLASTS

Using preilluminating flashes at 2°C prior to continuous excitation of isolated chloroplasts at -80°C, a period-four oscillation with maxima at the 0th, 4th and 8th flashes was observed in the amplitude of the thermoluminescence band Z_v. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatments greatly diminished the Z_v band. On the other hand, its amplitude was not considerably affected by the addition of inhibitory concentration of DCMU. On the basis of the results, the possible origin of the Z_v band is discussed.     

CHARACTERIZATION OF A MICROSECOND INTERMEDIATE IN THE LASER FLASH PHOTOLYSIS OF SMALL PHYTOCHROME FROM OAT

Abstract— Irradiation of small phytochrome from oat in its P_r form with 15 ns laser pulses of different wavelengths(605–655 nm) gave rise to a difference absorption with maxima at 400 and 685 nm for the first detectable transient. Bleaching of a 660 nm band was observed, non-recuperable up to 1 ms. The transient absorption has a lifetime of 70±15 μs at 273 K. The transient is tentatively identified as lumi-R and the conformation of its chromophore is postulated to be more extended than that of P_r. A deviation from the exponential decay of the lumi-R absorption at 284 and 300 K and the lack of observable enhancement of the far-red absorption within 1 ms are interpreted in terms of the appearance of still other intermediates on this time scale between lumi-R and P_fr phytochrome.     

RAPID REDUCTION OF P-700 PHOTOOXIDIZED BY A FLASH AT LOW TEMPERATURE IN SPINACH CHLOROPLASTS

Abstract—Excitation of chloroplasts at low temperature (down to 4.2 K) by short laser flashes causes largely reversible absorption changes which are attributed to the photooxidation of P-700, the primary electron donor of Photosystem I. At temperatures below 100 K the dark re-reduction of P-700⁺ is biphasic. with half-times of about 122μs and 1.7 ms. The relative contribution of the two phases varies with temperature with the fast phase becoming dominant at the lowest temperatures (˜90% at 5 K). The results are interpreted in terms of tunnelling of the electron from one or two primary accepting sites back to P-700, a process which is largely dominant over the process of charge stabilisation.     

EXCITATION OF CHLOROPLASTS IN Euglena gracilis IN THE ABSENCE OF LIGHT

Abstract— Challenging Euglena gracilis —a unicellular microorganism that contains chloroplasts—with phenylacetaldehyde induces malondialdehyde formation, sustained red emission and Hill activity. In chloroplasts, phenylacetaldehyde appears to undergo peroxidase catalyzed oxidation to formic acid and triplet benzaldehyde; the latter or, less likely, a precursor thereof promotes lipid peroxidation. Triplet benzaldehyde and/or the excited species formed in lipid peroxidation transfer energy to the chlorophylls. This explanation also applies to spinach chloroplasts preparations, thus accounting for the previous unexplained observation that phenylacetaldehyde induced sustained red emission and Hill activity. A homogeneous picture is now available regarding the intracellular generation of excited states and concomitant excitation of built-in structures.     

TEMPERATURE DEPENDENCE OF CHLOROPHYLL(IDE) SPECTRAL SHIFTS and PHOTOACTIVE PROTOCHLOROPHYLLIDE REGENERATION AFTER FLASH IN ETIOLATED BARLEY LEAVES

Abstract— Absorbance spectroscopy at 77 K was used to investigate the effect of temperature on in vivo chlorophyllide shifts and photoactive protochlorophyllide regeneration after a saturating flash, which transformed all protochlorophyllide to chorophyllide. Photoactive protochlorophyllide present in darkness was stable up to 40°C. The rate of Shibata shift and protochlorophyllide regeneration after flash were strongly temperature dependent in the range 0–25°C. At 0°C, the shift was still observed but no regeneration occurred. Only slight effects were observed in the range 25–40°C. At all temperatures, the process of protochlorophyllide regeneration was significantly slower than the Shibata shift. The final chlorophyll shift from 672 to 674 nm was observed up to 40°C. The implication of these results concerning the pigment-protein interactions during the Shibata shift are discussed.     

TEMPERATURE DEPENDENCE OF CHLOROPHYLL FLUORESCENCE FROM THE LIGHT HARVESTING COMPLEX II OF HIGHER PLANTS

Abstract— Chlorophyll fluorescence spectra of LCHII, the light harvesting complex of photosystem II, have been recorded in the aggregated and trimeric forms for a range of temperatures from 293 to 4 K. At least five long-wavelength emitters in the 682–702 nm region with different temperature dependencies were found in the spectra of the aggregates. At 293 K the yield of LCHII trimers was higher than aggregates by a factor of 4, but, upon lowering the temperature, a fluorescence rise was observed which was much stronger for LCHII aggregates than for LCHII trimers, so that at 4 K their yields were comparable. The implications of these data in terms of the function of LCHII are discussed.     

TEMPERATURE DEPENDENCE OF INDUCTION OF CYCLOBUTANE-TYPE PYRIMIDINE PHOTODIMERS IN HUMAN FIBROBLASTS BY 313 nm LIGHT

The rate of cyclobutane-type pyrimidine photodimerization with 313 nm light was determined in confluent cultures of xeroderma pigmentosum cells of group A. A new method was developed for the determination of sodium borohydride reduced thymine-thymine (TT) and cytosine-thymine (CT) dirners by high pressure liquid chromatography. It was found that the yield of dimerization andtheratioof CT/TT depended on the irradiation temperature in the physiological fluence range of 2.25 to 15 kJ m⁻². Both were significantly higher at 37 than at 0°C.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

THE MECHANISM OF DELAYED LIGHT PRODUCTION BY PHOTOSYNTHETIC ORGANISMS AND A NEW EFFECT OF ELECTRIC FIELDS ON CHLOROPLASTS*,‡

Abstract— Green plants, after illumination, emit light at times far too long to be fluorescence. This delayed light is closely connected with the process of photosynthesis and seems to be one of the few ways of studying the first steps in that process. In this paper we argue that there are at least 3 or maybe 4 mechanisms producing delayed light. (1) The delayed light in the range of 1–100 msec seems to come from the recombination of electrons and holes. The photosynthetic unit must absorb 2 quanta for this process. (2) At longer times the delayed light can come from thermal fluctuations lifting an electron from the level of ferredoxin to that of chlorophyll. The unit need only absorb 1 light quantum for this kind of delayed light. (3) Similarly, a part of the long-time delayed light comes from the untrapping of holes. (4) A part of the delayed light emitted at times longer than a few minutes seems to involve molecular oxygen. Finally, we shall describe a new phenomenon involving the effect of electric fields on chloroplasts, that we feel will be helpful in understanding the untrapping mechanisms of delayed light production.     

聚二甲基硅氧烷甲苯溶液特性粘数的温度依赖性

按照Flory-Fox的稀溶液理论,一个线型高分子在θ-溶剂中的特性粘数和它的分子量有下列关系:     

SPECTRAL DEPENDENCE OF A SINGLE AND A SUBSEQUENT SECOND LIGHT PULSE INDUCING BARLEY LEAF UNFOLDING

The unfolding of etiolated barley leaves was induced by two short pulses of light separated by a dark interval. For certain wavelengths of light the effect of the “second light pulse was enhanced when the pulse was given after a 300–2000 s dark interval as compared with its effect when the two light pulses were given simultaneously. We investigated the spectral dependence of the effect of the first, inducing light flash and of the effect of the second Light flash given after a 500 s dark interval. The spectral actinity for the effect of the first flash showed phytochrome involvement. The spectral actinity for the effect of the second light flash, however, was shifted towards shorter wavelengths and the inductive action of red and far red light was attenuated.     

THE TEMPERATURE DEPENDENCE OF Pfr ACTION GOVERNS THE UPPER TEMPERATURE LIMIT FOR GERMINATION IN LETTUCE

In most cultivars of lettuce (Lactuca saliva L,), red light acting through the red/far-red reversible phytochrome system promotes full germination within the20–30°C range, but at progressively higher temperatures germination declines sharply. The relationship between this upper ternperature limit for germination and the temperature dependence of phytochrome action was investigated in Grand Rapids lettuce. In fresh seeds the GT₅₀ (temperature giving half maximal germination) was ca 29–30°C. In these seeds, escape from far-red reversibility did not occur at 35°C, a temperature above the GT₅₀, but occurred rapidly at 27°C, a temperature below the upper limit. Increasing periods of dark pretreatment at high temperature (35°C) or increasing concentrations of the germination inhibitor coumarin caused a progressive decline in the GT₅₀, Escape from photoreversibility did not occur at 27°C in seeds in which the GT₅₀ had been reduced to less than 25°C by coumarin or by prolonged high temperature pretreatment. These results indicate that there is a close correlation between the position of the upper temperature limit for germination, and the temperature dependence of phytochrome action. We conclude that factors that alter the upper temperature limit for germination do so by changing the temperature dependence of phytochrome action.     

AN ANALYSIS OF A TRIPLET EXCITON MODEL FOR THE DELAYED LIGHT IN CHLORELLA*

Abstract— The time dependence of the delayed light in the green alga Chlorella pyrenoidosa has been examined quantitatively in the 1 to 12 msec range after excitation with light pulses (A = 6328 Å) of 100 μsec and 4.5 msec duration. We have confirmed the data of Tollin, Ruby, and Bertsch et al., on the time course of the delayed light in the msec range. New experiments, with 100 μsec flash excitation, on the time dependence of the delayed light emitted by Chlorella treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DMU), hydroxylamine, methyl violgen, and various combinations of these chemicals are presented. Also, data on the dependence of the delayed light intensity on the intensity of the excitation light in the 1.5 and 5.0 msec range are reported. The square law dependence, reported by Jones, in the 140 and 250 msec range is confirmed in the 1.5 and 5.0 msec range at very low light levels. The experimental data on delayed light has been analyzed in terms of a model which incorporates triplet exciton fusion. The following major points result from this analysis: (1) A triplet exciton kinetic model can explain both the time dependence and the excitation intensity dependence of the delayed light emitted by Chlorella. (2) The density of triplet excitons predicted by the model from the observed delayed light intensity is much less than that which can be detected by flash photolysis measurements. Therefore, the failure of such measurements to detect triplet states in vivo does not disprove the model. (3) The possibility of changes in the rate of electron transfer reactions of photosynthesis is included in the kinetic model. The predictions from the model are compared with the effects of chemical additives on the time dependence of the delayed light decay. (4) The proposed triplet exciton model predicts that the delayed light intensity may, under certain specific conditions, be affected by a magnetic field. The negative result of an attempt to observe this effect is reported and discussed. (5) It is concluded that the proposed triplet ‘fusion’ model is a valid alternative to the electron-hole recombination model.     

PHOTOCHEMISTRY OF THE REACTION CENTERS OF SYSTEM II UNDER REPETITIVE FLASH GROUP EXCITATION IN ISOLATED CHLOROPLASTS

Abstract— Absorption changes induced in isolated chloroplasts by excitation with repetitive flash groups have been measured at 690 nm, indicating the photochemical turnover of chlorophyll-a_II (Chl-αn), and at 480 nm and 513 nm respectively, reflecting via electrochromic effect the formation of a transmembrane electric field. The data are compared with measurements of oxygen evolution. In chloroplasts with practically fully intact oxygen evolving capacity it was found: 1. The initial amplitude of the 690 nm absorption change induced by the second flash as a function of the time t_v between the first and second flash of a group increases with a half life of about 35 µs. On the other hand, the average oxygen yield due to the second flash as a function of the time t_v rises with a half life of about 600 µs (and a kinetics in the ms-range of minor extent), confirming the data of Vater et al. (1968). 2. Under far red background illumination, where contributions due to PS I in the µs-range can be excluded, the difference spectrum in the red of the absorption changes induced by the first flash corresponds with that of the absorption changes induced by the second flash fired 200 µ after the first flash. 3. The pattern of the absorption changes at 690 nm induced by repetitive double flash groups at t_v= 200 µs does not markedly change in normal chloroplasts by the presence of DBMIB?. Similar 690 nm absorption changes occur in trypsin treated chloroplasts, independent of the presence of DCMU. 4. The fast regeneration in the µs-range of Chl-a_n is also observed in the third flash of a triple flash group at a time t_v= 200 µs between the flashes of the group. 5. The initial amplitudes of the absorption changes with a decay kinetics slower than 100 µs induced by the second flash at 480 nm and 513 nm, respectively, as a function of the time t, between the first and second flash of a group, are characterized by a recovery half-time of about 600 µs, confirming earlier measurements at 520 nm (Witt and Zickler, 1974). On the basis of these results it is inferred that there does exist a photoreaction of Chl-α_n., with an electron acceptor, referred to as X_a, other than the ‘primary’ plastoquinone acceptor X320, if X320 persists in its reduced state. Under conditions of X320 being in the reduced state, this photochemical reaction was shown to be highly dissipative with respect to charging up the watersplitting enzyme system Y. Furthermore, this Chl-a_n-photoreaction with X_a does not lead to a vectorial transmembrane charge separation, which is stable for more than a few microseconds. Different models for the functional and structural organization of PS II are discussed.