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.     

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.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

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.     

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.     

THE EFFECT OF PHOSPHORYLATION UNCOUPLERS AND ELECTRON TRANSPORT INHIBITORS UPON SPECTRAL SHIFTS AND DELAYED LIGHT EMISSION OF PHOTOSYNTHETIC BACTERIA

Abstract— Delayed light emission (measured 4 msec after excitation) and the light-induced red shifts of the bacteriochlorophyll and carotenoid absorption bands of chromatophores of Rhodopseudomonas spheroides were inhibited by a variety of reagents. These included anti-mycin A, NQNO, CCCP, desaspidin, quinacrine, chlorpromazine, 2,4-dinitrophenol, gramicidin D, Triton X-100 and valinomycin in the presence of potassium, cesium or ammonium ions. Delayed light emission was enhanced by orthophenanthroline, ethanol, succinate and glutathione.
Delayed light emission from chromatophores of Rhodospirillum rubrum was attenuated during photophosphorylation but restored approximately to its initial value in the presence of oligomycin. Since the delayed light and band shifts are inhibited under conditions which tend to deplete or block the formation of high energy phosphorylation intermediate, it is suggested that the presence of a high energy intermediate is a prerequisite for the appearance of each of the three phenomena.     

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.     

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

阴离子对邻甲基苯胺化学聚合的影响

苯胺在酸性溶液中经化学和电化学氧化所得聚合物有良好的导电性和很高的稳定性。由于导电高聚物的不溶和不熔,使许多研究工作难以深入,实际应用也受到限制,在导电高聚物方面存在的这些实际问题促使了对可溶性导电高聚物合成的研究。通过化学法合成可溶性聚(o-,m-)甲苯胺的研究结果表明,甲基在苯环上的位置对聚苯胺衍生物的电导率、溶解性等都有较明显的影响。最近,通过电化学聚合合成了聚邻甲基苯胺。     

ACTION OF DETERGENTS AND ELECTROPHORESIS ON SPINACH CHLOROPLASTS UNDER HIGH ALKALINE CONDITIONS

Abstract. The alkaline solubilization treatment (AUT) procedure followed by column electrophoresis is an easy and effective method for the preparation and isolation of PS I complexes. The subchloroplast particles prepared by the action of the detergents Triton or Triton/SDS differ in that, in the latter case, only low molecular weight polypeptides are observed. Similarly, the liquid He EPR spectra are almost identical except for the absence of a g ˜ 2.03 line in the case of the Triton/SDS treated sample and the significant absence of 1.86 and 1.89 inflections in either spectra.     

SUPPRESSION OF LIPID PHOTOPEROXIDATION BY QUERCETIN AND ITS GLYCOSIDES IN SPINACH CHLOROPLASTS

Abstract— Quercetin, quercitrin and rutin suppressed lipid photoperoxidation in spinach chloroplasts in the presence of 100 μ M carbonylcyanide m -chlorophenylhydrazone (CCCP) or 100 μ M methyl viologen (MV). Fifty percent inhibition of lipid peroxidation by quercetin was observed between 30 and 50 μ M . Concentrations of quercetin and rutin higher than 100 μ M were required to obtain 50% inhibition. Ouercitrin was more effective than rutin in the suppression of lipid photoperoxidation.
Photooxidation of the flavonols by chloroplasts in the presence of MV was suppressed by superoxide dismutase (SOD) more than 90%, and the rates of the oxidation decreased in order of quercetin, quer citrin and rutin suggesting that the reactivity of the flavonols with O₂-decreased in that order. The photooxidation of the flavonols by CCCP-poisoned chloroplasts was partially suppressed by SOD. Radicals generated in the course of lauroyl peroxide degradation also oxidized the flavonols and the oxidation was insensitive to SOD. In these experiments, oxidation rate of quercetin was faster than those of its glycosides. The results obtained suggest that flavonols can function as antioxidants in chloroplasts by scavenging both O₂-and the radicals formed during lipid peroxidation.     

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.     

ON THE ORIGIN OF GLOW PEAKS IN EUGLENA CELLS, SPINACH CHLOROPLASTS AND SUBCHLOROPLAST FRAGMENTS ENRICHED IN SYSTEM I OR II

Abstract— The origin of glow peaks (thermoluminescence) was investigated in isolated spinach chloroplasts and Euglena cells by pretreatment with various concentrations of 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU)?, different light intensities, and after mild heating at various temperatures. Experiments are also reported on subchloroplast fractions enriched in pigment systems I (PSI) or II (PSII) (prepared under conditions to reduce destruction of membranes by excessive detergent contact). These results provide the following, most likely, suggestion for the origin of glow peaks: (1) Z peak originates in metastable states; it is insensitive to DCMU, temperature (320–328 K), and appears only when other peaks are saturated (10 Wm^-2). (2) Peak I involves the use of a reducing entity A (plastoquinone) beyond Q (the primary electron acceptor of pigment system II, PSII), or, of a high “S” state (charge accumulator) of oxygen evolving system; its intensity is dramatically reduced by low concentrations (1 μM) of DCMU, and, there is more of it in PSII than in PSI particles. (3) Peak II is due to reaction of Q^- with the “S” states of the oxygen evolving system; its intensity increases upon the addition of low concentrations of DCMU, at the expense of peak I; it is most sensitive to mild heating, and there is more of it in PSII than in PSI particles. (4) Peak III was not studied here as it was not resolved in most of our preparations. (5) Peak IV is from both pigment system I and II; it is sensitive to heating (>50°C), is somewhat sensitive to DCMU, and is present in both PSI and PSII particles. (6) Peak V is from PSI; it is least sensitive to mild heating, and it is enriched in PSI particles. The present studies have extended our knowledge regarding the origin of glow peaks in spinach chloroplasts and Euglena cells; in particular, the involvement of the charge accumulating “S” states of oxygen evolution (for peaks I and II) and of system I (for peak V) are emphasized in this paper.     

INVOLVEMENT OF THYLAKOID MEMBRANE-DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

Photoinhibition of the light-regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light-induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (¹O_2,¹Δ_g) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light-induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe-S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by ¹O₂, which is photoproduced primarily by the Fe-S centers of thylakoids and diffuses into the stroma.     

EXCITATION OF MESOPHYLL AND BUNDLE SHEATH CHLOROPLASTS OF Atriplex repanda IN THE ABSENCE OF LIGHT

Abstract— Mesophyll and bundle sheath chloroplasts isolated from Atriplex repanda cells promote oxygen consumption by isobutyraldehyde or phenylacetaldehyde. In all cases, a red emission and reduction of tetrazolium blue was observed. Addition of horseradish peroxidase greatly increases the reduction of the dye. In the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, the reduction of the Hill acceptor was fully suppressed. This suppression was abolished when 2, 6-dichlorophenolindophenol and ascorbate were added to the systems. These results indicate that, in mesophyll and bundle sheath chloroplasts, chlorophylls can be efficiently excited in the absence of light and an electron flow through the photosystems can be promoted.     

THE CHEMISTRY AND BIOLOGY OF BACTERIAL LIGHT EMISSION

Abstract—Bioluminescent bacteria may be isolated from sea water, and grown on a medium containing fish (or meat or yeast) extract. Cells harvested at the peak of luminescence can be lysed osmotically, releasing into the medium the soluble enzyme bacterial luciferase, which catalyzes the bioluminescent oxidation of reduced riboflavin 5′-phosphate and long chain aldehyde by molecular oxygen. Luciferase is the simplest possible heterpolymeric protein, with an α (catalytic, 42,000 daltons)-β (regulatory, 37,000 daltons) dimeric structure. Luciferase is not constitutive; it is induced by a substance produced by the bacteria themselves and excreted into the medium. Control also involves repression (glucose) and cyclic nucleotides. Recent work has resulted in the characterization of an intermediate in the light emitting reaction postulated to be luciferase-bound 4a-peroxy-dihydro FMN. The final steps in the in vitro light-emitting reaction involve reaction of this peroxy intermediate with aldehyde in a mixed function oxidase-type reaction, yielding an excited luciferase-flavin and long chain acid. The excited state is postulated to be the luciferase-bound 4a-hydroxy-dihydro-FMN. Although the identity of the in vivo aldehyde, its localization and its metabolism is unknown, studies with mutants which fail to synthesize aldehyde suggest that the 14 carbon fatty acid is a precursor. Moreover, although bacterial luciferase is highly soluble (200 mg ml^-1 in aqueous solution) there is recent evidence from our laboratory and others that its function may involve the cytoplasmic membrane. The function of light emission is of particular interest since a considerable amount of energy is involved; assuming a quantum yield of 10%, the cell foregoes the production of about 60 ATP molecules per photon. A fully induced cell emits about 10⁴ quanta/s and about 20% (!) of the oxygen consumption of the cell has been estimated to go via the light emitting pathway. One function is in light organs of higher organisms, where they occur as symbionts. The inducible (and repressible) nature of the luminescent system may be appreciated in terms of ecological options; the bacteria may be biologically very versatile. Induction by an inducer produced by the bacteria themselves would occur only under conditions where it accumulates, as in a luminous organ of a host. In the open ocean such an accumulation does not occur; the luminous system would thus not be synthesized and energy loss via luminescence is averted, allowing the bacteria to compete in an alternate “life style”.     

PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6-diaminodurene/ascorbate and 3,6-diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO^-₂ reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP⁺ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO^-₂, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO^-₃ and NO^-₂ have no effect on photosystem I-mediated NADP⁺ reduction, NO^-₂ (10 m M ) inhibits ferricyanide-mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO^-₂ (1 m M ), but only slightly by NO^-₃. Nitrite reductase is inhibited by p -chloromercuribenzoate ( p CMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.