Photoinduction kinetics of electrical potential in a single chloroplast as studied with micro-electrode technique.

1. Using single chloroplasts of Peperomia metallica the kinetics of light-induced potential changes were studied. Three kinetic components (the initial fast rise, the decay in the light and the decay in the dark) were found to be characterized by time constants 4, 220 and 60 ms, respectively at light intensity 5000 1x and temperature 18 degrees C. After flash excitation the potential kept on rising for about 10 ms. Cooling of the medium down to 5 degrees C had no effect on the duration of potential rise after the flash. 2. Variations in the medium temperature in the range 2-23 degrees C had little effect on photoresponse magnitude but resulted in significant acceleration of decay in the light. 3. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (5-)0(-6) M) resulted in suppression of the magnitude of the photoresponse but was not accompanied by any change in the rate of initial rise of potential. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea-inhibited photoresponse could be restored and even enhanced by subsequent addition of N-methylphenazonium methosulfate (10(-4) M). N-Methylphenazonium methosulfate essentially influenced the time course and light-intensity curves of photoresponse. 4. The chloroplast photoresponses were of different time-courses when elicited by red (640 nm) or far red (712 nm) light. This fact as well as an enhancement effect of combined illumination by two intermittent light beams indicate on the interaction of two photosynthetic pigment systems when the photoelectric response was formed. 5. An imposed electrical field resulted in stimulation or suppression of chloroplast photoresponse depending on the polarity of the field. No indications for the existance of "reversal potential" for photoelectric response were obtained. 6. A kinetic scheme of photoresponse formation is proposed, which includes two sequential photochemical reactions of photosynthesis.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 °;K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth ～ 8 gauss) which decays with a half-time of 5.0 ms at 35 °;K. The half-time of decay is independent of temperature in the range of 10–77 °;K. This reversible signal can be eliminated by preillumination of the sample at 35 °;K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to — 150 mV prior to freezing. In the presence of ferricyanide (20–50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 °;K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680⁺ and the primary electron acceptor.     

Kinetics of luminescence in the 10−6–10−4-s range in Chlorella

The decay of luminescence in the 6–600-μs range following a microsecond flash has been studied in Chlorella. The decay is highly polyphasic; three kinetic components are outlined, in confirmation of the results of K. L. Zankel (1971, Biochim. Biophys. Acta 245, 373–385).Extrapolation of the decay to zero dark time suggests that a unique metastable species C^?₊, resulting from photochemical charge separation in the System II reaction center, is the substrate of the recombination reaction which gives rise to luminescence.The fast (5–10 μs) and medium (50–70 μs) phases of the decay denote different stabilization steps, preceding relaxation of the centers by electron and proton transduction to the photosynthetic chain.NH₂OH specifically inhibits the fast phase and enhances the medium phase. This effect is explained by assuming that the fast phase results from electron transfer from the water splitting system Z to the oxidized primary donor Y.3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), in the presence of NH₂OH elicits another fast phase. It is believed that DCMU affords a parasitic stabilization of C^?₊ by forming a complex with Q^?.     

Induction kinetics of delayed light emission in spinach chloroplasts

Induction curves of the delayed light emission in spinach chloroplasts were studied by measuring the decay kinetics after each flash of light. This study differs from previous measurements of the induction curves where only the intensities at one set time after each flash of light were recorded. From the decay kinetics after each flash of light, the induction curves of the delayed light emission measured 2 ms after a flash of light were separated into two components: one component due to the last flash only and one component due to all previous flashes before the last one. On comparing the delayed light induction curves of the two components with the fluorescence induction curves in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and in chloroplasts treated with hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the component due to the last flash only is found to be dependent on the concentration of open reaction centers and the component due to all previous flashes except the last is dependent on the concentration of closed reaction centers. This implies that the yield of the fast decaying component of the delayed light emission is dependent on the concentration of open reaction centers and the yield of the slow decaying component is dependent on the concentration of closed reaction centers.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

The effect of magnesium and phosphorylation of light-harvesting chlorophyll ab-protein on the yield of P-700-photooxidation in pea chloroplasts

The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA₈₂₀) in the microsecond time range. The extent of ΔA₈₂₀ induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg²⁺ free medium (plus 15 mM K⁺) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg²⁺ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.     

On the mechanism of the PMS-effected quenching of chloroplast fluorescence

Evidence is presented which suggests that N-methylphenazonium methosulfate suppresses the fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts by two mechanisms: (i) indirectly, by catalyzing the buildup of the phosphorylating potential X_E across the thylaknid membrane; (ii) directly, by interacting with excited chlorophyll molecules.Arguments in support of direct quenching are as follows: (i) N-methylphenazonium methosulfate is an efficient quencher of the fluorescence of chlorophyll a in methanol; (ii) the dark-irreversible portion of the light-induced fluorescence lowering in the presence of N-methylphenazonium-methosulfate increases with the concentration of the cofactor, (iii) N-methylphenazonium methosulfate lowers the fluorescence of chloroplasts at an excitation that is too weak to allow formation of X_E.Ascorbate-reduced N-methylphenazonium methosulfate (PMS-SQ) is a more efficient direct quencher of chloroplast fluorescence than oxidized PMS because the thylakoid membrane is more permeable to the reduced species. The permeability to these quenchers is enhanced by the light-induced protonation of the membrane, and suppressed by added Mg²⁺. Different permeability barriers appear to exist for the direct and for the X_E-mediated quenching by N-methylphenazonium methosulfate, since the latter is known to be insensitive to the presence of Mg²⁺.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Studies on the mechanism of ADRY agents (agents accelerating the deactivation reactions of water-splitting enzyme system Y) on thermoluminescence emission

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p), known to be the most powerful ADRY agent (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439), on thermoluminescence has been investigated. Two thermoluminescence bands were analyzed: (a) the emission peaking at about 20–30°C caused by warming up of untreated chloroplasts, illuminated with a single 5 μs flash at room temperature and frozen rapidly to 77 K; and (b) the band emitted in the range of ?10 up 10°C after warming of chloroplast suspensions containing 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) which were illuminated with a single 5 μs flash at ?15°C and frozen rapidly at 77 K. These bands were attributed to the recombination of the B ^?S₂(S₃) and X-320 ^?S₂ states, respectively (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). It was found that: (1) The B ^?S₂(S₃) band is markedly diminished at very low ANT-2p concentrations of less than one molecule per 2000 chlorophylls. (2) The inhibition of the X-320 ^?S₂ band requires significantly higher concentrations of ANT-2p (50% peak reduction at one ANT-2p molecule per 100 chlorophylls). (3) Preflashing at room temperature before cooling to ?15°C diminishes the X-320 ^?S₂ band significantly in the presence of ANT-2p, while almost no effect is observed in its absence. (4) The state X-320 ^?S₂ decays monoexponentially with a half-lifetime of 2 min at ?15°C in the absence of ANT-2p. In the presence of one ANT-2p molecule per 800 chlorophylls the decay becomes biphasic with half-lifetimes of 0.5 and 2 min and an amplitude ratio of 2:3, respectively. The results obtained can be explained consistently by the function of ANT-2p as an ADRY agent acting as a mobile species within the thylakoid membrane at room temperature. At subzero temperatures, a ‘fixed-place’ mechanism appears to be operative. The implications for the ADRY effect and thermoluminescence are discussed.     

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.     

The effect of cations and membrane permeability modifying agents on the dark kinetics of the photoelectric response in isolated chloroplasts

The kinetics of the photoelectric response induced by saturating light pulses were studied in isolated chloroplasts of Peperomia metallica as a function of K⁺- and Mg²⁺-concentrations in the medium in the absence and presence of ionophores for K⁺ and divalent cations. The dark decay of the potential generated in the light is found to be accelerated upon an increase in K⁺- or Mg²⁺-concentrations in the presence of valinomycin and A23187. An acceleration of the decay phase in the flash-induced response is also observed immediately after preillumination of the chloroplast. It is concluded that the dark kinetics of the potential decay after short and long light exposures are controlled by two different processes with rate constants of about 20 and 0.2 s^?1, respectively.     

A diffusional analysis of the temperature sensitivity of the Mg2+-induced rise of chlorophyll fluorescence from pea thylakoid membranes

The kinetics of the chlorophyll fluorescence rise induced by adding 20 mM MgCl₂ to a suspension of isolated pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) have been examined experimentally and theoretically as a function of temperature. The application of similarity arguments and particle aggregation theory to the experimental results suggests that at the first approximation, the salt-induced time-dependent fluorescence changes may be described by the diffusion-controlled lateral movement of Photosystem II pigment-protein complexes. From an analysis of the temperature dependence of the fluorescence changes, estimates obtained for the lateral diffusion coefficients were 1.85 · 10^?12–3.08 · 10^?11 cm²/s over the temperature range 10°C ? T?30°C.     

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts IV. The effects of electron-transport conditions on violaxanthin availability

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electrontransport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than for cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear r flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity.2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (≤ μM) had no effect on availability, whereas low levels of 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50% decrease at 1 μM). Furthermore, availability in 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone.3. Violaxanthin availability was zero in the dark and increased in the light to a maximum of 67% of the total violaxanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.     

Reversible uncoupling of energy transfer between phycobilins and chlorophyll in Anacystis nidulans. Light stimulation of cold-induced phycobilisome detachment

Phycobilin fluorescence of Anacystis nidulans grown at 28°C increases substantially upon cooling below 10°C. A maximal increase is found around ?5°C and amounts to 300%, with almost complete reversibility upon re-warming. Illumination with actinic light leads to considerable stimulation of the cold-induced phycobilin fluorescence increase. Analysis of the light stimulation phenomenon reveals: (1) Actinic illumination shifts the fluorescence-temperature characteristic by about 3°C upwards on the T-axis. At temperatures below 5°C the light stimulating effect becomes smaller again and fluorescence-temperature characteristics measured at high and low light intensity converge around ?5°C. (2) In the 13-8°C region a large (up to 100%) light-induced phycobilin fluorescence increase is observed, while only negligible changes occur in the dark. (3) 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea (DCMU) as well as uncouplers inhibit the light stimulation, which hence depends on coupled electron transport.In agreement with previous work (Schreiber, U. (1979) FEBS Lett. 107, 4–9) it is concluded that illumination enhances cold-induced phycobilisome detachment by increasing the net negative charge at the outer surface of the thylakoid membrane. The possible role of a fluid → ordered transition of membrane lipids (Murata, N. and Fork, D.C. (1975) Plant Physiol. 56, 791–796) is discussed.     

Slow fluorescence quenching of Type A chloroplasts. Resolution into two components

The divalent-cation-specific ionophore A23187 is used to define two components of the slow fluorescence quenching of type a spinach chloroplasts: ionophore-reversible and ionophore-resistant quenching. Ionophore-reversible quenching predominates at relatively low light intensities and approaches saturation as light levels are increased. It is sensitive to uncouplers and to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and is dark reversible. At high light intensities the bulk (> 80%) of slow fluorescence quenching is ionophore-resistant. Ionophore-resistant quenching is stimulated by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at pH 7.6 and by both CCCP and methylamine at pH 9.0. It is insensitive to DCMU and is not reversed in subsequent darkness. Taken together, the two components account for all quenching observed in Type A chloroplasts.Ionophore-reversible quenching is identified with the Mg²⁺-mediated fluorescence quenching described by Krause (Biochim. Biophys. Acta (1974) 333, 301–313) and by Barber and Telfer (in Membrane Transport in Plants (Dainty, J., and Zimmermann, U., eds.), pp. 281–288, Springer-Verlag, Berlin, 1974). Ionophore-resistant quenching, a first-order process requiring high light, resembles the quenching reported by Jennings et al. (Biochim. Biophys. Acta (1976) 423, 264–274).The resolution of the fluorescence quenching phenomenon into two distinct components reconciles the apparently contradictory observations of these earlier investigations.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by picosecond laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a.A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.