Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the watersplitting activity in photosynthesis

Plant materials (intact leaves, chloroplasts or subchloroplast particles) preilluminated at a low temperature (e.g. −60°C) were rapidly cooled to −196°C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as Z_v, A, B₁, B₂ and C bands. The A, B₁, B₂ and C bands appeared at constant temperatures, −10, +25, +40 and +55°C, respectively, being independent of the illumination temperature, but the Z_v band appeared at a variable temperature slightly higher than the illumination temperature. The B₁ and B₂ bands were absent in the thermoluminescence profiles of samples devoid of the oxygenevolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

Complementarity of ATP-induced and light-induced absorbance changes around 515 nm

A comparative study of the light-induced and the ATP-induced changes of P-515 absorbance gave the following results: (1) Following light activation of the latent ATP-hydrolase, ATP can induce a ΔA(515) of about the same size as that observed either in continuous light or by a saturating light flash. The ATP-induced ΔA(515) is stable in the dark as long as ATP is hydrolysed. (2) Any preceding ATP-induced ΔA(515) reduces the size of a consequent light-induced ΔA(515), and vice versa. The total P-515 absorbance change which can be induced by ATP and light is constant; there is strict complementarity of ATP- and light-induced ΔA(515). (3) The suppression of the flash-induced ΔA(515) by a preceding ATP-induced ΔA(515) is accompanied by an about 15-fold acceleration of the overall dark-decay rate, which is not further accelerated by addition of 0.2 μM valinomycin. (4) Adopting the kinetic model of Schapendonk (Doctoral Thesis, Wageningen, 1980) it is concluded that the apparent acceleration of the overall dark-decay rate results from a specific elimination of the slowly decaying ‘Reaction II’ component. ATP hydrolysis is suggested to produce and to maintain the Reaction II-type electrochromic pigment shift in the dark. (5) The data offer an alternative explanation to the prevailing notion that increased proton conductance via the activated ATPase is the main cause for the apparent acceleration of the overall decay rate of the flash-induced ΔA(515) following preillumination or under ‘phosphorylating conditions’. (6) On the basis of the presented data it is argued that the total number of available sites which can produce a Reaction II-type electrochromic pigment shift is strictly limited. Consequently, the notion of a localized ATP- or light-induced field is favored. The properties of this localized field would suggest a close link to energy-dependent changes at the coupling factor complex and to the electrogenic reactions coupled with cyclic photophosphorylation.     

pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

External electric field effects on prompt and delayed fluorescence in chloroplasts

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.     

Flash-induced photophosphorylation in chloroplasts with activated ATPase

Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K⁺ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower threshold.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

Adenosine triphosphate-generated transmembrane electric potential in chloroplasts

Utilizing oxonol VI as a transmembrane electric potential indicating dye, chloroplasts are shown to develop rapid transient light-induced and ATP-induced potentials. Following the large transient signal smaller steady-state potentials are maintained with either driving system. The ATP-induced potential in the dark depends upon preactivation of the light-triggered ATPase of the chloroplasts, and is inhibited by uncouplers, ionophores such as valinomycin, and energy-transfer inhibitors such as tentoxin, Dio-9 or DCCD. Nigericin increased the signal of both the light- and the ATP-induced reactions. The fact that relatively large transient membrane potentials are induced by either a dark-to-light transition or ATP in the dark provides an explanation for previously observed phenomena such as early kinetics of photophosphorylation and the ATP-induced luminescence.     

Potentiometric titration of Photosystem II fluorescence decay kinetics in spinach chloroplasts

The fluorescence yield of chloroplasts reflects the redox state of the electron acceptor of the Photosystem II reaction center, with increasing yield as the acceptor is reduced. Chemical reductive titrations of fluorescence yield in chloroplasts at room temperature indicate two distinct midpoint potentials, suggesting the possibility of Photosystem II electron acceptor heterogeneity. We have carried out a potentiometric titration of the fluorescence decay kinetics in spinach chloroplasts using a continuous mode-locked dye laser with low-intensity excitation pulses and a picosecond-resolution single-photon timing system. At all potentials the fluorescence decay is best described by three exponential components. As the potential is lowered, the slow phase changes 30-fold in yield with two distinct midpoint potentials, accompanied by a modest (3-fold) increase in the lifetime. The titration curve for the slow component of the fluorescence decay of spinach chloroplasts is best characterized by two single-electron redox reactions with midpoint potentials at pH 8.0 of +119 and ?350 mV, with corresponding relative contributions to the fluorescence yield of 49 and 51%, respectively. There is little change in the fast and middle components of the fluorescence decay. We found that the oxidized form of the redox mediator 2-hydroxy-1,4-naphthoquinone preferentially quenches the fluorescence, causing an anomalous decrease in the apparent midpoint of the high-potential transition. This effect accounts for a significant difference between the midpoint potentials that we observe and some of those previously reported. The selective effect of reduction potentials on particular fluorescence decay components provides useful information about the organization and distribution of the Photosystem II electron acceptor.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.     

Relation between slow delayed light emission and acid-base triggered luminescence in chloroplasts

Acid-base triggered luminescence in relation to slow delayed light emission (> 3 s) was studied in chloroplasts. After analyzing their time courses, the acid-base induced luminescence curve was found to return to the original curve of delayed light emission. Peaks of the acid-base triggered luminescence induced after various darkness periods following preillumination decreased parallel to the time course of delayed light emission without base treatment. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea enhanced both the delayed light emission and acid-base induced luminescence, while carbonyl cyanide m-chlorophenylhydrazone inhibited both. Several photophosphorylation uncouplers inhibited the acid-base induced luminescence without any substantial effect on the delayed light emission. It is concluded that the acid-base triggered luminescence is not caused by the reversion of electrons from remote intermediates on the reducing side of Photosystem II. The possibility of the presence of an activation pathway for the acid-base triggered luminescence which differs from that of the delayed light emission is also discussed.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Iodination of chloroplasts. I. Properties of iodinated chloroplasts

Spinach chloroplasts exposed to iodide can be washed free of the bulk of the iodide. In the presence of lactoperoxidase and H₂O₂, iodide can be introduced into chloroplasts in high amounts and in non diffusible forms. The resultant particles, which have been named iodochloroplasts, extrude their iodide upon stimulation by light. The form and the amount of extruded iodide bears a definite relationship to the amount of incident light. A flash of marginally effective light is additive to the next such flash even after a lapse of 10 min of darkness. These and other properties of iodochloroplasts may make them of great use in the study of intermediate 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.     

Identification of the 120 μs phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH

After a 500 μs laser flash a 120 μs phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Biophys. Acta 376, 329–344, indicates that their EPR signal II_f which they suppose to be due to Z⁺, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 μs phase. We explain our 120 μs phase by the intrinsic back reaction of the excited reaction center in the presence of Z⁺, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333–342. The redox state of Z⁺ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the μs region are compared with those obtained in the ms region.     

Synergism of triggered luminescence by simultaneous treatment of pH transition and potassium addition in chloroplasts

KCl-induced luminescence in relation to slow delayed light emission (> 3 s) and pH shift-triggered luminescence was studied in preilluminated chloroplasts. An activation pathway for KCl-induced luminescence similar to that for acid-base-triggered luminescence but different from that for delayed light emission is suggested.When the chloroplasts were subjected to a small amount of pH transition together with a simultaneous addition of KCl, a synergistic enhancement of triggered luminescence was observed. The synergism was not observed when the pH transition was increased. The results are interpreted according to the protonation model for stimulated luminescence.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.