Correlation between the activity of membrane-bound ATPase and the decay rate of flash-induced 515-nm absorbance change in chloroplasts in intact leaves,assayed by means of rapid isolation of chloroplasts

(1) The relationship between activation of the membrane-bound ATPase and the stimulation of dissipation of the flash-induced membrane potential by preillumination was studied in intact spinach leaves by measuring the ATPase activity of rapidly isolated chloroplasts and the decay of the flash-induced 515-nm absorbance change (ΔA₅₁₅) in intact leaves. (2) The decay of ΔA₅₁₅ was accelerated by preillumination. The ΔA₅₁₅ decay in leaves treated with N,N′-dicyclohexylcarbodiimide (DCCD) became slower and was not accelerated by preillumination. However, treatment with DCCD did not lower the intensity of delayed fluorescence. (3) Membrane-bound ATPase of chloroplasts which were rapidly isolated from the preilluminated leaves (90 s preparation time) showed a higher activity (over 200 μmol P_i/mg chlorophyll per h in the case of 2-min preillumination) than that of chloroplasts isolated from dark-adapted leaves. (4) The acceleration of ΔA₅₁₅ decay and the activation of ATPase showed similar dependences on illumination time in intact leaves. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea, carbonyl cyanide p-chlorophenylhydrazone and DCCD inhibited the activation of ATPase and the acceleration of the ΔA₅₁₅ decay by preillumination. (5) The ATPase activity of chloroplasts isolated from illuminated leaves showed a single exponential decay (‘dark inactivation in vitro’). The ATPase activity induced by illuminating the leaves became lower as the dark interval between illumination and the isolation of chloroplasts was increased (‘dark inactivation in vivo’). The time course of the decay of activity had a lag and showed a sigmoidal curve when plotted semilogarithmically. The decay had an apparent half-time of 25 min. (6) The recovery of the accelerated ΔA₅₁₅ decay in preilluminated leaves to the original slow rate showed a sigmoidal decay similar to that of the activity of ATPase in intact leaves with a half-time of about 23 min in the dark. (7) It was concluded that the decay rate of ΔA₅₁₅ reflected the chloroplast ATPase activity in intact leaves and that the ion conductance of thylakoid membrane was mainly determined by the H⁺ flux through the ATPase, the activity of which was increased after the formation of the high-energy state.     

The kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flashinduced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5×10^–8 mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H⁺ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.     

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane

Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W · m^–2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W · m^–2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W · m^–2, the ratio was approximately 35% lower than that of plants grown at 60 W · m^–2. The amount of both cytochromeb ₅₆₃ and cytochromef was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.     

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts.

Intense illumination isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg2+ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowing in the dark and is inhibited by N,N'-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.     

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts

Intense illumination of isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg²⁺ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowly in the dark and is inhibited by N,N′-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.     

亚硫酸氢钠在低光强下对叶绿体循环光合磷酸化的促进作用

我们在70年代曾发现喷洒低浓度的亚硫酸氢钠能提高多种作物叶片的光合作用速率(沈允钢等1980)。近年来,谭实和沈允钢(1987)观察到喷洒低浓度的亚硫酸氢钠不但能增加作物叶片的光合作用速率,而且同时增加其呼吸作用速率,并初     

The energy level associated with the light-triggered Mg-dependent ATPase in spinach chloroplasts

Light-induced Mg²⁺-ATPase activity of chloroplasts and the pH difference (ΔpH) across the thylakoid membrane maintained by this activity are measured simultaneously under varying conditions of preillumination time and dark decay time. It is shown that with increasing ATPase activity, ΔpH reaches a maximal level which is determined by the degree of uncoupling of the thylakoid membrane.     

Activation of the non-electrochromic component in the 518 nm absorbance change by photosystem I

The flash-induced absorbance change measured at 518 nm (P515) in intact chloroplasts consists of at least 4 kinetically different components. Here the non-electrochromic component, either called phase d or reaction 3, is studied in some detail. The effect of DCMU, DQH₂ and DBMIB on the amplitude of reaction 3 and the turnover of cytochrome f and P700 have been monitored, suggesting an involvement of photosystem 1 in the activation of the non-electrochromic absorbance change. This is confirmed by the parallel oscillation pattern found in P700 rereduction and the amplitude of reaction 3.     

Induction kinetics of photosystem I-activated P700 oxidation in plant leaves and their dependence on pre-energization

Absorbance changes ΔA ₈₁₀ were measured in pea (Pisum sativum L., cv. Premium) leaves to track redox transients of chlorophyll P700 during and after irradiation with far red (FR) light under various preillumination conditions in the absence and presence of inhibitors and protonophorous uncoupler of photosynthetic electron transport. It was shown that cyclic electron transport (CET) in chloroplasts of pea leaves operates at its highest rate after preillumination of leaves with white light and is strongly suppressed after preillumination with FR light. The FR light-induced suppression was partly released during prolonged dark adaptation. Upon FR illumination of dark-adapted leaves, the induction of CET was observed, during which CET activity increased to the peak from the low level and then decreased gradually. The kinetics of P700 oxidation induced by FR light of various intensities in leaves preilluminated with white light were fit to empirical sigmoid curves containing two variables. In leaves treated with a protonophore FCCP, the amplitude of FR light-induced changes ΔA ₈₁₀ was strongly suppressed, indicating that the rate of CET is controlled by the pH gradient across the thylakoid membrane.     

A proposed mechanism for the stimulatory effect of bicarbonate ions on ATP synthesis in isolated chloroplasts

The effect of bicarbonate ions on induction of Mg²⁺-ATPase activity, on the N-ethylmaleimide inhibition of phosphorylation and on energy-dependent adenine nucleotide exchange has been examined with pea seedling chloroplasts. Incubation of chloroplasts with N-ethylmaleimide in the presence of 15 millimolar bicarbonate in the light results in enhanced inhibition of ATP synthesis when the preillumination pH is maintained between 7.0 and 7.5. Bicarbonate also enhances Mg²⁺-ATPase activity when it is included in the light-triggering stage at pH 7.0. The conditions (medium pH, bicarbonate concentration, etc.) for demonstrating the bicarbonate-induced enhancement of the N-ethylmaleimide inhibition and ATPase activity are similar to those required for the direct effect of bicarbonate on phosphorylation. Bicarbonate, under the same conditions, does not affect adenine nucleotide exchange (binding or release). It is concluded that the stimulatory effect of bicarbonate on ATP synthesis may be related to its ability to alter directly the conformation of the chloroplast coupling factor under conditions (suboptimal pH) where the enzyme shows minimal activity.     

Evidence for an electrogenic and a non-electrogenic component in the slow phase of the P515 response in chloroplasts

The flash-induced P515 absorbance change in intact chloroplasts consists of a fast and a slow phase. There is disagreement in the literature over the origin of the slow phase. Here we argue that the flash-induced slow phase in P515 absorbance change is composed of two different components. One component is most probably due to the electrogenic Q-cycle associated with the cytochrome b/f complex. The second component has decay kinetics that are much slower than the electrogenic reactions. We suggest that the second component is due to a non-electrogenic reaction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DQH₂ durohydroquinone - MV methylviologen - P515 Absorbance change at 518 nm     

Evidence for light-dependent reversible conformational change in spinach chloroplast CF1

We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF₁) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg²⁺-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the K_m of CF₁ for both ADP and P_i. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF₁ may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF₁ which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination.     

Plastid development in primary leaves of Phaseolus vulgaris. Development of plastid adenosine triphosphatase activity during greening.

The etioplasts of dark-grown bean leaves showed ATPase (adenosine triphosphatase) activity which had a pH optimum of 8.5, was stimulated by dithiothreitol and unaffected by light-triggering. Bean chloroplasts showed a low activity of dark-induced ATPase with a pH optimum of 8.5 and a substantial amount of light-triggered activity with a pH optimum of 8.0. The light-triggered activity depended on dithiothreitol and Mg2+ and was promoted by phenazine methosulphate. Light-triggered ATPase activity was completely inhibited by 20mum-dicyclohexylcarbodi-imide. Etioplasts developed light-triggered ATPase activity in response to 30 min illumination of the etiolated leaves. During the 48 h of light-induced greening of dark-grown leaves there was a 70% increase of the chloroplast ATPase activity found after light-triggering and a 30% fall in the dark-induced activity, both expressed on a per leaf basis. As the larger part of these changes occurred during the first 30 min of illumination, it is concluded that most or all of the chloroplast ATPase was present in the etioplast, a conclusion identical with that of Lockshin et al. (1971) for maize. During 48 h of greening there was a tenfold increase in the amount of thylakoid membrane in the leaf together with an 83% fall in the ATPase activity per m2 of thylakoid membrane, measured after light-triggering.     

Ion-Stimulated Stomatal Opening Induced by Preillumination in Epidermal Strips of Commelina communis

The effects of preillumination were investigated on ion-stimulated stomatal opening of epidermal strips isolated from Commelina communis L. leaves, which are dark-starved 24 hours or more. The rate and the extent of ion-stimulated stomatal openings were increased by preexposure of epidermal strips to light. The evidences are interpreted as indicating that the energy induced by preillumination can be conserved in guard cells for considerable time periods and then used for a delayed stomatal opening in the presence of higher concentration of potassium or sodium ions. Action spectrum showed two peaks, one in blue and one in the red light region. The ratio of the blue peak to the red peak is 1.2; which is the smallest reported value in action spectra of stomatal movements. 3-(4-chlorophenyl)-1,-1-Dimethylurea suppressed the ion-stimulated stomatal opening induced by the preillumination. We conclude that the photosynthetic electron transport system, containing photosystem II, in guard cell chloroplasts is a basic system of energy acquirement for stomatal opening.     

Changes in the Ability of Photophosphorylation and Activities of Surface-Bound Adenosine Triphosphatase and Ribulose Diphosphate Carboxylase of Chloroplasts Isolated from the Barley Leaves Senescing in Darkness

Dark-induced aging of detached primary leaves of 11-day-old barley seedlings brings about a significant decline in the rates of ferricyanide [Fe(CN)₆]^3? reduction and photophosphorylations of isolated chloroplasts. Ferricyanide-supported noncyclic photophosphorylation is somewhat more susceptible to leaf aging than phenazine methosulfate (PMS)-supported cyclic phosphorylation. Non-latent membrane-bound adenosine triphosphatase (ATPase) and ribulosediphosphate carboxylase (RuDPCase) lose about half of their initial activities after 24 h, while during this period the electron transport and photophosphorylation activities are much less affected. Also, the loss of RuDPCase is almost complete, while chloroplasts still exhibit a significant level of [Fe(CN)₆]^3? reduction and photophosphorylations after 7 days of dark incubation. This would suggest that the enzymatic dark reactions are more sensitive to aging stress than the primary photochemical reactions of chloroplasts. Studies on the effect of divalent cations such as Mg²⁺ and Ca²⁺ on non-latent ATPase activity revealed that the dark stressed aging of detached leaves brings about a time dependent alteration in the response of this enzyme to Mg²⁺, but not to Ca²⁺. The former showed inhibitory as well as stimulatory response, whereas the latter always caused the usual stimulation. Addition of kinetin (50 μM) ensured retention of [Fe(CN)₆]^3? reduction, photophosphorylations and RuDPCase activity in chloroplasts during leaf aging, but it failed to preserve the initial loss in the activity of the non-activated membrane-bound ATPase.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Light Activation of Mg-dependent Adenosine 5'-Triphosphatase in Isolated Euglena Chloroplasts

Enhancement of Mg²⁺-dependent ATPase activity in Euglena gracilis chloroplasts by light in the presence of a sulfhydryl compound has been demonstrated. A number of uncouplers and energy transfer inhibitors were studied for their effects on the light enhancement of ATPase activity simultaneously with their effects on photophosphorylation. Results suggest that the light-enhanced ATPase activity in Euglena chloroplasts is an energy-initiated process and that the energy is supplied through electron flow upon illumination of the chloroplasts. However, by studying the difference in their response toward the various uncouplers and inhibitors, it seems that the two processes (photohydrolysis of ATP and photophosphorylation) share only the latter part of their energy-transferring pathway.     

The relation of the 515 nanometers absorbance change to adenosine triphosphate formation in chloroplasts and digitonin subchloroplast particles

The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K⁺ accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K⁺, addition of Na⁺, or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg²⁺ and inorganic phosphate. NH₄Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change.