ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack. These observations, in addition to a slight increase of charge density of the surface—as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies—and partial unstacking of the membranes—as monitored by digitonin method and 540 nm light scattering changes—after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.     

ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants. Dependence on structural properties of the membranes

The ATP-induced quenching of chlorophyll fluorescence in chloroplasts of higher plants is shown to be inhibited when the mobility of the protein complexes into the thylakoid membranes is reduced. Its occurrence also requires the presence of LHC complexes and the ability of the membranes to unstack.These observations, in addition to a slight increase of charge density of the surface-as indicated by 9-aminoacridine fluorescence and high salt-induced chlorophyll fluorescence studies-and partial unstacking of the membranes-as monitored by digitonin method and 540 nm light scattering changes-after phosphorylation, suggest that the ATP-induced quenching of chlorophyll fluorescence could reflect some lateral redistribution of membrane proteins in the lipid matrix of the thylakoids.Abbreviations ATP adenosine triphosphate - 9-AA 9-aminoacridine - Chl chlorophyll - EDTA ethylenediaminetetraacetate - GDA glutaraldehyde - Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - LHC light-harvesting chlorophyll a/b complex PS photosystem     

Circularly polarized chlorophyll luminescence reflects the macro-organization of grana in pea chloroplasts

Circular polarization of luminescence (CPL; Steinberg IZ (1978) Annu Rev Biophys Bioeng 7: 113–137) was applied to study pea chloroplasts in different structural states. The structural changes of chloroplasts were induced by variation of osmotic pressure, concentration of magnesium-ions or photoinhibition. Both large CPL and psi-type circular dichroism (psi, polymerization and salt induced) signals appeared in the presence of granal macrostructure and were sensitive to structural changes of the grana. The relation was studied between the amount of CPL expressed as an emission anisotropy factor g _em and amplitudes of the red psi-type CD bands. The positive psi-type CD band was not directly correlated with g _em possibly due to a large contribution of circular intensity differential scattering to the measured CD spectra. However, a linear correlation between the amplitude of the negative psi-type CD band and g _em was found. The CPL signal of pea chloroplasts was attributed to a psi-type origin, which is observed in macroaggregates with densely packed chromophores with a long-range chiral order, and directly depends on the level of macroorganization. With the use of CPL-based microscopy, the long-range packing of LHC II particles can be studied in individual chloroplasts in future. In addition, the CPL method in general allows the study of the macro-organization of grana in green leaves, where conventional light-transmission methods fail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intramembranous particles and chlorophyll complexes in chloroplasts.

The size and population density of large and small particles from freeze-fractured chloroplasts of three wild-type algae and of normal spinach were determined. Computer analyses of low-temperature absorption spectra of chloroplast preparations from these species were performed, and a possible correlation between the occurrence of seven chlorophyll complexes and the aforementioned properties of the intramembranous particles was studies. It was found that only single-sized particles occur in a species containing neither chlorophyll b nor chlorophyll a-685 complexes. The three remaining species carry particles of two sizes, termed large and small particles. However, from quantitative considerations it is concluded that the chlorophyll content of none of the various pigment complexes is related to the size and the population density of the studied particles. If such a relationship exists, it seems likely to be due to the carrier moiety of the chorophyll b-chlorophyll a-685 complex.     

Studies on chlorophyll fluorescence in chloroplasts. I. Effects of dithionite on fluorescence of chlorophyll A in isolated spinach chloroplasts

Effects of dithionite on the time-course of fluorescence emitted from chlorophyll a in isolated spinach chloroplasts were studied. Addition of dithionite markedly shortened the induction period of fluorescence and increased the steady-state level of fluorescence. However, a small but distinct induction, comparable to that observed in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea, was always observed in the presence of dithionite. When the fluorescence change was determined in the presence of DCMU, preincubation of the chloroplasts with dithionite for a prolonged period further shortened, but only slowly, the induction period. However, addition of DCMU during the incubation period abolished most of the effects of dithionite in reducing the induction period. The results obtained were interpreted in terms of the reduction by dithionite of endogenous electron carriers associated with photosystem 2.     

ATP-induced increase in chlorophyll fluorescence. Characterization of rapid and slow induction phases

The biphasic rise of chlorophyll fluorescence induced in the dark (following activation of the latent ATP-ase) upon ATP-hydrolysis was investigated in detail, yielding the following main results: (1) The rapid phase is independent of artificial reductants or redox mediators. On the contrary, the slow phase requires such additions. (2) The slow phase is selectively eliminated by substances which collapse the transmembrane proton gradient, while the rapid phase may even be stimulated. (3) The ratio of rapid-to-slow phase is favored by a high degree of chloroplast integrity. The same factors which favor the rapid phase appear to be essential for a pronounced ‘slow electrogenic reaction’ in the flash-induced P 515 absorbance change. (4) For the rapid phase of the ATP-induced fluorescence increase, neither a ΔpH nor a Δψ are obligatory intermediates. (5) Hydroxylamine at about 5 · 10^?3 M causes a preferential stimulation of the rapid phase by about a factor 2. (6) There is selective inhibition of the slow phase by DBMIB, dinitrophenylether of iodonitrothymol, Bathocuproine and HQNO (2-heptyl-4-hydroxy quinoline-N-oxide) which are known to block at the level of the Cyt $\text{b}\text{f}$ FeS-complex. (7) The rapid phase is not affected by presence of 5 mM ferricyanide; however, there is substantial suppression if in addition a lipophilic redox mediator, like diamino-durene, is present. It is concluded that the two components of the reverse coupling reactions, reflected by the biphasic ATP-induced fluorescence rise, involve different coupling intermediates and different types of reverse electron flow. The rapid component appears to reflect close interaction between the coupling factor and a redox component in the vicinity of Photosystem II.     

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.     

Comparison of ATP-induced and DCMU-induced increases of chlorophyll fluorescence

A comparative study of the ATP-induced and the DCMU-induced increases of dark chlorophyll fluorescence after activation of the latent ATPase gave the following results: (1) The ATP-induced fluorescence rise exceeds the DCMU-induced rise by an amount equivalent to the rapid component of the biphasic ATP-induced change. There is complementarity between the slow component and any preceding DCMU-induced fluorescence rise. (2) Up to 10^?4 M DCMU (3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea)), with the slow component being completely suppressed, the rapid ATP-induced phase is unaffected. It becomes eliminated, though, with an I₅₀ of about 3 · 10^?4 M. (3) No binary oscillations in dependence of the number of preilluminating flashes are observed for the rapid ATP-induced fluorescence increase. Under identical conditions such oscillations are found upon DCMU-addition. (4) The amplitude of the rapid ATP-induced fluorescence rise is unaffected by closure of Photosystem II reaction centers in presence of DCMU and NH₂OH by a single saturating flash (removal of about 50% of total quenching). With further flashes and gradual complete removal of quenching, the rapid ATP-induced change is eliminated with a two-step dependency. It is concluded that the rapid phase of the ATP-induced increase in fluorescence reflects reverse electron flow at non-B-type reaction centers, while the slow phase is linked to reverse electron flow at B type centers. On the basis of these results a model is proposed for heterogeneous interactions between the ATPase and B-type and non-B-type electron-transport chains. ‘Direct coupling’ appears to be possible between CF₀-CF₁ and those electron-transport chains which are located in the stroma-exposed margin region of the grana stacks (PS IIβ units with non-B-type properties).     

Intramembranous particles and chlorophyll complexes in chloroplasts

The size and population density of large and small particles from freeze-fractured chloroplasts of three wild-type algae and of normal spinach were determined.Computer analyses of low-temperature absorption spectra of chloroplast preparations from these species were performed, and a possible correlation between the occurrence of seven chlorophyll complexes and the aforementioned properties of the intramembranous particles was studied.It was found that only single-sized particles occur in a species containing neither chlorophyll b nor chlorophyll a-685 complexes. The three remaining species carry particles of two sizes, termed large and small particles. However, from quantitative considerations it is concluded that the chlorophyll content of none of the various pigment complexes is related to the size and the population density of the studied particles. If such a relationship exists, it seems likely to be due to the carrier moiety of the chlorophyll b · chlorophyll a-685 complex.     

The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. 1. Isolated chloroplasts

Nonphotochemical quenching of chlorophyll fluorescence in plants is indicative of a process that dissipates excess excitation energy from the light-harvesting antenna of photosystem II. The major fraction of quenching is obligatorily dependent upon the thylakoid DeltapH and is regulated by the de-epoxidation state of the xanthophyll cycle carotenoids associated with the light-harvesting complexes. Basic principles of enzyme kinetics have been used to investigate this process in isolated chloroplasts. The extent of quenching was titrated against the estimated thylakoid lumen pH, and a sigmoidal relationship was obtained with a Hill coefficient of 4.5 and a pK of 4.7. Upon de-epoxidation, these parameters changed to 1.6 and 5.7, respectively. Antimycin A suppressed quenching, increasing the Hill coefficient and reducing the pK. The rate of induction of quenching fitted second-order kinetics with respect to illumination time, and the rate constant was dependent upon the DeltapH, the de-epoxidation state, the presence of antimycin, and also the presence of dibucaine, a quenching enhancer. All these data are consistent with the notion that quenching is caused by a conformational transition in a chloroplast thylakoid protein; this transition shows cooperativity with respect to proton binding, and is controlled by de-epoxidation state and various exogenous reagents.