Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al³⁺ showed similar but less significant effects on the fluorescence yields. Zn²⁺ and Cd²⁺ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM.

In accordance with the results of our previous study concerning the effects of Mg²⁺ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophylla of pigment system II to that of pigment system I.     

Studies on photosystem I. Characteristics of "310 material" isolated from spinach chloroplasts

Exposure of osmotically shocked chloroplasts to dilute pyridine and sonic oscillation results in the extraction of a small molecular-weight factor. Purification of the factor was accomplished using gel filtration chromatography. Due to the spectral nature of the purified species (λ_max at 310 nm) the factor was named “310 material.”Physiologically, the 310 material was found to inhibit a variety of ferredoxin-dependent photoreductions catalyzed by isolated spinach chloroplasts but stimulate both pseudocyclic photophosporylation and the ferredoxin-independent photoreduction of mammalian cytochrome c. The latter reaction was found to involve, at least partially, the formation of a Superoxide radical. Dark-reduction studies have further established that the 310 material is an autooxidizable electron carrier.Chemically, the 310 material is a water-soluble, low molecular-weight phenolic-type compound; possibly a derivative of coumaric acid. No proteinaceous material is observed in physiologically active preparations of 310 material.Based on these findings, it is concluded that the isolated 310 material acts on the reducing side of Photosystem I at or near the site of reduction of ferredoxin and competes with ferredoxin for the reducing power generated by the Photosystem I reaction center. The exact physiological role of the 310 material in the intact photosynthetic system, however, remains unknown.The similarities between the 310 material and a variety of other factors previously isolated from chloroplasts are discussed.     

Studies on chlorophyll fluorescence in chloroplasts III. Effect of artificial electron donors for photosystexn 2 on reoxidation of the fluorescence quencher, Q, in spinach chloroplasts

1. Effects of various reducing reagents including dithionite,whichserve as artificial electron donors for photosystem 2,on therecovery of fluorescence induction in the presence of3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU) during the darkincubation of preilluminated chloroplastswere investigated.
2. The dark recovery of fluorescence induction was not affectedby the addition of the p-phenylenediamine-ascorbate couple,the hydroquinone-ascorbate couple or manganese. Incubation ofchloroplasts with dithionite caused gradual suppression of thedark recovery.
3. Preillumination of chloroplasts caused partialinhibition ofthe recovery of fluorescence induction.
4. At lowintensities of excitation light, the fluorescence yieldincreasedvery slowly and continuously, and never reached asteady state.This continuous increase in fluorescence yieldunder weak lightwas due to photoinhibition of the dark recovery.A techniquewas devised to determine the steady state yieldof fluorescence,without the intervention of photoinhibition,at weak light intensities.The steady state yield of fluorescencein the presence of DCMUwas suppressed at lower excitation intensities.This drop inthe fluorescence yield was not altered by the presenceof addedreducing reagents but was suppressed after long preincubationof chloroplasts with dithionite.
5. The delayed fluorescencewith a decay time of seconds was affectedby dithionite butnot by other reductants.
6. Results are discussed in terms ofreoxidation of the reducedprimary electron acceptor, Q, bythe oxidized primary electrondonor for photosystem 2. A modelof the electron transport associatedwith photoreaction 2 isproposed to account for the experimentalresults obtained.

(Received February 27, 1973; )     

Enrichment of Photosystem I reaction center chlorophyll from spinach chloroplasts

The reaction center chlorophyll of Photosystem I in spinach chloroplasts was highly enriched. Preparations having 5–9 chlorophylls per 1 P700 were obtained by treating the Photosystem I particles prepared by digitonin treatment of chloroplasts with wet diethyl ether. All P700 present in the extracted particles was found to be photoactive, undergoing oxidation upon illumination.     

Studies on the slow fluorescence decline in isolated chloroplasts.

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar. At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission. Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sigmoidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116-125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.     

A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts.

A detailed study of the photo-induced decline in chlorophyll a fluorescence intensity (Kautsky phenomenon) in coupled isolated chloroplasts from a high level (P) to a low stationary level (S) is presented. 1. A linear relationship between P leads to S quenching and intrathylakoid H+ concentration was found. When the light-induced proton gradient was abolished by uncoupling, the fluorescence emission at room temperature was lowered proportionally to increased H+ concentration in the medium. 2. Fluorescence spectra at -196 degrees C of samples frozen at the P and S states showed no significant differences in the Photosystem I/Photosystem II ratio of fluorescence emission. Furthermore, freezing to -196 degrees C reversed the P leads to S quenching. This indicates that the P leads to S quenching is not related to an increase of spillover of excitation energy from Photosystem II to Photosystem I. 3. When Mg2+ was added to thylakoids suspended in a medium free of divalent cations, the inhibition of spillover required lower Mg2+ concentrations (half saturation at 0.6 mM). Increased proton concentration in the medium also inhibited spillover. 4. The results are interpreted in terms of two sites of Mg2+ and H+ effects on excitation deactivation in Photosystem II. One site is located on the outer face of the thylakoid membrane; action of both Mg2+ and H+ at this side diminishes spillover. The second site is located on the inner face of the membrane; as Mg2+ is displaced there by protons, a non-photochemical quenching of Photosystem II fluorescence is induced, which is manifested by the P leads to S decline.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Studies on the slow fluorescence decline in isolated chloroplasts

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar.At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission.Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll light-harvesting antenna.     

The relationship between cation-induced fluorescence and membrane stacking in isolated spinach chloroplasts

In order to study the relationship between Mg⁺⁺-induced fluorescence and membrane stacking, trypsin was used as a probe. Trypsin treatment diminished to a high degree the light-induced variable fluorescence and membrane stacking. Mg⁺⁺ markedly increased the fluorescence yield near 680 nm and membrane stacking. Pretreatment of chloroplasts with Mg⁺⁺ eliminates the effect of trypsin on cation-induced fluorescence change but not on the membrane stacking. The results presented in this contribution support the evidence that the cation-induced membrane stacking and the fluorescence yield are not linked     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of phosphorylation

We have used single-photon timing with picosecond resolution to investigate the effect of phosphorylation on the fluorescence decay from broken spinach chloroplasts. Phosphorylation of spinach thylakoids causes a quenching of the slow decay phase (equivalent to a quenching of variable fluorescence) and an increase in the yield of the middle phase decay component. In addition, phosphorylation alters the intensity dependence of fluorescence in a manner which indicates a decreased antenna size of Photosystem II. The observed changes are indicative of a State 1-State 2 transition and show a clear reversal when the membranes are dephosphorylated.