Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

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 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.     

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.     

Effect of abscisic acid on activities of photosystems 1 and 2 in french bean chloroplasts

Biologia Plantarum - Abscisic acid (ABA) had no significant immediate effect on the activities of Photosystems PS 1 (DPIP/Ascorbate → MV) and PS 2 (H2O → K3[Fe(CN)6]), when added during...     

FTIR spectroscopy shows structural similarities between photosystems II from cyanobacteria and spinach.

Photosystem II (PSII), an essential component of oxygenic photosynthesis, is a membrane-bound pigment protein complex found in green plants and cyanobacteria. Whereas the molecular structure of cyanobacterial PSII has been resolved with at least medium resolution [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W. & Orth, P. (2001) Nature (London) 409, 739-743; Kamiya, N. & Shen, J.R. (2003) Proc. Natl Acad. Sci. USA 100, 98-103], the structure of higher plant PSII is only known at low resolution. Therefore Fourier transform infrared (FTIR) difference spectroscopy was used to compare PSII from both Thermosynechococcus elongatus and Synechocystis PCC6803 core complexes with PSII-enriched membranes from spinach (BBY). FTIR difference spectra of T. elongatus core complexes are presented for several different intermediates. As the FTIR difference spectra show close similarities among the three species, the structural arrangement of cofactors in PSII and their interactions with the protein microenvironment during photosynthetic charge separation must be very similar in higher plant PSII and cyanobacterial PSII. A structural model of higher plant PSII can therefore be predicted from the structure of cyanobacterial PSII.     

Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts

The complete primary structure of m-type thioredoxin from spinach chloroplasts has been sequenced by conventional sequencing including fragmentation, Edman degradation and carboxypeptidase digestion. As already reported [Tsugita, A., Maeda, K. & Schürmann, P. (1983) Biochem. Biophys. Res. Commun. 115, 1-7] these thioredoxins contain the same active-site sequence as thioredoxins from other sources. Based on the amino acid sequence thioredoxin mc contains 103 residues, has a relative molecular mass of 11425 and a molar absorption coefficient at 280 nm of 19 300 M-1 cm-1. The spinach thioredoxin mc has an overall homology of 44% with the thioredoxin from Escherichia coli mainly due to differences in the N-terminal and C-terminal regions.     

Further evidence for stroma lamellae as a source of Photosystem 1 fractions from spinach chloroplasts

The mechanism of digitonin action on spinach chloroplasts was investigated by thin sectioning. Evidence is presented which shows that digitonin continues to modify membranes for many minutes after the addition of the fixative glutaraldehyde. However, the action of digitonin can be stopped by simultaneous fixation and dilution of the detergent. Such experiments indicate that the initial action of digitonin is to release stroma lamellae which in turn yield a Photosystem 1 fraction. This interpretation is further supported by a significant correlation between the chlorophyll a/chlorophyll b ratio and the ratio of stroma to grana lamellae in spinach chloroplasts.     

Photoreduction of sulfur dioxide by spinach leaves and isolated spinach chloroplasts

Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed.     

Observation and characterisation of a transient in the yield of chlorophyll fluorescence in intact spinach chloroplasts

A transient in chlorophyll fluorescence, which is associated with a transient in 9-aminoacridine fluorescence and a perturbation in the rate of oxygen evolution, has been observed in intact spinach chloroplasts. The results indicate that changes in the redox state of Q are, at least partially, responsible for the transient in chlorophyll fluorescence. The size of the transient is highly dependent upon the concentration of inorganic phosphate and upon the pH of the medium. The properties of the transient are consistent with the suggestion that it reflects changes in the levels of stromal intermediates during induction.Abbreviations BES NN-Bis(2-hydroxyethyl)2-aminoethanesulphonic acid dihydroxyacetone-P(DHAP): dihydroxyacetone phosphate glycerate-3-P (PGA): glycerate-3-phosphate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-Morpholino)ethanesulphonic acid - Pi inorganic phosphate - qE quenching of chlorophyll fluorescence by the energisation of the thylakoid membrane - qQ quenching of chlorophyll fluorescence by oxidised Q, the electron acceptor of photosystem 2 - ribose-5-P (R5P) ribose-5-phosphate - Rbu-5-P ribulose-5-phosphate     

Role of beta-carotene in the reaction centres of photosystems I and II of spinach chloroplasts prepared in non-polar solvents.

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl4, n-hepatane) and the beta-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b6 in the ratio 1 : 2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP+ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn2+ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of beta-carotene in photochemistry separate from its roles in energy transfer and photoprotection. Removal of the fraction of beta-carotene closely associated with the Photosystem I reaction centre caused the rate of NADP+ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of beta-carotene, photochemistry can still be observed, however the specific association of beta-carotene with the reaction centre is required for maximal rates. We propose that beta-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state beta-carotene and chlorophyll in the first excited state.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics