Two new components of 9 and 14 kDa from spinach photosystem I complex

Two formerly-uncharacterized subunits of 9 kDa and 14 kDa were found in spinach PSI complex. The 9 kDa subunit was released upon removal of antenna chlorophyll complex, whereas the 14 kDa subunit was tightly bound to the core complex. We determined the N-terminal amino acid sequence of the 9 kDa, and an internal sequence of the 14 kDa subunit after protease treatment, since the N-terminus of the latter protein was blocked. These partial sequences suggested that both subunits are new PSI components.     

Tentative identification of the apoproteins of iron-sulfur centers of Photosystem I

A newly purified Photosystem (PS) I particle is described, with still active iron-sulfur acceptors: A, B and X. Apart from the apoprotein of P700, 3 other main polypeptides of this particle are located at 20, 17 and 10 kDa, and two minor ones are detectable at 16.5 and 8 kDa. Both in vivo ³⁵S labeling and carboxymethylation with iodo[¹⁴C]acetate show that most of the cysteine residues are located in the 8-kDa band. The amino acid composition of this band reveals important common features with small iron-sulfur proteins of the ferredoxin type.     

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.     

Natural diterpenes from <Emphasis Type="Italic">Croton ciliatoglanduliferus</Emphasis> as photosystem II and photosystem I inhibitors in spinach chloroplasts

In our search for new natural photosynthetic inhibitors that could lead to the development of “green herbicides” less toxic to environment, the diterpene labdane-8α,15-diol (1) and its acetyl derivative (2) were isolated for the first time from Croton ciliatoglanduliferus Ort. They inhibited photophosphorylation, electron transport (basal, phosphorylating and uncoupled) and the partial reactions of both photosystems in spinach thylakoids. Compound 1 inhibits the photosystem II (PS II) partial reaction from water to Na⁺ Silicomolibdate (SiMo) and has no effect on partial reaction from diphenylcarbazide (DPC) to 2,6-dichlorophenol indophenol (DCPIP), therefore 1 inhibits at the water splitting enzyme and also inhibits PS I partial reaction from reduced phenylmetasulfate (PMS) to methylviologen (MV). Thus, it also inhibits in the span of P₇₀₀ to Iron sulfur center X (F_X). Compound 2 inhibits both, the PS II partial reactions from water to SiMo and from DPC to DCPIP; besides this, it inhibits the photosystem I (PS I) partial reaction from reduced PMS to MV. With these results, we concluded that the targets of the natural product 2 are located at the water splitting enzyme, and at P₆₈₀ in PS II and at the span of P₇₀₀ to F_X in PS I. The results of compounds 1 and 2 on PS II were corroborated by chlorophyll a fluorescence.     

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles fromSynechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes adsorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center F_x reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers F_x and F_A/F_B, and (iii) reduction of photo-oxidized P700⁺ by reduced cytochromec ₅₅₃. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Isolation and characterization of cDNA clones encoding a 18.8 kDa polypeptide,the product of the gene psaL,associated with photosystem I reaction center from spinach

Several cDNA clones encoding subunit XI of photosystem I reaction center (PSI-L) have been isolated from two gt11 expression libraries based on polyadenylated RNA of spinach seedlings illuminated for 4 and 16 h, respectively. The precursor polypeptide made from these recombinant DNAs in vitro can be efficiently imported into isolated spinach chloroplasts. It is correctly processed to the size of the authentic polypeptide and integrates into the photosystem I assembly. The 834 nucleotide sequence of the longest cDNA insert encodes a precursor polypeptide of 24 kDa (216 residues) and a mature protein of probably 18.8 kDa (169 residues). Hydropathy analysis suggests that the polypeptide contains two transmembrane segments. The protein appears to originate in a single-copy gene in spinach and to be decoded from RNA species of ca. 900 bases.     

Photosystem I reaction centers fromChlamydomonas and higher plant chloroplasts

A photosystem I reaction center has been isolated fromChlamydomonas chloroplasts and compared with the photosystem I reaction center from higher plants. While the higher plant reaction center is active in cytochrome 552 photooxidation, theChlamydomonas preparation was not active unless salts were included in the assay medium or the pH was lowered to 5. Subunit III-depleted photosystem I reaction center from higher plants is also inactive in cytochrome 552 photooxidation in the absence of salts. As with theChlamydomonas reaction center, salts induced its activity. Subunit I of the photosystem I reaction center has tentatively been identified as the binding site of cytochrome 552.     

Shikonin isovalerate: An inhibitor of photosystem II in Chlorogloeopsis fritschi

Shikonin isovalerate, extracted from the roots of the desert plant Arnebia decumbens, was tested for its effect on photosynthetic electron transport system of Chlorogloeopsis fritschii. The ferricyanide-Hill reaction with water and DPC as electron donors was inhibited completely with 10^-5 M shikonin isovalerate. The photoreduction of DCPIP through photosystem II was only slightly inhibited. Photosystem I from durohydroquinone to methyl viologen was not affected using 10^-6 M shikonin isovalerate. The same concentration caused 49% inhibition of cyclic photophosphorylation. These results suggest that shikonin isovalerate inhibits photosynthetic electron flow at the plastoquinone pool.Abbreviations DCMU 3-(3,4-dichlorophenyl)-N,N-dimethyl urea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-P-benzoquinone - DCPIP 2–6-dichlorophenolindophenol - DPC Diphenylcarbazide - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Effect of gibberellic acid on photosynthesis and glycollate dehydrogenase in Anacystis nidulans

Gibberellic acid at 10^-4 Mxxx was optimal for enhancement of growth, O₂ evolution, photosystem II and I and the activity of glycollate dehydrogenase of Anacystis nidulans. A stimulatory effect was observed on photosystem II. Other concentrations of gibberellic acid were inhibitory to O₂ evolution and photosystem I. Syntheses of phycocyanin, phycoerythrin and -carotene were significantly enhanced after 48 h incubation with gibberellic acid at 10^-3 Mxxx but the chlorophyll content began to increase 3 h after adding 10^-4 Mxxx gibberellic acid.The author is with the Department of Biological Sciences, Faculty of Science, University of Science and Technology, Irbid, Jordan.     

Intrathylakoidal crystal appearance during the vital cycle of spinach chloroplasts

Summary The entire vital cycle of chloroplasts was followed in spinach leaves, in order to determine the plastidial situations leading to the build-up of intrathylakoidal crystals. This kind of inclusion was noticed in the juvenile ontogenetic stages as well as in the senescent chloroplast stage, while intrathylakoidal crystals were not present in the mature organelles. The appearance of such crystals, which are known to consist of ribulose 1,5-diphosphate carboxylase, is interpreted as a consequence of an imbalance between enzyme level and membrane function.Supported by a C.N.R. grant.     

Light saturation response of inactive photosystem II reaction centers in spinach

Oxygen evolving photosystem II particles were exposed to 100 and 250 W m^–2 white light at 20°C under aerobic, anaerobic and strongly reducing (presence of dithionite) conditions. Three types of photoinactivation processes with different kinetics could be distinguished: (1) The fast process which occurs under strongly reducing (t _1/21–3 min) and anaerobic conditions (t _1/24–12 min). (2) The slow process (t _1/215–40 min) and (3) the very slow process (t _1/2>100 min), both of which occur under all three sets of conditions.The fast process results in a parallel decline of variable fluorescence (F _v) and of Hill reaction rate, accompanied by an antiparallel increase of constant fluorescence (F _o). We assume that trapping of Q_A in a negatively charged stable state, (Q_A ^–)_stab, is responsible for the effects observed.The slow process is characterized by a decline of maximal fluorescence (F _m). In presence of oxygen this decline is due to the well known disappearance of F _v which proceeds in parallel with the inhibition of the Hill reaction; F _o remains essentially constant. Under anaerobic and reducing conditions the decline of F _m represents the disappearance of the increment in F _o generated by the fast process. We assume that the slow process consists in neutralization of the negative charge in the domain of Q_A in a manner that renders Q_A non-functional. The charge separation in the RC is still possible, but energy of excitation becomes thermally dissipated.The very slow photoinactivation process is linked to loss of charge separation ability of the PS II RC and will be analyzed in a forthcoming paper.Abbreviations F chlorophyll a fluorescence - F _o, F _v, F _m constant, variable, maximum fluorescence - F _o, F _v, F _m the same, measured in presence of dithionite (F _v suppression method) - PS II photosystem II - RC reaction centre (P680. Pheo) - P680 primary electron donor - Pheo pheophytin, intermediary electron acceptor - Q_A, Q_B the primary and secondary electron acceptor - Z, D electron donors to P680 - (Q_A)_stab, (Q_A H)_stab hypothetical modifications of Q_A resulting from photoinactivation - O-, A- and R-conditions aerobic, anaerobic and strongly reducing (presence of dithionite) conditions - MES 2-(N-morpholine) ethanesulphonic acid - DCPIP 2,6-dichlorphenolindophenol - GGOC mixture of glucose, glucose oxidase and catalase - DT-20 oxygen-evolving PS II particles     

Synchronization of Anacystis nidulans

A new technique of short alternating lightdark periods was successfully used to synchronize the blue-green alga Anacystis nidulans. Oxygen evolution during the cell cycle is characterized by a maximum in the middle of the cycle and by a minimum at the time of division, a pattern very similar to that found in synchronized green algae.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Three iron-sulfur proteins encoded by three ORFs in chloroplasts and cyanobacteria

A brief review is presented on the gene products of frxA, frxB and frxC found in chloroplasts. The product of frxA shows high sequence homologies to bacterial 2[4Fe-4S] ferredoxins, but it functions as iron-sulfur centers A and B in Photosystem I, transferring electrons to [2Fe-2S] ferredoxin. This protein is located on surface of the thylakoid membranes in a state being covered by two other proteins. Proteins homologous to frxB product are found in mitochondrial respiratory Complex I and the product of frxB may function in chlororespiration, but at present no clear function of this protein is known. The frxC gene product is found to function in light-independent chlorophyll synthesis as one of the subunits of protochlorophyllide reductase and is reviewed in comparison to nitrogenase. Several problems and future research direction in these areas are also presented.     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.     

The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I,not photosystem II

Maximum quantum yields (QY) of photosynthetic electron flows through PSI and PSII were separately assessed in thylakoid membranes isolated from leaves of Cucumis sativus L. (cucumber) that had been chilled in various ways. The QY(PSI) in the thylakoids prepared from the leaves treated at 4° C in moderate light at 220 mol quanta·m^–2·s^–1 (400–700 nm) for 5 h, was about 20–30% of that in the thylakoids prepared from untreated leaves, while QY(PSII) decreased, at most, by 20% in response to the same treatment. The decrease in QY(PSI) was observed only when the leaves were chilled at temperatures below 10° C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4° C for 5 h, the quantum flux density that was required to induce 50% loss of QY (PSI) was ca. 50 umol quanta·m^–2·s^–1. When the chilling treatment at 4° C in the light was conducted in an atmosphere of N₂, photoinhibition of PSI was largely suppressed, while the damage to PSII was somewhat enhanced. The ferricyanide-oxidised minus ascorbate-reduced difference spectra and the light-induced absorbance changes at 700 nm obtained with the thylakoid suspension, indicated the loss of P700 to extents that corresponded to the decreases in QY(PSI). Accordingly, the decreases in QY(PSI) can largely be attributed to destruction of the PSI reaction centre itself. These results clearly show that, at least in cucumber, a typical chillingsensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - P700 primary electron donor of PSI - PPFD photosynthetically active photon flux density - QY quantum yield We are grateful to invaluable comments by Prof. S. Katoh, K. Hikosaka and the members of our laboratory. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science, and Culture, Japan, to I. Terashima (#03740342 and #04640621).