Exchange of Metabolites in Cyanophora paradoxa and its Cyanelles*

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic CO₂-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert ¹⁴C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for ¹⁴C-labelled sorbitol and ³H₂O were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements of the solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.     

The nucleotide sequence of five ribosomal protein genes from the cyanelles ofCyanophora paradoxa: Implications concerning the phylogenetic relationship between cyanelles and chloroplasts

Summary The nucleotide sequences of the ribosomal protein genesrps18, rps19, rpl2, rpl33, and partial sequence ofrpl22 from cyanelles, the photosynthetic organelles of the protistCyanophora paradoxa, have been determined. These genes form two clusters oriented in opposite and divergent directions. One cluster contains therpl33 andrps18 genes; the other contains therpl2, rps19, andrpl22 genes, in that order. Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles,Euglena gracilis and land plant chloroplasts, andEscherichia coli, using parsimony or maximum likelihood methods. In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site. The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than betweenE. gracilis chloroplasts and land plant chloroplasts.     

On the developmental dependence between Cyanophora paradoxa and Cyanocyta korschikoffiana in symbiosis

The prokaryote Cyanocyta korschikoffiana was isolated from the eukaryote Cyanophora paradoxa. The synthesis of several thylakoid proteins in these cyanelles is influenced by light and darkness and is sensitive to cycloheximide, the inhibitor of the eukaryotic host's translation. The possibility of a direct coordination between the translations of the host and of the cyanelles is discussed.Abbreviations CHM treatment addition of cycloheximide - CPN chlorophylline - PBN phycobiline - SDS-PAGE sodium-dodecylsulphate-polyacrylamide gelelectrophoresis     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Strong homology between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of two species of Acetabularia and the occurrence of unusual codon usage

Summary The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c ₁ subunits of the ubiquinol-cytochrome c ₂ oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.Abbreviations BSA bovine serum albumin - Rb Rhodobacter - Rps Rhodopseudomonas     

Photomovement in Cyanophora paradoxa

Photomovement has been studied in the symbiontic association of the colorless flagellate, Cyanophora paradoxa Korschikoff with the cyanelles, Cyanocyta korschikoffiana. There is no phototactic orientation in this organism, but a photokinetic effect. In addition, the cells show a pronounced step-up photophobic response (however no or only a weak step-down response). The phobic response is mediated by a subset of the photosynthetic pigments located in the symbiontic cyanelles. It is linked to the noncyclic photosynthetic electron transport chain but it is independent of the photosynthetic generation of a proton gradient and the ATP synthesis linked to it.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropylbenzo quinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

Effect of dehydration and high light on photosynthesis of two C3 plants (Phaseolus vulgaris L. and Elatostema repens (Lour.) Hall f.)

The effect of drought on the photosynthetic functioning of two C₃ plants, Phaseolus vulgaris and Elatostema repens, has been examined. Leaf net CO₂ uptake measured in normal air was negligible at a leaf water deficit of about 30% while the calculated leaf intercellular CO₂ concentration (Ci) was unchanged. However, both the maximal photosynthetic capacity (CO₂-dependent O₂ evolution) and apparent quantum yield, measured in the presence of saturating CO₂ levels (5 to 14%), only started to decrease within the range of 25 to 30% leaf water deficit. This shows that the drought-induced inhibition seen in normal air is not caused by an inhibition of the photosynthetic mechanism, and that in this case Ci values can be misleading. Both 77 K and room-temperature fluorescence measurements indicate that the functioning of the photosystem-II reaction centre is hardly modified by water shortage. Furthermore, an analysis of photochemical chlorophyll fluorescence quenching shows, in the absence of CO₂, that O₂ can be an efficient acceptor of photosynthetic energy, even in severly dehydrated plants which do not show net CO₂ uptake in normal air. In these plants, O₂ is probably reduced mainly via Mehler-type reactions. High-light treatment given at low O₂ increases photoinhibition as measured by the decrease of apparent quantum yield in dehydrated P. vulgaris, whereas, interestingly, 1% O₂ protects dehydrated E. repens against high-light damage. The two plants could have different protective mechanisms depending upon the O₂ level or different photoinhibitory sites or mechanisms.Abbreviations and symbols Ca, Ci ambient and calculated intercellular CO₂ concentration - Fm, Fo, Fv maximum, initial and variable fluorescence emission - LWD leaf water deficit - PPFD photosynthetic photon flux density - PSII photosystem II - qQ photochemical quenching of chlorophyll fluorescence     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate     

Hydrogen Peroxide Inhibits Photosynthetic Electron Transport in Cells of Cyanobacteria

The effect of H₂O₂ on photosynthetic O₂ evolution and photosynthetic electron transfer in cells of cyanobacteria Anabaena variabilis and Anacystis nidulans was studied. The following experiments were performed: 1) directly testing the effect of exogenous H₂O₂; 2) testing the effect of intracellular H₂O₂ generated with the use of methyl viologen (MV); 3) testing the effect of inhibiting intracellular H₂O₂ decomposition by salicylic acid (SA) and 3-amino-1,2,4-triazole (AT). H₂O₂ inhibited photosynthetic O₂ evolution and light-induced reduction of p-benzoquinone (BQ) + ferricyanide (FeCy) in the Hill reaction. The I₅₀ value for H₂O₂ was 0.75 mM. Photosynthetic electron transfer in the cells treated with H₂O₂ was not maintained by H₂O₂, NH₂OH, 1,5-diphenylcarbazide, tetraphenylboron, or butylated hydroxytoluene added as artificial electron donors for Photosystem (PS) II. The H₂O CO₂, H₂O MV (involving PSII and PSI) and H₂O BQ + FeCy (chiefly dependent on PSII) electron transfer reactions were inhibited upon incubation of the cells with MV, SA, or AT. The N,N,N",N"-tetramethyl-p-phenylenediamine MV (chiefly dependent on PSI) electron transfer was inhibited by SA and AT but was resistant to MV. The results show that H₂O₂ inhibits photosynthetic electron transfer. It is unlikely that H₂O₂ could be a physiological electron donor in oxygenic photosynthesis.     

Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO₂ photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. α,β-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO₂-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H₂O → ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.     

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae,Codium andCaulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO₂-dependent and ferricyanide-dependent O₂ evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O₂ evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO₂-dependent O₂ evolution in both species, but this was partially restored in theCodium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when theCodium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen.Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as inCodium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar,Caulerpa chloroplasts could not regain their capacity for CO₂-dependent O₂ evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids.Caulerpa chloroplasts were less sensitive to detergent than those ofCodium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

‘Evolution of Photosynthesis’ (1970), re-examined thirty years later

I have re-examined my 1970 article ‘Evolution of Photosynthesis’ (Olson JM, Science 168: 438–446) to see whether any of my original proposals still survive. My original conviction that the evolution of photosynthesis was intimately connected with the origin of life has been replaced with the realization that photosynthesis may have been invented by the Bacteria after their divergence from the Archea. The common ancestor of all extant photosynthetic bacteria and cyanobacteria probably contained bacteriochlorophyll a, rather than chlorophyll a as originally proposed, and may have carried out CO₂ fixation instead of photoassimilation. The first electron donors were probably reduced sulfur compounds and later ferrous iron. The common ancestor of all extant reaction centers was probably similar to the homodimeric RC1 of present-day green sulfur bacteria (Chlorobiaceae) and heliobacteria. In the common ancestor of proteobacteria and cyanobacteria, the gene for the primordial RC1 was apparently duplicated and one copy split into two genes, one for RC2 and the other for a chlorophyll protein similar to CP43 and CP47 in extant cyanobacteria and chloroplasts. Homodimeric RC1 and homodimeric RC2 functioned in series as in the Z-scheme to deliver electrons from Fe(OH)⁺ to NADP⁺, while RC1 and/or RC2 separately drove cyclic electron flow for the production of ATP. In the line of evolution leading to proteobacteria, RC1 and the chlorophyll protein were lost, but RC2 was retained and became heterodimeric. In the line leading to cyanobacteria, both RC1 and RC2 replaced bacteriochlorophyll a with chlorophyll a and became heterodimeric. Heterodimeric RC2 further coevolved with a Mn-containing complex to utilize water as the electron donor for CO₂ fixation. The chlorophyll–protein was also retained and evolved into CP43 and CP47. Heliobacteria are the nearest photosynthetic relatives of cyanobacteria. The branching order of photosynthetic genes appears to be (1) proteobacteria, (2) green bacteria (Chlorobiaceae plus Chloroflexaceae), and (3) heliobacteria plus cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro synthesis of the cyanelle proteins of Cyanophora paradoxa by isolated cyanelles and cyanelle RNA

Cyanelles isolated from the alga Cyanophora paradoxa Korschikoff synthesized cyanelle proteins in vitro. This synthesis was stimulated by light and totally inhibited by chloramphenicol. Cycloheximide had only a small inhibitory effect. Electrophoretic separation of the labelled soluble cyanelle proteins yielded at least 20 discrete polypeptides. The RNA isolated from the cyanelles and the whole cells was successfully translated in a rabbit reticulocyte-lysate system.Abbreviations poly(A)^-RNA, poly(A)⁺RNA nonadenylated, polyadenylated RNA; - SDS sodium dodecyl sulfate     

Inhibition of photosynthesis and respiration by batatasins

Effects of batatasins I, III and V, phenolic growth inhibitors occuring in dormant bulbils of Dioscorea batatas Decne., on photosynthetic reactions of chloroplasts from spinach (Spinacia oleracea L.) and on respiration of mitochondria from potatoes (Solanum tuberosum L.) were investigated. In chloroplasts, the batatasins effectively inhibited CO₂-dependent oxygen evolution and electron flow from water to acceptors such as dichlorophenolindophenol, ferricyanide and methylviologen. Photosystem-I dependent electron transport from ascorbate to oxygen was stimulated. The proton conductivity of thylakoid membranes was increased and phosphorylation was uncoupled from electron transport. Inhibition of electron transport with water as electron donor appeared to precede uncoupling. In mitochondrial, batatasin I did not much inhibit succinate-dependent O₂ uptake in the absence of ADP, but caused strong inhibition in the presence of ADP. Batatasins III and V inhibited oxygen uptake irrespective of the presence or absence of ADP. Inhibition of chloroplast and mitochondrial reactions by batatasins was shown to be reversible.Abbrevations B-I batatasin I, 6-hydroxy-2,4,7-trimethoxyphenanthrene - B-III batatasin III, 3,3-dihydroxy-5-methoxybibenzyl - B-V batatasin V, 2-hydroxy-3,4,5-trimethoxybibenzyl - Chl chlorophyll - MV methylviologen - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PVP polyvinylpyrrolidone     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid