Search

To examine the effect of mitochondrial function on photosynthesis, wild-type and transgenic Nicotiana tabacum with varying amounts of alternative oxidase (AOX) were treated with different respiratory inhibitors. Initially, each inhibitor increased the reduction state of the chloroplast electron transport chain, most severely in AOX knockdowns and least severely in AOX overexpressors. This indicated that the mitochondrion was a necessary sink for photo-generated reductant, contributing to the 'P700 oxidation capacity' of photosystem I. Initially, the Complex III inhibitor myxothiazol and the mitochondrial ATP synthase inhibitor oligomycin caused an increase in photosystem II regulated non-photochemical quenching not evident with the Complex III inhibitor antimycin A (AA). This indicated that the increased quenching depended upon AA-sensitive cyclic electron transport (CET). Following 12 h with oligomycin, the reduction state of the chloroplast electron transport chain recovered in all plant lines. Recovery was associated with large increases in the protein amount of chloroplast ATP synthase and mitochondrial uncoupling protein. This increased the capacity for photophosphorylation in the absence of oxidative phosphorylation and enabled the mitochondrion to act again as a sink for photo-generated reductant. Comparing the AA and myxothiazol treatments at 12 h showed that CET optimized photosystem I quantum yield, depending upon the P700 oxidation capacity. When this capacity was too high, CET drew electrons away from other sinks, moderating the P700+ amount. When P700 oxidation capacity was too low, CET acted as an electron overflow, moderating the amount of reduced P700. This study reveals flexible chloroplast-mitochondrion interactions able to overcome lesions in energy metabolism.

A natural-artificial hybrid system was constructed to enhance photophosphorylation. The system comprises chloroplasts modified with optically matched quantum dots (chloroplast-QD) with a large Stokes shift. The QDs possess a unique optical property and transform ultraviolet light into available and highly effective red light for use by chloroplasts. This favorable feature enables photosystem II contained within the hybrid system to split more water and produce more protons than chloroplasts would otherwise do on their own. Consequently, a larger proton gradient is generated and photophosphorylation is improved. At optimal efficiency activity increased by up to 2.3 times compared to pristine chloroplasts. Importantly, the degree of overlap between emission of the QDs and absorption of chloroplasts exerts a strong influence on the photophosphorylation efficiency. The chloroplast-QD hybrid presents an efficient solar energy conversion route, which involves a rational combination of a natural system and an artificial light-harvesting nanomaterial.

The initial product of photosynthesis is NADPH (dihydronicotinamide adenine dinucleotide phosphate), which is produced from the oxidized form (NADP+) by reduction with two electrons and one proton released from Photosystem I (PSI) via ferredoxin. The proton gradient generated across the thylakoid membrane produces a proton-motive force, which is utilized to synthesize ATP by the use of ATP synthase. NADPH is used as a hydride source in the Calvin–Benson cycle to produce sugars by photosynthesis. In addition to NADP+, PSI can reduce O2 by two electrons with two protons to produce hydrogen peroxide (H2O2), which can be used as a fuel in H2O2 fuel cells. This Minireview focuses on artificial photosynthetic systems to produce the products of natural photosynthesis, such as ATP, NAD(P)H and H2O2 from NAD+ and O2 with water using solar energy, respectively. ATP was produced by use of an artificial photosynthetic membrane, composed of a photosynthetic reaction center mimic that pumps protons into the interior of the liposome, where F-type ATP synthase was incorporated. Solar-driven catalytic water splitting produces hydrogen, which can reduce NAD+ to NADH with an iridium complex catalyst in a slightly alkaline solution at room temperature. H2O2 has been produced by the combination of four-electron oxidation of H2O with four protons to evolve O2 and two-electron/two-proton reduction of O2 under sun-light irradiation. H2O2 can also be produced by direct reaction of H2 and O2 by the combination of an iridium complex catalyst and flavin coenzyme.

Enhancing solar energy conversion efficiency is very important for developing renewable energy, protecting the environment, and producing agricultural products. Efficient enhancement of photophosphorylation is demonstrated by coupling artificial photoacid generators (PAGs) with chloroplasts. The encapsulation of small molecular long-lived PAGs in the thylakoid lumen is improved greatly by ultrasonication. Under visible-light irradiation, a fast intramolecular photoreaction of the PAG occurs and produces many protons, remarkably enhancing the proton gradient in situ. Consequently, compared to pure chloroplasts, the assembled natural-artificial hybrid demonstrates approximately 3.9 times greater adenosine triphosphate (ATP) production. This work will provide new opportunities for constructing enhanced solar energy conversion systems.

Prototypes of natural biosystems provide opportunities for artificial biomimetic systems to break the limits of natural reactions and achieve output control. However, mimicking unique natural structures and ingenious functions remains a challenge. Now, multiple biochemical reactions were integrated into artificially designed compartments via molecular assembly. First, multicompartmental silica nanoparticles with hierarchical structures that mimic the chloroplasts were obtained by a templated synthesis. Then, photoacid generators and ATPase-liposomes were assembled inside and outside of silica compartments, respectively. Upon light illumination, protons produced by a photoacid generator in the confined space can drive the liposome-embedded enzyme ATPase towards ATP synthesis, which mimics the photophosphorylation process in vitro. The method enables fabrication of bioinspired nanoreactors for photobiocatalysis and provides insight for understanding sophisticated biochemical reactions.

Since solar light energy is the source of all renewable biological energy, the direct usage of light energy by bacterial cell factory has been a very attractive concept, especially using light energy to promote anaerobic fermentation growth and even recycle low-energy carbon source when energy is the limiting factor. Proteorhodopsin(PR), a light-driven proton pump proven to couple with ATP synthesis when expressed heterogeneously, is an interesting and simple option to enable light usage in engineered strains. However, although it was reported to influence fermentation in some cases, heterogeneous proteorhodopsin expression was never shown to support growth advantage or cause metabolic shift by photophosphorylation so far. Hereby, we presented the first experimental evidence that heterogeneously expressed proteorhodopsin can provide growth advantage and cause ATP-dependent metabolism shift of acetate and lactate changes in Escherichia coli at anaerobic condition. Those discoveries suggest further application potential of PR in anaerobic fermentation where energy is a limiting factor.

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field. Biotechnol. Bioeng. 2015;112: 429–437. © 2014 Wiley Periodicals, Inc.

Leaf respiration continues in the light but at a reduced rate. This inhibition is highly variable, and the mechanisms are poorly known, partly due to the lack of a formal model that can generate testable hypotheses. We derived an analytical model for non-photorespiratory CO₂ release by solving steady-state supply/demand equations for ATP, NADH and NADPH, coupled to a widely used photosynthesis model. We used this model to evaluate causes for suppression of respiration by light. The model agrees with many observations, including highly variable suppression at saturating light, greater suppression in mature leaves, reduced assimilatory quotient (ratio of net CO₂ and O₂ exchange) concurrent with nitrate reduction and a Kok effect (discrete change in quantum yield at low light). The model predicts engagement of non-phosphorylating pathways at moderate to high light, or concurrent with processes that yield ATP and NADH, such as fatty acid or terpenoid synthesis. Suppression of respiration is governed largely by photosynthetic adenylate balance, although photorespiratory NADH may contribute at sub-saturating light. Key questions include the precise diel variation of anabolism and the ATP : 2e⁻ ratio for photophosphorylation. Our model can focus experimental research and is a step towards a fully process-based model of CO₂ exchange.

Unraveling of all of the information contained in proteomes poses a tremendous chemical challenge, which is balanced by the promise of potentially transformational knowledge. Mass spectrometry offers an unprecedented arsenal of tools for diverse proteomic investigations. Recently, it was demonstrated that ultraviolet light can be utilized to initiate unique and potentially useful fragmentations in peptides and proteins. Either nonspecific dissociation or highly specific dissociation at engineered chromophoric sites is possible following photon absorption. The level of specificity and control over fragmentation in these experiments is greater than with other fragmentation methods. Novel techniques made possible by this technology are poised to make substantial contributions to the field of proteomics.

Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.

Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (Pi) concentration in the upper layer and high salinity and high Pi concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using 31P-NMR spectroscopy. At night this species migrated to the lower phosphate-rich layer and took up inorganic phosphate (Pi) which then was accumulated as polyphosphate (PPi) by an increase in the chain length of PPi During the daytime this species migrated to the phosphate-depleted surface water and utilized the accumulated PPi for photophosphorylation by decreasing the chain length of PPi During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as Pi But the cells did not convert Pi to PPi presumably due to their lack of ATP. After the second day of the experiment, conversion of Pi to PPi at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of Pi and conversion to PPi at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PPi in the metabolism of this species appears to be as a phosphate pool which regulates the level of Pi and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate-rich lower layer and the photic upper layer in stratified waters. 31P-NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms.

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy-rich phosphate donor in biological energy conversion, were followed in the mid-1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane-bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy-requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H(+)-PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H(+)-PPases differing in K(+) sensitivity were established. Over 200 H(+)-PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H(+)-PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high-molecular-weight polyphosphates (HMW-polyP). Here we will focus attention on the H(+)-PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures.

We demonstrated that a significant volume of H(2) gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H(2) gas was produced without CCCP. The role of CCCP in enhancing photobiological H(2) production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O(2) in the light. As a result, anaerobiosis during the stage of photobiological H(2) evolution was established, preventing severe O(2) inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of DeltapH across the thylakoid membrane and thus enhances the accessibility of electron and H(+) to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H(2) evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H(2) evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.

Use of the Flagyl selection procedure [Schmidt et al. (1977) Proc. Natl Acad. Sci. USA, 74, 610-614] led to the isolation of a nuclear mutant of Chlamydomonas reinhardtiidesignated thm-24. This mutant displays normal electron transport rates in vitro, possesses high latent ATPase activity bound to the thylakoid membrane, but is incapable of photophosphorylation. Decay of the transmembrane potential, as indicated by the kinetics of the 520-nm absorption change after illumination, is unusually slow and markedly biphasic. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of purified thylakoid membrane. shows mutant thm-24 to be lacking a number of polypeptides including those previously designated 4.1, 4.2 and 8.1. Treatment of purified thylakoid membranes of wild-type and mutant algae, using the chloroform-release procedure of Beechey et al. [(1975) Biochem. J. 748, 533–537] resulted in the removal of ATPase activity from each strain. In wild-type cells, the ATPase activity was of heterogeneous enzymatic origin; fractionation of the chloroform-release extracts by non-denaturing polyacrylamide gel electrophoresis yielded three distinct bands displaying ATPase activity, designated ATPases I, II and III. In contrast, extracts from membranes of mutant thm-24 yielded only one ATPase-containing fraction, co-migrating with ATPase I from wild-type. Use of electrophoretic, immunological and enzymatic methods established a correspondence of the polypeptide subunits of ATPases II and III and those of spinach coupling factor, CF1. ATPase I from either algal strain was shown to be structurally distinct from higher plant CF1 and to C. reinhardtii ATPases II and III.

Spraying a 1–2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.) Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%, respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes. (Managing editor: Ping HE)

Respiration in all cells depends upon synthesis of ATP by the ATP synthase complex, a rotary motor enzyme. The structure of the catalytic moiety of ATP synthase, the so-called F(1) headpiece, is well established. F(1) is connected to the membrane-bound and ion translocating F(0) subcomplex by a central stalk. A peripheral stalk, or stator, prevents futile rotation of the headpiece during catalysis. Although the enzyme functions as a monomer, several lines of evidence have recently suggested that monomeric ATP synthase complexes might interact to form a dimeric supercomplex in mitochondria. However, due to its fragility, the structure of ATP synthase dimers has so far not been precisely defined for any organism. Here we report the purification of a stable dimeric ATP synthase supercomplex, using mitochondria of the alga Polytomella. Structural analysis by electron microscopy and single particle analysis revealed that dimer formation is based on specific interaction of the F(0) parts, not the F(1) headpieces which are not at all in close proximity. Remarkably, the angle between the two F(0) part is about 70 degrees, which induces a strong local bending of the membrane. Hence, the function of ATP synthase dimerisation is to control the unique architecture of the mitochondrial inner membrane.

Photosynthesis is inhibited by high temperatures that plants are likely to experience under natural conditions. Both increased thylakoid membrane ionic conductance and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) deactivation have been suggested as the primary cause. The moderately heat-tolerant crop Pima S-6 cotton ( Gossypium barbadense ) was used to examine heat stressinduced inhibition of photosynthesis. Previous field-work indicated that moderate heat stress ( T = 35‐45 ∞ ∞ ∞ C) is associated with very rapid leaf temperature changes. Therefore, a system was devised for rapidly heating intact, attached leaves to mimic natural field heat-stress conditions and monitored Rubisco activation, carbon-cycle metabolites, thylakoid ionic conductance, and photosystem I activity. As a proxy for NADPH and stromal redox status the activation state of NADP-malate dehydrogenase (NADP-MDH) was measured. In dark-adapted cotton leaves, heating caused an increase in thylakoid permeability at temperatures as low as 36 ∞ ∞ ∞ C. The increased permeability did not cause a decline in adenosine 5 ¢¢ -triphosphate (ATP) levels during steady-state or transient heating. Rapid heating caused a transient decline in ribulose 1,5-bisphosphate without a decrease in Rubisco activation. Sustained heating caused a decline in Rubisco activation and also oxidized the stroma as judged by NADP-MDH activation and this is hypothesized to result from increased cyclic photophosphorylation, explaining the maintenance of ATP content in the face of increased thylakoid membrane ion leakiness.

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.

Wheat (Triticum aestivum cv. Sonalika) plants were grown with three different concentrations of salicylic acid (SA; 50/500/1000 μM) for 7 days and the effects on the level of thylakoid photochemical activities were examined. SA treatment stimulated photosystem II-catalyzed electron flow in all concentrations tested. Photosystem I-associated electron transport activity was stimulated at low concentrations of SA (50 μM) but at higher concentrations (500 and 1000 μM) the electron transport activity was drastically attenuated. Thylakoids isolated from the leaves of seedlings grown with high concentrations of SA (500 and 1000 μM) showed a substantial reduction in uncoupler (NH4Cl)-mediated stimulation in electron flow. In addition, they failed to support ADP-dependent stimulation of electron transport activity and induced a significant reduction in ATPase activity. Incubation of isolated thylakoids with SA, however, had no effect on thylakoid photofunction, indicating no direct effect of SA on photoelectron transport activity. Furthermore, high concentrations of SA specifically reduce the thylakoid cytochrome f554 level. The results suggest that SA, depending on its concentration, imparts differential effects on the photofunction of thylakoids. A low concentration of SA favours photosynthetic activity while the high concentration induces drastic attenuation of photosynthetic activity because of the decline in cytochrome f554.

Background: F1Fo ATP synthase is the fundamental means of cellular energy production in animals, plants, and almost all microorganisms. ATP is generated by oxidative or photophosphorylation in memb...

The nitrogen fixing cyanobacterium Nostoc muscorum has been found to be sensitive to the herbicide SAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)-3-(2H) pyridazinon] at 30-45 microM within 15 min. The toxicity was more severe in combined nitrogen-free (Ncomb-free) medium than in a combined nitrogen medium; this enhancement was reversible by supplementation of the medium with 3 mM glucose or 5 microM ATP, serving as carbon and/or energy source in this organism. A mutant of this cyanobacterium resistant to 3 mM SAN 6706 has been isolated and characterized to perform nitrogenase activity in exogenous ATP supplemented Ncomb-free medium. However, it exhibited a moderate growth in combined nitrogen media in the absence of external ATP. The resistance factor is higher than 100. Simultaneously, this strain possesses a cross-resistance to methylamine, a well-known inhibitor of photophosphorylation, irrespective of the exogenous ATP supply. The behavior of the mutant suggests a defective phosphorylation in its photosynthetic system.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.

Tricolorin A, (11S)-11-hydroxyhexadecanoic acid 11-O-α-L-rhamnopyranosyl-(1→ 3)-O-α-L-{2-O-(2S-methylbutanoyl)-4-O-(2S-methylbutanoyl)}-rhamnopyranosil-(1→ 2)-O-β-D-glucopyranosil-(1→ 2)-β-fucopyranoside-(1,3-lactone), the major phytogrowth inhibitor isolated from Ipomoea tricolor Cav. (Convolvulaceae) was found to be a potent uncoupler (U 50 =0.33 μM) of photophosphorylation in spinach chloroplasts. Tricolorin A inhibited H + -uptake and adenosine 5'-triphosphate (ATP) synthesis, and stimulated basal and phosphorylating electron flows. Using a combination of two well-known fluorescent ApH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine, the uncoupling behavior of tricolorin A was also demonstrated for submitochondrial particles. Polarographic data showed that high concentrations (20 μM) of tricolorin A inhibited photosystem II (PSII) electron flow at the level of plastoquinone B (Q B ). Chlorophyll (Chl) a fluorescence analysis showed that tricolorin A induced accumulation of Q A - and strongly decreased the electron transport capacity, suggesting that the target of this molecule was located at the Q B level. The macrocyclic lactone-type structure of this allelopathic agent proved to be an important structural requirement for uncoupling activity since its hydrolysis caused loss of the inhibitory potential.

The chloroplast ATP synthase utilises the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. This multi-subunit thylakoid membrane-bound enzyme consists of a proton channel, CF 0 , and an extrinsic catalytic sector, CF 1 . Stimulated by the elucidation of a three-dimensional partial structure of the mitochondrial enzyme, substantial progress has been made to understand the catalytic mechanism and interesting hypotheses have been proposed about the molecular mechanism of energy coupling. The review discusses the present state of knowledge concerning the structure, molecular genetics, catalytic mechanism, energy coupling and regulation of this important enzyme involved in photophosphorylation.

The major phytotoxic compounds (encecalin, euparin and demethylencecalin) isolated from Helianthella quinquenervis (Hook) A Gray (Asteraceae) were evaluated on different photosynthetic activities in chloroplasts isolated from spinach leaves. ATP synthesis, proton uptake and electron flow (basal, phosphorylating and uncoupled) were inhibited by encecalin and demethylencecalin in a concentration dependent manner, therefore acting as Hill reaction inhibitors. Encecalin and demethylencecalin did not affect photosystem I (electron transport from diaminodurene to methylviologen), but they inhibited photosystem II (from water to 2,5-dibromo-3-methyl-6-isopropyl-1,4-p-benzoquinone). Since these compounds inhibited electron flow in the photosystem II partial reactions from water to silicomolybdate and from diphenylcarbazide to dichlorophenol-indophenol, the site of inhibition was located in the span from P680 to QA of the electron transport chain. Euparin, inhibited ATP synthesis, proton uptake and basal and phosphorylating electron transports, but it has not effect on uncoupled electron flow from water to methylviologen. Mg2+-ATPase activity from bound membrane thylakoid chloroplasts was also inhibited by this compound. These results suggested that euparin inhibited phosphorylation in chloroplasts, acting as an energy-transfer inhibitor. © 1998 Society of Chemical Industry.

During protein import into mitochondria, matrix-localized mitochondrial hsp70 (mhsp70) interacts with the inner membrane protein Tim44 to pull a precursor across the inner membrane. We have proposed that the Tim44-mhsp70 complex functions as an ATP-dependent "translocation motor" that exerts an inward force on the precursor chain. To clarify the role of ATP in mhsp70-driven translocation, we tested the effect of the purified ATP analogues AMP-PNP and ATP gamma S on the Tim44-mhsp70 interaction. Both analogues mimicked ATP by causing dissociation of mhsp70 from Tim44. ADP did not disrupt the Tim44-mhsp70 complex, but did block the ATP-induced dissociation of this complex. In the presence of ADP, mhsp70 can bind simultaneously to Tim44 and to a peptide substrate. These data are consistent with a model in which mhsp70 first hydrolyzes ATP, then associates tightly with Tim44 and a precursor protein, and finally undergoes a conformational change to drive translocation.

The rate of both ATP synthesis and hydrolysis catalysed by the thiol-modulated and activated ATP synthase from spinach is measured as a function of all substrates including the protons inside the thylakoid lumen. The most important findings are: (1) sigmoid kinetics with respect to H in + , (2) hyperbolic kinetics with respect to ADP, ATP and phosphate, with K m for phosphate and ADP decreasing upon increasing H in + , (3) binding of ADP and phosphate in random order and competitive to ATP. Simulation of the complete set of experimental data is obtained by a kinetic model featuring Boyer's binding-change mechanism.

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to 200 nmol ATP · min-1 · mg protein-1 were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal ΔpH attainable at 40°C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40°C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of ΔpH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96→Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by ΔpH/ΔΨ transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

ATP synthesis and phosphorylating (coupled) electron flow of freshly lysed pea chloroplasts were inhibited by piquerol A and diacetyl piquerol, the latter being more active. H + -uptake, basal and uncoupled electron transport were not affected by either compound, indicating that they act as inhibitors of photophosphorylation at the H + -ATPase complex.

Photoinhibition of photosynthesis and its reactivation was studied in the cyanobaterium A. nidulans in the presence of the respiratory inhibitor sodium azide, the uncouplers carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the photosystem I elicitor phenazine methosulphate (PMS). Inhibition of dark respiration by azide increased the susceptibility of the cyanobacterium to photoinhibition. Both FCCP and CCCP also remarkably affected the process of photoinhibition in A. nidulans. The PMS at lower photoinhibitory light intensity partially protected A. nidulans from photoinhibition. The recovery from photoinhibition in the presence of azide or FCCP was slow and normal photosynthesis could not be resumed even after a longer period of incubation under suitable reactivating condition. Thus dark respiration has a key function in the process of photoinhibition of photosynthesis and its reactivation in the cyanobacterium A. nidulans.

The incorporation of 14C-leucine into the total-protein fraction of needles of Norway spruce (Picea abies [L.] Karst.) during short time incubation was used as a measure of protein synthesis in the light and in the dark. Light saturation curves, obtained for needles of different ages (new flush and 1 and 2 years old) or at different seasons (summer-winter) followed the Michaelis-Menten algorithm, exhibiting marked differences with regard to light saturation (Vmax) and the half-saturation constant (K5. 2). The light saturation curves of ATP level (mg g−1 fresh weight) and of leucine incorporation into protein (nmol mg−1 h−1) matched each other, suggesting that photophosphorylation may be decisive for the rate of protein synthesis in the light. This is confirmed by the action spectrum of leucine incorporation. which resembled an action spectrum of leaf photosynthesis, and also by partial inhibition of protein synthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of non-cyclic photophosphorylation. Light stimulated protein synthesis showed pronounced seasonal fluctuations with a summer maximum. Furmigation of 5 years old spruce trees for 3 months with SO2 in combination with O3 and/or NO2 caused a distinct enhancement of the protein synthesis rate in the light and, at a reduced absolute level, also in the dark. A similar result was obtained for 40 to 70 years old spruce stands when healthy and sick trees were compared: the latter being afflicted by the novel type of forest decline, which is characterized by yellowish bronze discolouration of sun-exposed older needles and partial loss of older needle generations (3 to 4 years old). The 1 year old needles of the unhealthy trees showed a markedly increased 14C-leucine incorporation rate which, in the dark, was even more pronounced than in the light. Stress-physiological mechanisms, which could possibly explain this stimulation, are discussed.

SUMMARY The effect of photoacclimation on cyclic (CPP), non-cyclic (NCPP), and oxidative phosphorylation was investigated in three algal species: Isochrisis galbana, Scenedesmus quadricauda and Synechococcus leopoliensis. The relative contribution of the three phosphorylative pathways to the incorporation of 32P into soluble and particulate cellular pools was assessed. To block NCPP we used 1 μm dichlorophenyl-dimethylurea (DCMU) which does not interrupt CPP. Oxidative phosphorylation was blocked by purging oxygen with N2. In all three species the ratio of CPP to the total photophosphorylation (PP) increased in the process of photoacclimation to high light. We suggest that the observed increase in CPP in high-light-adapted cells allows bypassing the relatively slow enzymatic reactions in NCPP while channelling excess electrons to the faster CPP. This redistribution of energy increases ATP supply for the augmented metabolic requirements of fast growing, high-light-adapted cells.

The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis--termed rotary catalysis--is involved.

Enhancement in the rates of photosystem (PS) II-catalysed O2 evolution was observed with thylakoids isolated from cotyledonary leaves of Brassica juncea seedlings raised in medium containing 200 mM NaCl compared to controls. The nature of this enhancement in PS II activity by NaCl treatment has been investigated. The pH profile showed an optimal PS 11(H2OFeCN) activity at pH 7.5 in thylakoids of both controls and NaCl-raised seedlings. However, the thylakoids from NaCl-raised plants showed significantly higher PS II activity at all the pHs (from 6.0 to 9.0) with maximal difference occurring at alkaline pHs. Besides the enhanced electron transport rates, light-induced proton uptake was about 30% higher in the thylakoids from NaCl-raised plants than in the control thylakoids. The 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-induced enhancement in the room temperature fluorescence was significantly higher in thylakoids from NaCl-grown plants compared to controls, suggesting more efficient PS II photochemical reactions in thylakoids from NaCl-raised seedlings. The analysis of the changes in the fluorescence yield of Chl also showed an enhancement in Fv level in NaCl-raised plants, suggesting that NaCl treatment stimulates the photochemistry of PS II. Analysis of light saturation kinetics of PS II [H2O2,6-dichloro-p-benzoquinone (DCBQ) or phenylenediamine (PD)] activity showed that the extent of stimulation by NaCl under rate-limiting light intensities was greater than at rate-saturating intensities. This result indicates that NaCl treatment not only alters the electron transport rates but also the relative quantum yield. Incubation of thylakoids at pH 9.0 inhibited FeCN-supported PS II activity more drastically in control than in NaCl-treated thylakoids. Recovery from alkaline-pH-induced inhibition occurred when the thylakoids were resuspended at pH 7.5. Reversal in PS II activity was significantly higher in the thylakoids of NaCl-raised plants than in the control thylakoids. These results suggest that the seedlings raised at moderately elevated levels of NaCl have thylakoids with enhanced stability and PS II photochemical functions.

Growth and photosynthetic performance were assessed for the Finnish wheat Triticum aestivum L. var. APU under a cool (13/10�C day/night) and a warm (30/25�C day/night) regime. Plants exhibited a certain degree of acclimation to warm growth conditions. This acclimation appeared to involve enhanced performance of both photosystem II and whole-chain electron transport. Enhanced thermal stability of photophosphorylation was also observed in warm-grown plants. Wiley Online Library

The cryoprotective efficiency of sucrose, proline and glycerol for chloroplast membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) was determined after freeze-thaw treatment in media containing the predominant inorganic electrolytes of the chloroplast stroma. In most cases, the protective capacity of equimolar concentrations of the cryoprotectants followed the order sucrose > proline > glycerol. The lower the freezing temperature the less cryoprotectant was necessary for comparable preservation of the capacity of photosynthetic electron transport. Likewise, the cryoprotective efficiency of sucrose for cyclic photophosphorylation and light-induced proton gradient increased with decreasing freezing temperature. In contrast, while proline effectively stabilized these membrane reactions at mild and moderate freezing temperatures, it was much less efficient at more severe freezing stress. Cryoprotection of photophosphorylation and proton gradient formation at given initial concentrations of glycerol was largely independent of the freezing temperature. While dissociation of the peripheral part of chloroplast coupling factor (CF1) during freeze-thaw treatment cannot be prevented in the presence of lower initial concentrations of proline and glycerol and. at mild freezing temperatures, of sucrose, the latter may stabilize this protein complex at least under more severe freezing conditions. The differences in the cryoprotective efficiency of the solutes are discussed relative to their non-ideal activity-concentration profiles, solution properties and penetration behaviour across the thylakoid membrane.

The diurnal cycling of leaf water potential (Ψleaf) in field-grown sunflower (Helianthus annuus) was used to investigate the cause of water deficitinduced limitation of net photosynthesis. Daily midafternoon decreases in Ψleaf of up to 1.5 MPa and in net photosynthesis of up to 50% were typical for irrigated sunflower during seed filling. These midafternoon values were lowered an additional 0.6 to 0.8 MPa by prolonged drought treatment. There was a nearly linear relationship between the decline in net photosynthesis and reductions in leaf conductance over the course of the day. Thus, it was unexpected to find that the low, midafternoon rates of photosynthesis were associated with the highest intercellular CO2 concentrations. These and other observations suggest that the daily decline in photosynthesis represents a ‘down regulation’ of the biochemical demand for CO2 that is coordinated with the diurnally developing need to conserve water, thus establishing a balanced limitation of photosynthesis involving both stomatal and non-stomatal factors. There were no indications that either short term (i.e. diurnal declines in Ψleaf) or long term (i.e. drought treatment) water deficits caused any damage or malfunctioning of photosynthesis. Rather, both the daily declines in photosynthesis and the nearly 25% decrease in leaf area induced by prolonged drought appeared to be well-controlled adaptive responses by field-grown sunflower plants to limited water availability.

Solar tracking has been described in several agronomic and wild species, yet variation within a given species has not been reported. To address that issue, photoperiod leaflet reorientation in response to vectorial light was measured in three alfalfa (Medicago sativa L.) germplasm pools selected from the cultivar Ladak 65. Dark-leaf germplasm responded to all photosynthetic photon flux densities (PPFD) (100 to 700 μmol/m2/s) with no detectable lag period. Following a 24-h dark treatment only high PPFD (600 to 700μmol/m2/s) elicited reorientation. Pale-leaf germplasm responded to intermediate (300 to 400 μmol/m2/s) and high PPFD in a similar manner; however, the low (100-125μmol/m2/s) PPFD treatment had little effect on reorientation. The 24-h dark treatment decreased reorientation at all PPFD. Small-leaf germplasm responded equally to all PPFD and was not affected by the 24-h dark treatment. The results suggest that available energy levels may influence the reorientation response. Carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of photophosphorylation, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of non-cyclic photosynthetic electron transport, reduced reorientation only in the pale-leaf type. These results suggest that stored energy reserves were adequate to maintain leaflet reorientation in the absence of photosynthesis in all but the pale-leaf germplasm. In that germplasm photosynthetically generated energy may be required to support reorientation.

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and Pi were measured using either cold ATP, [gamma-32P]ATP or [14C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, [gamma-32P]ATP was bound, hydrolyzed and 32Pi was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP-binding kATP = 2 . 10(5) M-1 s-1, ADP-release: kADP less than 10(-2)s-1, Pi-release: kPi = 0.1 s-1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s-1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP-binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP-release.

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF1 were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF1-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF1-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu2+ but not by Ca2+, Mg2+ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl2 and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF1-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF1-δ.

Upon EDTA treatment thylakoids lose the chloroplast coupling factor 1 (CF1) part of their ATP synthase, CF0CF1, this exposes the proton channel, CF0. The previously established ability of the CF1 subunit delta to block the proton leak through CF0 prompted us to study (a) the ability of complete CF1 and, for comparison, CF1 lacking the delta subunit to block proton leakage and thereby to reconstitute structurally some photophosphorylation activity of the remaining CF0CF1 molecules and (b) their ability to form functional enzymes (functional reconstitution). In order to discriminate between activities caused by added CF1 or CF1(-delta) and remaining CF0CF1, the former were inhibited by chemical modification of subunit beta by N,N'-dicyclohexyl carbodiimide (DCCD) and the latter by tentoxin. We found that added CF1 acted both structurally and functionally while added DCCD-treated CF1 (DCCD-CF1) acted only structurally. In contrast to previous observations, CF1(-delta) and DCCD-CF1(-delta) also acted structurally although the reduction of proton leakage was smaller than with DCCD-CF1. Hence there was no functional reconstitution without subunit delta present. Previous studies indicated that only a small fraction of exposed CF0 is highly conducting and that this small fraction is distinguished by its high affinity for added CF1. The results of this study point rather to a wider distribution of CF0 conductance states and binding affinities.

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.

In plants and cyanobacteria the ‘light reactions’ of photosynthesis use light energy to generate reducing power in the form of nicotinamide–adenine dinucleotide phosphate (NADPH) and metabolic energy as adenosine triphosphate (ATP), which are subsequently used in the ’dark reactions’ to drive the reduction of carbon dioxide and synthesis of carbohydrate. The light reactions, as conventionally defined, are actually a complex series of processes which include not only reactions in which light participates directly, but also closely associated reactions that indirectly depend on light. Keywords: light reaction; thylakoid membrane; photosystems; photosynthetic electron transfer; photophosphorylation

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O2 evolution by isolated chloroplasts equally (I50 approximately 2 microM), but is practically without effect on the O2 evolution coupled with SiMo reduction within the 0.2-10 microM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by QP, competing for electrons with FeCy and the cytochrome blf complex.

Cell metabolism requires transmembrane proton and electrochemical gradients to synthesize adenosine 5′-triphosphate (ATP), translocate ions, proteins and metabolites and regulate other vital activities. Keywords: adenosine-5′-triphosphate; chemiosmosis; photophosphorylation; oxidative phosphorylation; proton electrochemical potential gradient