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New and enhanced functions were potentially imparted to the plant organelles after interaction with nanoparticles. In this study, we found that ∼ 44% and ∼ 29% of the accumulated graphene in the rice leaves passively transported to the chloroplasts and thylakoid, respectively, significantly enhanced the fluorescence intensity of chloroplasts, and promoted about 2.4 times higher adenosine triphosphate production than that of controls. The enhancement of graphene on the photophosphorylation was ascribed to two reasons: One is that graphene facilitates the electron transfer process of photosystem II in thylakoid, and the other is that graphene protects the photosystem II against photo-bleaching by acting as a scavenger of reactive oxygen species. Overall, our work here confirmed that graphene translocating in the thylakoid promoted the photosynthetic activity of chloroplast in vivo and in vitro, providing new opportunities for designing biomimetic materials to enhance the solar energy conversion systems, especially for repairing or increasing the photosynthesis activity of the plants grown under stress environment.

The present study was conducted to examine the effects of increasing concentrations of chromium (Cr(6+)) (0, 25, 50, 100, and 200 μmol) on rice (Oryza sativa L.) morphological traits, photosynthesis performance, and the activities of antioxidative enzymes. In addition, the ultrastructure of chloroplasts in the leaves of hydroponically cultivated rice (O. sativa L.) seedlings was analyzed. Plant fresh and dry weights, height, root length, and photosynthetic pigments were decreased by Cr-induced toxicity (200 μM), and the growth of rice seedlings was starkly inhibited compared with that of the control. In addition, the decreased maximum quantum yield of primary photochemistry (Fv/Fm) might be ascribed to the decreased the number of active photosystem II reaction centers. These results were confirmed by inhibited photophosphorylation, reduced ATP content and its coupling factor Ca(2+)-ATPase, and decreased Mg(2+)-ATPase activities. Furthermore, overtly increased activities of antioxidative enzymes were observed under Cr(6+) toxicity. Malondialdehyde and the generation rates of superoxide (O2̄) also increased with Cr(6+) concentration, while hydrogen peroxide content first increased at a low Cr(6+) concentration of 25 μM and then decreased. Moreover, transmission electron microscopy showed that Cr(6+) exposure resulted in significant chloroplast damage. Taken together, these findings indicate that high Cr(6+)concentrations stimulate the production of toxic reactive oxygen species and promote lipid peroxidation in plants, causing severe damage to cell membranes, degradation of photosynthetic pigments, and inhibition of photosynthesis.

To investigate the effects of Nitrogen (N):phosphorus (P) ratio and solar ultraviolet radiation (UVR) on the growth and photophosphorylation of oceanic phytoplankton, the effects of UVR and N:P ratio on K. mikimotoi were evaluated. K. mikimotoi cells were cultured in artificial seawater (ASW) under five different N:P ratios (1:1, 16:1, 50:1, 100:1 and 200:1) for 15 days (phase 1: 1–5 day; phase 2: 6–10 day; phase 3: 11–15 days). Next, K. mikimotoi cultures were exposed to photosynthetically active radiation (PAR), PAB (PAR + UV-A + UV-B) and PA (PAR + UV-A), respectively. Finally, K. mikimotoi cells grown under the Redfield ratio (16:1) had the largest growth rate (0.257day−1), highest pigment concentrations (chlorophyll a (Chla): 0.465 × 10− 5 μg cell−1, carotenoid (Caro): 0.249 × 10−5 μg cell−1) and maximum superoxide dismutase (SOD) activity (0.9 U × 10−6 cell−1) compared with those grown under other N:P ratios. The UV-absorbing compounds’ (UVabc) absorbance was largest at the N:P ratio of 50:1 during the range of 310–360 nm, with a relatively higher value at the N:P ratio of 16:1. UV intensity was positively correlated with photoinhibition. K. mikimotoi cells grown at the N:P ratio of 16:1 showed the smallest photoinhibition compared to those grown at other N:P ratios under the PAR treatment with the largest r:k ratio; similar results were found for both the PA and PAB treatments. The Redfield ratio of 16:1 is optimal for the growth and photophosphorylation of K. mikimotoi. The weakest UVR-induced photoinhibition at this ratio may be attributed to the highest photosystem II (PSII) repair rate and SOD activity.

A model for abiotic photophosphorylation of adenosine diphosphate by orthophosphate with the formation of adenosine triphosphate was studied. The model was based on the photochemical activity of the abiogenic conjugates of pigments with the polymeric material formed after thermolysis of amino acid mixtures. The pigments formed showed different fluorescence parameters depending on the composition of the mixture of amino acid precursors. Thermolysis of the mixture of glutamic acid, glycine, and lysine (8:3:1) resulted in a predominant formation of a pigment fraction which had the fluorescence maximum at 525 nm and the excitation band maxima at 260, 375, and 450 nm and was identified as flavin. When glycine in the initial mixture was replaced with alanine, a product formed whose fluorescence parameters were typical to pteridines (excitation maximum at 350 nm, emission maximum at 440 nm). When irradiated with the quasi-monochromatic light (over the range 325-525 nm), microspheres in which flavin pigments were prevailing showed a maximum photophosphorylating activity at 375 and 450 nm, and pteridine-containing chromoproteinoid microspheres were most active at 350 nm. The positions and the relative height of maxima in the action spectra correlate with those in the excitation spectra of the pigments, which point to the involvement of abiogenic flavins and pteridines in photophosphorylation.

The molecular mechanism by which the membrane-embedded FO sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to FO c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H+ by coordinating the hydronium ion (H3O+) in the ion binding site of FO. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel FO probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.

17β-estradiol (E2) is considered to modulate the ATP synthase activity through direct binding to the oligomycin sensitive-conferring protein. We have previously demonstrated that E2 increases the amplitude of depolarization associated with the addition of ADP to energized mitochondria (i.e., to initiate a phosphorylative cycle) suggesting a direct action on the phosphorylative system of mitochondria. The purpose of the present study was to investigate the underlying mechanisms responsible for this effect. We show here that E2 modulates the activity of mitochondrial ATP synthase by promoting the intrinsic uncoupling ("slipping") of the ATP synthase. E2 depressed RCR, ADP/O ratio and state 3 respiration, whereas state 4 respiration was increased and VFCCP (uncoupled respiration) remained unaltered. In contrast to the stimulatory effect on state 4 respiration, state 2 respiration and Volig were not affected by E2. The effect of E2 appeared to be directed towards ATP synthase, since glutamate/malate respiration, uncoupled from the electron transport chain, was unaffected by E2. Apparently, E2 allows a proton back-leak through the Fo component of ATP synthase. This action of E2 is dependent on the presence of ATP, is more pronounced at high membrane potentials, and it is reversed by oligomycin (a Fo-ATP synthase inhibitor) but not by resveratrol (a F1-ATP synthase inhibitor). Altogether, our data provide a mechanistic explanation for the effect of E2 at the level of mitochondrial ATP synthase.

When visible light is excess, the photosynthetic machinery is photoinhibited. The extent of net photoinhibition of photosystem II (PSII) is determined by a balance between the rate of photodamage to D1 and some other PSII proteins and the rate of the turnover cycle of these proteins. It is widely believed that the protein turnover requires much energy cost. The aims of this study are to (1) evaluate the energy cost of PSII repair, (2) measure the benefit in terms of photosynthetic gain realized by the repairing of the photodamaged PSII, and (3) know whether acclimation of photosynthesis to growth light affects the rates of the photodamage and repair. We grew spinach in high-light (HL) and low-light (LL) and measured the rates of D1 photodamage and repair in these leaves. We determined the rate constants of photodamage (k (pi)) and repair (k (rec)) by the PAM fluorometry in the presence or in the absence of lincomycin, an inhibitor of 70S protein synthesis. HL leaves showed smaller k (pi) and greater k (rec) than LL leaves. The energy cost of the repairing of the photodamaged D1 protein was0.5 % of ATP produced by photophosphorylation at PPFDs ranging from 400 to 1600 μmol m(-2) s(-1) and was greater in HL leaves than in LL leaves. The benefits brought about by the repair were more than from 35 to 270 times the cost at PPFDs ranging from 400 to 1600 μmol m(-2) s(-1). The benefits of HL leaves were greater than those of LL leaves because of the higher photosynthesis rates in HL leaves. Running a simple simulation of daily photosynthesis using the parameters obtained in this study, we discuss why the plants need to pay the cost of D1 protein turnover to repair the photodamaged PSII.

Bisulfite at low concentrations (L-NaHSO3) increases cyclic electron transport around photosystem I (PSI) and photosynthesis. However, little is known regarding the detailed contribution of cyclic electron transport to the promoted photosynthesis by L-NaHSO3. In the present work, we used tobacco mutant defective in ndhC-ndhK-ndhJ (ΔndhCKJ) to investigate the role of NAD(P)H dehydrogenase (NDH)-dependent cyclic electron transport around PSI in an increase in photosynthesis by L-NaHSO3. After the treatment of tobacco leaves with L-NaHSO3 (10 μmol L−1), the NDH-dependent cyclic electron transport, monitored by a transient post-illumination increase in Chl fluorescence and the amount of NDH, was notably up-regulated in wild type (WT). The NDH-dependent cyclic electron transport was severely impaired in ΔndhCKJ and was not significantly affected by treatment with L-NaHSO3. Accordingly, the NDH-dependent transthylakoid membrane proton gradient (ΔpH), as reflected by the slow phase of millisecond-delayed light emission (ms-DLE), was increased by L-NaHSO3 in WT, but not in ΔndhCKJ; the enhancement of cyclic photophosphorylation (PSP) activity by L-NaHSO3 was more obvious in WT than ΔndhCKJ. The accumulation of both superoxide and hydrogen peroxide was reduced in WT when subjected to L-NaHSO3 treatment, but not in ΔndhCKJ. Furthermore, the increase of photosynthetic O2 evolution rate by L-NaHSO3 was more significant in WT than in ΔndhCKJ. We therefore conclude that L-NaHSO3 alleviates the photo-oxidative damage by the enhancement of NDH-dependent cyclic PSP, thereby improving photosynthesis.

New perspectives on photophosphorylation have been offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis. New experimental data on the involvement of malate anions in ATP synthesis in an acid-base malate bath procedure has been reported on spinach chloroplast thylakoids as the model system. The data cannot be reconciled with the chemiosmotic theory but has been shown to be naturally explained by the torsional mechanism. The path of malic acid in the acid and base stages of the experiment has been traced, offering further strong support to the new paradigm. Classical observations in the field have been re-interpreted in the light of these findings. A new concept of ion translocation, energy transduction and coupling at the overall physiological level in photophosphorylation has been presented and a large number of novel experimentally testable predictions have been made and shown to arise as logical consequences of the new perspectives.

The mechanism of the fact that manganese deprivation and cerium addition affect the photochemical efficiency of plants is unclear. In this study, we investigated the improvement by cerium of the damage of the photochemical function of maize chloroplasts under manganese-deprived stress. Chlorophyll fluorescence induction measurements showed that the ratio of variable to maximum fluorescence (Fv/Fm) underwent great decreases under manganese deficiency, which was attributed to the reduction of intrinsic quantum efficiency of the photosystem II units. The electron flow between the two photosystems, activities of Mg(2+)-ATPase and Ca(2+)-ATPase, and rate of photophosphorylation on the thylakoid membrane of maize chloroplasts were reduced significantly by exposure to manganese deprivation. Furthermore, the inhibition of cyclic photophosphorylation was more severe than non-cyclic photophosphorylation under manganese deficiency. However, added cerium could relieve the inhibition of the photochemical reaction caused by manganese deprivation in maize chloroplasts. It implied that manganese deprivation could disturb photochemical reaction of chloroplasts strongly, which could be improved by cerium addition.

The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (10 W/m(2)) and a negative one at higher irradiances (10 W/m(2)). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.

The mechanism of the fact that Mn deficiency damages the photosynthesis of plants is not yet fully understood. The main aim of the study was to determine Mn deficiency effects in photophosphorylation and key enzymes of CO 2 assimilation of maize. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mn deficiency and to Mn administered in the Mn-deficient Hoagland’s media. The results showed that Mn deficiency was found to cause extensive declines in plant weight and chlorophyll a content, electron transport and oxygen-evolving rate, photophosphorylation rate, activities of Mg 2+ -ATPase, Ca 2+ -ATPase, Rubisco and Rubisco activase, and mRNA expressions of Rubisco and Rubisco activase of maize, but it only slightly affected chlorophyll b and carotenoid formation. However, Mn addition decreased the inhibition of the photosynthesis in maize caused by Mn deficiency.

Light-dependent binding of labeled ADP and ATP to noncatalytic sites of chloroplast ATP synthase and the effect of light-exposed thylakoid membrane preincubation with ADP or ATP on ATPase activity were studied. ADP binding during the preincubation was shown to inactivate the chloroplast ATPase, whereas ATP binding caused its activation. The rate and equilibrium constants of ATPase inactivation and activation were close to those of ADP and ATP binding to a noncatalytic site, with K (d) values of 38 and 33 μM, respectively. It is suggested that ADP- or ATP-binding to one of the noncatalytic sites affects the ATPase activity of chloroplast ATP synthase through a mechanism that modulates tightness of ADP binding to a catalytic site.

Effects of cerium (Ce) on photosynthetic pigments and photochemical reaction activity in soybean (Glycine max L.) under ultraviolet-B (UV-B) radiation stress were studied under laboratory conditions. UV-B radiation caused the decrease in chlorophyll content, net photosynthetic rate, Hill reaction activity, photophosphorylation rate and Mg(2+)-ATPase activity. Ce (III) (20 mg L(-1)) could alleviate UV-B-induced inhibition to these photosynthetic parameters because values of these photosynthetic parameters in Ce (III) + UV-B treatment were obviously higher than those with UV-B treatment alone. Dynamic changes of the above photosynthetic parameters show that Ce (III) could slow down the decrease rate of these photosynthetic parameters during a 5-day UV-B radiation and quicken the restoration during recovery period. The final restoration degree of five parameters mentioned above in leaves exposed to low level of UV-B radiation (0.15 W m(2)) was higher than that exposed to high level (0.45 W m(2)). Correlating net photosynthetic rate with other four parameters, we found that the regulating mechanisms Ce (ΠΙ) on photosynthesis under various level of UV-B radiation were not the same. The protective effects of Ce (III) on photosynthesis in plants were influenced by the intensity of UV-B radiation.

Photosynthesis is one of the most sensitive processes to lead, but the effects of lead on the transformation of light energy of plants are still not clearly understood. In the present paper, spinach was cultivated in the experimental fields and was sprayed with various concentrations of PbCl2 solution. We investigated the effects of lead on the activities of photochemical reaction and the key enzymes of carbon assimilation in spinach chloroplast. The results showed that Pb2+ treatment could significantly inhibit the Hill reaction activity of spinach chloroplast and photophosphorylation, and it had a more conspicuous effect on cyclic photophosphorylation than non-cyclic photophosphorylation. The activities of ATPase on the thylakoid membrane were severely inhibited under Pb2+-treated condition, and Ca2+ ATPase activity was affected more obviously than Mg2+ ATPase activity. Meanwhile, the activities of the key enzymes of carbon assimilation were also significantly reduced by Pb2+, especially Rubisco activase. The reduction of dry weight of spinach caused by Pb2+ was more significant than that of fresh weight. It implied that Pb2+ could disturb light energy transformation of chloroplast.

Chloroplast absorbs light energy and transforms it into electron energy, and then converts it into active chemical energy and stable chemical energy. In the present paper, we investigated the effects of Ce(3+), which has the most significant catalytic effects and similar characteristics with Ca(2+), on light energy conversion of spinach chloroplasts under Ca(2+)-deficient stress. The results illuminated that the Hill reaction activity, electron flow both photosystems and photophosphorylation rate of spinach chloroplasts reduced significantly under Ca(2+)-deficient condition, and activities of Mg(2+)-ATPase and Ca(2+)-ATPase on the thylakoid membrane were severely inhibited. Meanwhile, the activity of Rubisco, which is the key enzyme of photosynthetic carbon assimilation, was also prohibited. However, Ce(3+) decreased the inhibition of calcium deprivation the electron transport rate, the oxygen evolution rate, the cyclic and noncyclic photophosphorylation, the activities of Mg(2+)-ATPase, Ca(2+)-ATPase and Rubisco of spinach chloroplasts. All above implied that Ca(2+)-depletion could disturb light energy conversion of chloroplasts strongly, which could be reversed by Ce(3+).

A model for abiogenic photophosphorylation of ADP by orthophosphate to yield ATP was studied. The model is based on the photochemical activity of flavoproteinoid microspheres that are formed by aggregation in an aqueous medium of products of thermal condensation of a glutamic acid, glycine and lysine mixture (8:3:1) and contain, along with amino acid polymers (proteinoids), abiogenic isoalloxazine (flavin) pigments. Irradiation of aqueous suspensions of microspheres with blue visible light or ultraviolet in the presence of ADP and orthophosphate resulted in ATP formation. The yield of ATP in aerated suspensions was 10–20% per one mol of starting ADP. Deaeration reduced the photophosphorylating activity of microspheres five to 10 times. Treatment of aerated microsphere suspensions with superoxide dismutase during irradiation partially suppressed ATP formation. Deaerated microspheres restored completely their photophosphorylating activity after addition of hydrogen peroxide to the suspension. The photophosphorylating activity of deaerated suspensions of flavoproteinoid microspheres was also recovered by introduction of Fe3+-cytochrome c, an electron acceptor alternative to oxygen. On the basis of the results obtained, a chemical mechanism of phosphorylation is proposed in which the free radical form of reduced flavin sensitizer $$\left( {{\text{FlH}}^ \bullet } \right)$$ and ADP are involved.

The psbZ gene of Synechocystis sp. PCC 6803 encodes the approximately 6.6 kDa photosystem II (PSII) subunit. We here report biophysical, biochemical and in vivo characterization of Synechocystis sp. PCC 6803 mutants lacking psbZ. We show that these mutants are able to perform wild-type levels of light-harvesting, energy transfer, PSII oxygen evolution, state transitions and non-photochemical quenching (NPQ) under standard growth conditions. The mutants grow photoautotrophically; however, their growth rate is clearly retarded under low-light conditions and they are not capable of photomixotrophic growth. Further differences exist in the electron transfer properties between the mutants and wild type. In the absence of PsbZ, electron flow potentially increased through photosystem I (PSI) without a change in the maximum electron transfer capacity of PSII. Further, rereduction of P700(+) is much faster, suggesting faster cyclic electron flow around PSI. This implies a role for PsbZ in the regulation of electron transfer, with implication for photoprotection.

With a photocatalyzed characteristic, nanoanatase TiO2 under light could cause an oxidation-reduction reaction. Our studies had proved that nano-TiO2 could promote photosynthesis and greatly improve spinach growth. However, the mechanism of nano-TiO2 on promoting conversion from light energy to electron energy and from electron energy to active chemistry energy remains largely unclear. In this study, we report that the electron transfer, oxygen evolution, and photophosphorylation of chloroplast (Chl) from nanoanatase-TiO2-treated spinach were greatly increased under visible light and ultraviolet light illumination. It was demonstrated that nanoanatase TiO2 could greatly improve whole chain electron transport, photoreduction activity of photosystem II, O2-evolving and photophosphorylation activity of spinach Chl not only under visible light, but also energy-enriched electron from nanoanatase TiO2, which entered Chl under ultraviolet light and was transferred in photosynthetic electron transport chain and made NADP+ be reduced into NADPH, and coupled to photophosphorylation and made electron energy be transformed to ATP. Moreover, nanoanatase h+, which photogenerated electron holes, captured an electron from water, which accelerated water photolysis and O2 evolution.

ATP synthesis assisted by the F1F0-ATP synthase enzyme is considered in the context of chemical thermodynamics. It is shown that the proton gradient itself has no energy. For this reason, ATP synthesis with the participation of protons’ gradient has no privileges in comparison with direct ATP synthesis.

In our search for new natural photosynthetic inhibitors that could lead to the development of "green herbicides" less toxic to environment, the diterpene labdane-8alpha,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 P(700) 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 P(680) in PS II and at the span of P(700) to F(X) in PS I. The results of compounds 1 and 2 on PS II were corroborated by chlorophyll a fluorescence.

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the gamma subunit of the ATP synthase in the light enhances the rate of Mg2+-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH4Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.

In order to understand the pathway involved in the chemical enhancement of photosynthetic rate, sodium bisulfite (NaHSO3) and benzyladenine (BA), a growth regulator, were applied to strawberry plants. The influence of these compounds on gas exchange and millisecond delayed light emission (ms-DLE) was investigated using 2-month-old plants. Results showed the net photosynthetic rate (A) in leaves was promoted by both NaHSO3 and BA. Stomatal conductance (g) and transpiration rate (E) were significantly increased only by BA, while intercellular CO2 concentration (Ci) was significantly decreased by NaHSO3. The enhancement of A by NaHSO3 and BA was only a short-term effect, lasting approximately 5 days for NaHSO3 and 30 h for BA. Plants treated with NaHSO3, BA or NaHSO3 + BA, showed no significant fluctuations in carboxylation efficiency (CE), photorespiration (RP) or dark respiration (RD). These results suggest that the influences of NaHSO3 and BA on gas exchange particularly A, could be via different mechanisms: the enhancement of A by the application of low concentrations of NaHSO3 appears to be associated with increased cyclic electron flow, while BA enhancement of A is at least partially due to increased g and/or E.

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Ilowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO3)2. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 µM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 µM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves.

In this work, we studied theoretically the effects of diffusion restrictions and topological factors that could influence the efficiency of energy coupling in the heterogeneous lamellar system of higher plant chloroplasts. Our computations are based on a mathematical model for electron and proton transport in chloroplasts coupled to ATP synthesis in chloroplasts that takes into account the nonuniform distribution of electron transport and ATP synthase complexes in the thylakoids of grana and stroma. Numerical experiments allowed the lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids to be simulated under different metabolic conditions (in the state of photosynthetic control and under conditions of photophosphorylation). This model also provided an opportunity to simulate the effects of steric constraints (the extent of appression of thylakoids in grana) on the rates of non-cyclic electron transport and ATP synthesis. This model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: 1) slowing down of non-cyclic electron transport due to a decrease in the intra-thylakoid pH, and 2) retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana. Numerical experiments for model systems that differ with respect to the arrangement of thylakoids in grana allowed the effects of osmolarity on the photophosphorylation rate in chloroplasts to be explained.

Mass coral bleaching is linked to elevated sea surface temperatures, 1-2 degrees C above average, during periods of intense light. These conditions induce the expulsion of zooxanthellae from the coral host in response to photosynthetic damage in the algal symbionts. The mechanism that triggers this release has not been clearly established and to further our knowledge of this process, fluorescence rise kinetics have been studied for the first time. Corals that were exposed to elevated temperature (33 degrees C) and light (280 mumol photons m(-2) s(-1)), showed distinct changes in the fast polyphasic induction of chlorophyll-a fluorescence, indicating biophysical changes in the photochemical processes. The fluorescence rise over the first 2000ms was monitored in three species of corals for up to 8 h, with a PEA fluorometer and an imaging-PAM. Pocillopora damicornis showed the least impact on photosynthetic apparatus, while Acropora nobilis was the most sensitive, with Cyphastrea serailia intermediate between the other two species. A. nobilis showed a remarkable capacity for recovery from bleaching conditions. For all three species, a steady decline in the slope of the initial rise and the height of the J-transient was observed, indicating the loss of functional Photosystem II (PS II) centres under elevated-temperature conditions. A significant loss of PS II centres was confirmed by a decline in photochemical quenching when exposed to bleaching stress. Non-photochemical quenching was identified as a significant mechanism for dissipating excess energy as heat under the bleaching conditions. Photophosphorylation could explain this decline in PS II activity. State transitions, a component of non-photochemical quenching, was a probable cause of the high non-photochemical quenching during bleaching and this mechanism is associated with the phosphorylation-induced dissociation of the light harvesting complexes from the PS II reaction centres. This reversible process may account for the coral recovery, particularly in A. nobilis.

This study investigated the effects of tetracycline on photophosphorylation, electron transport and P/O ratio of spinach chloroplasts. When chloroplast preparations were treated with low concentrations of tetracycline, non-cyclic and cyclic photophosphorylation activities increased, electron transport rates and P/O ratios improved, chloroplast ms-DLE also improved, and the Mg(2+)-ATPase activity of CF1 increased in comparison to the control. These results indicate that spinach chloroplasts are sensitive to tetracycline. Next, we used the fluorescence emission spectra of CF1 to examine the possible binding sites for tetracycline. The fluorescence emission spectra of CF1 treated with glutaraldehyde, NEM and TNBS, which interact with CF1 across its whole structure, at the γ subunit and at the β subunit, respectively, were compared with that of control CF1. The peak sites of the various fluorescence emission spectra were the same, but the peak values for CF1 treated with glutaraldehyde, NEM and TNBS were lower than that of control CF1. The peak value of CF1 treated with 50 µM tetracycline was very similar to that of CF1 treated with NEM. The above results indicate that the acting site of tetracycline may be at or near the γ subunit of CF1, and allows the creation of a model in which tetracycline binding strengthens the subunit interactions of ATP synthase, enlarges the proton motive force across the thylakoid membrane, and allows the excess proton motive force to increase ATP formation and improve the P/O ratio.

A thalium chloride-resistant (TlCl(r)) mutant strain and a sodium chloride-resistant (NaCl(r)) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K(+)) analog, and nitrosoguanidine (NTG), respectively. The TlCl(r) mutant strain was found to be defective in K(+) transport and showed resistance against 10 microM TlCl. However, it also showed sensitivity against NaCl (LD(50), 50 m M). In contrast, neither wild-type A. variabilis nor its NaCl(r) mutant strain could survive in the presence of 10 microM TlCl and died even at 1 microM TlCl. The TlCl(r) mutant strain exhibited almost negligible K(+) uptake, indicating the lack of a K(+) uptake system. High K(+) uptake was, however, observed in the NaCl(r) mutant strain, reflecting the presence of an active K(+) uptake system in this strain.DCMU, an inhibitor of PS II, inhibited the K(+) uptake in wild-type A. variabilis and its TlCl(r) and NaCl(r) mutant strains, suggesting that K(+) uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention is further supported by the inhibition of K(+) uptake under dark conditions. Furthermore, the inhibition of K(+) uptake by KCN, DNP, and NaN(3) also suggests the involvement of oxidative phosphorylation in the regulation of an active K(+) uptake system. The whole-cell protein profile of wild-type A. variabilis and its TlCl(r) and NaCl(r) mutant strains growing in the presence of 50 m M KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlCl(r) mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K(+) and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaCl(r) mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt tolerance of the NaCl(r) mutant strain was owing to increased K(+) uptake and accumulation, whereas the salt sensitivity of the TlCl(r) mutant strain was owing to the lack of K(+) uptake and accumulation.

Application of NaHSO(3) solution at low concentrations (20-200 muM) to the culture medium enhanced photosynthetic oxygen evolution in cyanobacterium Synechocystis PCC6803 by more than 10%. The slow phase of ms-DLE was strengthened, showing that the transmembrane proton motive force related to photophosphorylation was enhanced. It was also observed that dry weight as well as ATP content under illuminated conditions were both increased after the treatment, indicating that low concentrations of NaHSO(3) could enhance the supply of ATP and thus increase biomass accumulation. In accord with the promotion in the photosynthetic oxygen evolution and ATP content, the transient increase in chlorophyll fluorescence after the termination of actinic light was increased; and meanwhile, the half-time of re-reduction of P700(+) in the presence of DCMU after a pulse light under background far-red light was shortened by approximately 30%, indicating that cyclic electron flow around PS I was accelerated by the treatment. Based on these results it is suggested that the increase in photosynthesis in Synechocystis PCC6803 caused by low concentrations of NaHSO(3) solution might be due to the stimulation of the cyclic electron flow around PS I and thus the increase in photophosphorylation.

The effects of the antioxidant Ambiol and 2-chlorethylphosphonic acid (2-CEPA) on individual concentrations and concentration ratios of phytohormones, photosynthesis and photophosphorylation rates, sucrose and starch content in tubers, and plant productivity were studied in potato (Solanum tuberosum L). Ambiol increased the ratio of indoleacetic acid (IAA) to abscisic acid (ABA), IAA/ABA, and that of zeatin (Z) and zeatin riboside (ZR) to ABA, (Z + ZR)/ABA. These effects were underlain by an increase in the content of auxins and cytokinins and a decrease in ABA. Unlike Ambiol, 2-CEPA increased the level of ABA, the effect being the most pronounced in the tubers. Ambiol increased the rates of photosynthesis and noncyclic photophosphorylation in chloroplasts isolated from potato leaves. The relation of this phenomenon to auxin and cytokinin accumulation, Ambiol- and 2-CEPA-induced changes in the hormonal balance of potato tubers, carbon metabolism, and plant productivity is discussed.

Photophosphorylation was discovered in chloroplasts by D. Arnon and coworkers, and in bacterial ‘chromatophores’ (intercytoplasmic membranes) by A. Frenkel. Initial low rates were amplified by adding electron-carrying compounds such as FMN, later shown to support the ‘pseudocyclic’ electron flow. ATP synthesis, and coupling to electron flow, was detected accompanying linear electron flow from H2O to either NADP+ or ferricyanide. Another pattern of electron flow supporting photophosphorylation was that of a cycle around Photosystem I (PS I). Isolation and analysis of the ATP synthase showed, as with mitochondrial and bacterial analogues, an intrinsic membrane complex (CF0) and an extrinsic complex (CF1). CF1 is a latent ATPase, activated additively by the high-energy state of the thylakoids, and by reduction of a disulfide bond on the gamma subunit. Once reduced, ATP synthesis occurs at lower energy levels. The search for an ‘intermediate’ linking electron flow and ATP synthesis led to the discovery of post-illumination ATP synthesis by thylakoids, where turnover occurs in the dark. Once interpreted by P. Mitchell’s chemiosmotic hypothesis, this led to the discovery of light-driven proton uptake into the thylakoid lumen, with accompanying Cl− intake and Mg2+ and K+ output. Chemiosmosis was confirmed in several ways, including ATP synthesis in the dark due to an acid-to-base transition of thylakoids, and photophosphorylation accomplished in artificial lipid vesicles containing both the proton-pumping bacterial rhodopsin and a mitochondrial ATPase complex. The now generally accepted chemiosmotic interpretation is able to clarify some other aspects of photosynthesis as well.

In 1893, Charles Barnes (1858–1910) proposed that the biological process for ‘synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light’ should be designated as either ‘photosyntax’ or ‘photosynthesis.’ He preferred the word ‘photosyntax,’ but ‘photosynthesis’ came into common usage as the term of choice. Later discovery of anoxygenic photosynthetic bacteria and photophosphorylation necessitated redefinition of the term. This essay examines the history of changes in the meaning of photosynthesis.

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557-567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483-493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a "proton gate" proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of deltamuH+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid-base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the deltapH of the acid-base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid-base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+-ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 5Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel.

A NaCl(r) mutant of the diazotrophic cyanobacterium Anabaena variabilis has been isolated by NTG mutagenesis and selection for NaCl resistance. The NaCl(r) strain has been characterized with respect to its mechanism of NaCl tolerance and regulation of Na(+) influx. NaCl(r) strain exhibits low Na(+) influx, accumulated high level of glycine betaine as a compatible solute, and persistent synthesis of SSPs at a higher rate than its wild-type counterpart. DCMU, an inhibitor of PS-II, inhibited Na(+) influx, suggesting that Na(+) influx is an energy-dependent process and that the energy is derived from photophosphorylation. This contention is further supported by the inhibition of Na(+) influx under dark conditions. The inhibition of Na(+) influx by KCN, DNP, NaN(3) also supports the involvement of oxidative phosphorylation in the regulation of active Na(+) influx. Thus, it appears that the synthesis of SSPs, accumulation of compatible solutes, and exhibition of low Na(+) influx in the NaCl(r) strain made this organism NaCl tolerant.

The rates of electron transfer in the presence of natural cofactors, ferredoxin and NADP, which were added in the amounts catalyzing noncyclic or cyclic electron transfer, were studied in thylakoids isolated from 17-day-old wheat seedlings. Upon excitation of both photosystems (PS) of photosynthesis, the potential rate of NADP reduction in thylakoids isolated from plants grown on nitrogen-free nutrient solution did not differ from that in thylakoids from the control plants. However, the P/2e ratio was significantly lower in thylakoids isolated from nitrogen-deficient plants. On the contrary, in the presence of DCMU, the rate of PSI-driven electron transfer from an artificial donor to NADP was considerably higher in these than in the control thylakoids. In the presence of ferredoxin under anaerobic conditions, the rate of phosphorylation coupled to cyclic electron transport was also significantly higher in thylakoids isolated from nitrogen-deficient plants, than in thylakoids isolated from control plants. Our data show that PSI-driven electron transport and cyclic photophosphorylation are activated in nitrogen-starved wheat plants, at least at the initial stages of starvation.

Spraying 1–2 mmol/L NaHSO3 on the leaf of wheat results in enhancement of photosynthesis in leaves for about 3 d. The amount of ATP has been increased and the millisecond delayed light emission of the leaves has been enhanced, showing that the transmembrane proton motive force related to photophosphorylation is increased. Spraying PMS (a cofactor catalyzing cycle photophosphorylation) and NaHSO3 separately or together on the leaves, 20% increase in photosynthesis has been observed in all the treatments. There is no additive effect when a mixture is applied, suggesting that the mechanism for NaHSO3 promotion of photosynthesis is similar to PMS, and both of them enhance the supply of ATP.

The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and Pi assumes that all states of the F1 portion of the ATP synthase have β subunits in “tight,” “loose,” and “open” conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional β subunit conformation, “closed,” observed in the rat liver structure overcomes these difficulties.

The pigment content and rates of primary photosynthetic reactions were determined in chloroplasts of 14-day-old pine (Pinus silvestris L.) seedlings grown in light and darkness. In addition, the functional activities were investigated in chloroplasts from dark-grown seedlings exposed to white, red (λ = 670 nm), and red + far-red (λ = 748 nm) light. Dark-grown seedlings were capable of performing the Hill reaction, noncyclic photophosphorylation, and phenazine methosulfate–supported photophosphorylation, although the reaction rates in chloroplasts from dark-grown plants were considerably lower than in preparations from light-grown plants. Light treatment of dark-grown seedlings rapidly activated the photoreduction of ferricyanide and photophosphorylation, while the additional accumulation of green pigments started only after a lag period of two hours. Preirradiation of dark-grown seedlings with red light stimulated the formation of pigments, especially chlorophyll b, as well as the functional activity of chloroplasts. When far-red light was applied after red-light exposure, the processes examined were inhibited. It is concluded that accumulation of the light-harvesting complex and functional activities of chloroplasts at the photosystem II level in pine seedlings are controlled by the phytochrome.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 micromol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the gamma and epsilon subunits of CF(1) play key roles in both regulation of activity and proton translocation.

The major theme of my work in photosynthesis has been electron transport in green plant thylakoids. In particular, we investigated the properties and the role of the NADP-reducing flavoprotein and its possible function in cyclic electron transport, the regulation by protons of electron transport, and the redox system of ascorbate and monodehydroascorbate (the ascorbate free radical). The function of this system in providing ATP in the stoichiometric amount needed for carbon assimilation, and the regulation of the alternative transfer of electrons to NADP and to the ascorbate free radical were among the achievements of my collaborators and myself. Specifically, the early conviction that cyclic phosphorylation was essential part in photosynthesis was shattered as far as higher plants are concerned, and replaced by a modified Mehler reaction providing additional ATP to run the Calvin cycle. The situation seems to be different in unicellular green algae, where quantitatively much l arger changes of the relative size of Photosystem (PS) I and PS II antennae during the so-called ‘state’ transitions have been reported, and these seem to be associated with a high activity of cyclic electron transport in state 2. Beyond the science, the friendly interactions with so many persons around the world sharing my interest in photosynthesis and in other aspects of human life have been most rewarding.

The inactivation of electron transport upon preillumination of isolated, stroma free thylakoids has been studied. Inactivation is defined here as the loss of activity which is not reversed upon relaxation of qE. It was found that both PS 2 and PS 1 dependent electron transport were inactivated, whilst the coupling of ATP synthesis to electron transport was not affected. The inactivation concerned both the transfer of excitation energy to the reaction centres, and the reaction centres themselves. Ascorbate protected against photoinactivation of the electron transport from H2O to NADP or to methylviologen, much less the electron transport depending only on PS 1. The protection by ascorbate required its well known action as a cofactor of de-epoxidation of violaxanthin and the consequent formation of qE: under conditions where de-epoxidation was inhibited (presence of DTT or uncouplers) qE was also suppressed and ascorbate protection was abolished. Ascorbate did not p rotect the thylakoids against inactivation caused by H2O2in the dark.The latter was shown to concern mostly PS 2 electron transport.

The effect of osmotic shock on the ultrastructure and functions of C-class pea chloroplasts has been examined. When incubated in a non-sucrose medium for 30 s or more, thylakoids were found to pass to a stable deformed state. This state was characterized by an altered orientation of thylakoids to each other with the lumen thickness remaining the same as in the normal state. Experiments with shorter incubation periods (10–20 s) revealed a swelling of thylakoids, which probably represented an intermediate stage. The deformation of the thylakoid system was accompanied by a decrease in the non-cyclic ATP synthesis but by an increase in the rate of cyclic photophosphorylation. Besides, the deformed thylakoids demonstrated an acceleration of the basal electron transport, as well a rise in the light-induced H+ and imidazol uptake. The data obtained are discussed in the light of membrane interactions fixing the configuration of a thylakoid.

Helminthosporol is one of the natural sesquiterpenoid toxins isolated and identified in the culture medium of the phytopathogenic ascomycete fungus Cochliobolus sativus. The effect of this phytotoxin was investigated on enzymatic activities, electron and ion transport in mitochondria, chloroplasts, and microsomes of plant. The results indicate that helminthosporol drastically affects the membrane permeability of these organelles to protons and substrate anions, inhibiting the mitochondrial oxidative phosphorylation, the photophosphorylation in chloroplasts, and the proton pumping across the cell plasma membrane. The 1,3-beta-glucan synthase activity, involved in defense mechanisms of plant cells against stress and damage, e.g., during pathogen attack, was also strongly inhibited by the toxin.

The relation between the fast phase of ms-DLE (delayed light emission measured with a phosphoroscope) and the proton released from water oxidation in spinach chloroplasts was studied in several aspects. When photophosphorylation was allowed to be coupled to the Hill reaction the intensity of the fast phase of ms-DLE of chloroplast was lowered more at 1 °C than at 25 °C, and the photophosphorylation rate within 40 ms of flashing light was higher at 1 °C than at 25 °C. Adding the e subunit of ATP synthase to the chloroplast preparation to block the leakage of protons through ATP synthase, the intensity of the fast phase of ms-DLE was enhanced, to a larger extent at 1 °C than at 25°C. When the ms-DLE was measured under isotonic conditions, the intensity of fast phase of ms-DLE enhanced by proton released from oxidation of water was more pronounced. The above results support the suggestion that under lower temperature and isotonic conditions, the proton released from water oxidation was liable to be localized and could enhance the intensity of the fast phase of ms-DLE more effectively.

Isolated spinach CF1 (chloroplast coupling factor 1) forms enzyme-bound ATP without any supply of energy in the presence of high concentrations of Pi [Feldman and Sigman (1982) J Biol Chem 257: 1676-1683]. The final amount of CF1-bound ATP synthesized was increased greatly by 1,2-propanediol, and moderately by methanol, ethanol, and dimethyl sulfoxide, but decreased by glycerol and octyl glucoside. Methanol and ethanol greatly increased the initial rate of ATP synthesis, while 1,2-propanediol increased it only moderately. Low concentrations (10-8 -10-6 M) of tentoxin, which inhibit ATPase activity of isolated CF1, did not affect enzyme-bound ATP synthesis. However, high concentrations (>10-5 M) of tentoxin, which stimulate ATPase activity of isolated CF1, enhanced the initial rate of CF1-bound ATP synthesis without significant effect on the final amount of ATP synthesized in the presence of medium ADP. The substrate of enzyme-bound ATP synthesized came largely from tightly bound ADP, not medium ADP, and tentoxin did not affect this substrate profile. Tentoxin did not affect the binding of medium ADP to high affinity sites on CF1.

Morphology and photophosphorylation of chromatophores from t Rhodospirillum rubrum have been investigated by dynamic light scattering (DLS) and in situ 31P-NMR measurement. Two components, designated as light and heavy fractions, with different average sizes and size distributions were detected by the DLS and can be separated by sucrose density gradient centrifugation. The light fraction has an average size of about 140 nm in diameter with a narrow distribution and shows a high activity of photophosphorylation. About 70 of ADP were found to be converted to ATP purely by the photophosphorylative reaction. In contrast, the heavy fraction has a broad size distribution centered around 350 nm and a low activity of photophosphorylation. Only about 50 of ADP was converted into ATP and AMP with a ratio of 7:3, indicating that most membrane-bound adenylate kinase are attached on the particles of the heavy fraction. Effect of physical disruption on the structural integrity of chromatophores has been examined by using sonication with various oscillating strengths. The result shows that the morphology of chromatophores for both light and heavy fractions is relatively stable to the disruption, while the photophosphorylative activity of the light fraction is very sensitive to the disrupting strength, suggesting that the internal structure of the purified chromatophores could be partially damaged by the disruption.

Nitrogen fixation is dependent on a source of ATP and the generation of a reductant at low enough red-ox potential to transfer electrons to nitrogenase. In Rhodospirillum rubrum, grown photoheterotrophically, ATP is produced by photophosphorylation, a process studied in great detail, but the source of reductant for nitrogenase is as yet unidentified. In this report we have studied the effect on nitrogen fixation when the energization of the chromatophore membranes was changed, by decreasing the light intensity or by addition of uncouplers. When the light intensity was lowered a pronounced decrease in nitrogenase activity was observed although there was no decrease in the ATP/ADP ratio. The inhibition observed was not due to ADP-ribosylation, as the same effect was observed in a mutant devoid of the enzymes in the metabolic regulatory cascade operating in R. rubrum and some other diazotrophs. Even at low concentrations of the uncouplers used, a drastic decrease in the ATP/ADP ratio was observed. However, this decrease in the ATP/ADP ratio did not cause a decrease in nitrogenase activity. At higher concentrations of uncouplers, nitrogenase activity decreased but the ATP/ADP ratio remained essentially at a constant low level. These results support a model in which reduction of the electron donor(s) to nitrogenase in R. rubrum is coupled to the energization of the chromatophore membranes.

Effect of quality, quantity and minimum duration of light on the process of recovery was investigated in the photoinhibited cells of the green alga Chlamydomonas reinhardtii. Complete and rapid reactivation of photosynthesis took place in diffuse white light of 25 μmol m(-2) s(-1). The recovery was partial (10%) in the dark. Far red (725 nm), red (660 nm) and blue light (480 nm) in the range of 10 to 75 μmol m(-2) s(-1) did not enhance the process of reactivation. Photoinhibited cells incubated in dark for 15 min when exposed for 5 min to diffuse light (25 μmol m(-2) s(-1)) showed complete reactivation. Even exposure of 15 min dark incubated photoinhibited cells to photoinhibitory light (2500 μmol m(-2) s(-1)) for 5 s fully regained the photosynthesis. The study indicated a very precise and triggering effect of light in the process of reactivation. The dark respiratory inhibitor KCN and uncouplers FCCP and CCCP increased the susceptibility of C. reinhardtii to photoinhibition and also prevented photoinhibited cells to reactivate fully even after longer period of incubation under suitable reactivating conditions. Of the various possibilities envisaged to assign the role of dark respiration in recovery process, supply of ATP by mitochondrial respiration appeared sound and pertinent.

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H2O2 was light dependent with a production of 1.2 μmol·g FW−1·h−1 in sea water (pH 8.2) at an irradiance of 700 μmol photons m−2·s−1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW−1·h−1) but at pH 9.0 the production was 5.0 μmol·g FW−1·h−1. The production of H2O2 was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H2O2 was checked and found to be seven times higher at 08.00 hours than other times of the day. The H2O2 concentration in the cultivation tank (density: 2 g FW·l−1) reached the highest value (3 μM) at 11.00 hours. Photosynthesis was not influenced by H2O2 formation. The H2O2 is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW−1·h−1 at pH 8.2 and 90 mmol·g FW−1·h−1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H2O2 production. The H2O2 production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F1 supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP. The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k+1) and the reverse dissociation step (k-1). k-1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement of k-1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP. k+1 was affected little by NADH oxidation. The dissociation constant (KdATP) increased many orders of magnitude during the transition from nonenergized to energized states.