[Effects of Medium Viscosity Increasing Agents on ATP Synthesis in Chloroplast Thylakoids].

The effect of an increase in the medium viscosity on cyclic photophosphorylation in chloroplast thylakoids and on Ca2+ -dependent ATP hydrolysis by the chloroplast coupling factor CF, was studied. With 0.1-0.2 mM ADP used it was found that the rate of ATP synthesis decreases after addition of various agents that increase the medium viscosity (sucrose, dextran 40 or polyethylene glycol 6000 provided that these agents cause neither uncoupling nor electron transport inhibition in the absence of ADP. Dextran and polyethylene glycol inhibited ATP synthesis by 50% when their concentrations were much lower (6-10%) than that of sucrose (30-40%), while 50% inhibition of Ca2+ -dependent ATP hydrolysis by CFI-ATPase was observed at higher concentrations of dextran and polyethylene glycol (9-13%) and lower concentrations of sucrose (about 20%). For ADP, the effective Michaelis constant (KM) was shown to increase 2-3-fold with the increasing viscosity; meanwhile the maximal rate of cyclic photophosphorylation remained virtually unchanged. The dependence of K(M) on the medium viscosity can serve as a criterion for the process of diffusion-controlled photophosphorylation. Possible mechanisms of ADP and ATP diffusion are discussed.

Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation.

Cryopreservation can be a safe and cost-effective tool for the long-term storage of plant germplasm. In Arabidopsis, the ability to recover from cryogenic treatment was lost as growth progressed. Growth could be restored in 48-h seedlings, whereas 72-h seedlings died after cryogenic treatment. Why seedling age and survival are negatively correlated is an interesting issue. A comparative transcriptomics was performed to screen differentially expressed genes between 48- and 72-h seedlings after exposure to cryoprotectant. Among differentially expressed genes, oxidative stress response genes played important roles in cryoprotectant treatment, and peroxidation was a key factor related to cell survival. Seedlings underwent more peroxidation at 72-h than at 48-h. A comprehensive analysis indicated that peroxidation injured membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Furthermore, the apoptosis-like events were found in cryogenic treatment of Arabidopsis seedlings. 48- and 72-h seedlings underwent different degrees of membrane lipid peroxidation during cryoprotectant treatment, and reducing the injury of oxidative stress was an important factor to successful cryopreservation. This study provided a novel insight of genetic regulatory mechanisms in cryopreservation, and established an excellent model to test and evaluate the effect of exogenous antioxidants and conventional cryoprotectants in plant cryopreservation.

Exposure to Cr(VI) induces organ dependent MSI in two loci related with photophosphorylation and with glutamine metabolism.

Chromium (Cr), as a mutagenic agent in plants, has received less attention than other metal pollutants. To understand if Cr induces microsatellite instability (MSI), Pisum sativum seedlings were exposed for 28 days to different concentrations of Cr(VI) up to 2000mgL(-1), and the genetic instability of ten microsatellites (SSRs) was analyzed. In plants exposed to Cr(VI) up to 1000mg L(-1), MSI was never observed. However, roots exposed to 2000mgL(-1) displayed MSI in two of the loci analyzed, corresponding to a mutation rate of 8.3%. SSR2 (inserted in the locus for plastid photosystem I 24kDa light harvesting protein) and SSR6 (inserted in the locus for P. sativum glutamine synthetase) from Cr(VI)-treated roots presented alleles with, respectively, less 6bp and more 3bp than the corresponding controls. This report demonstrates that: (a) SSRs technique is sensitive to detect Cr-induced mutagenicity in plants, being Cr-induced-MSI dose and organ dependent (roots are more sensitive); (b) two Cr-sensitive loci are related with thylakoid photophosphorylation and with glutamine synthetase, respectively; (c) despite MSI is induced by Cr(VI), it only occurs in plants exposed to concentrations higher than 1000mgL(-1) (values rarely found in real scenarios). Considering these data, we also discuss the known functional changes induced by Cr(VI) in photosynthesis and in glutamine synthetase activity.

Toxic effects of erythromycin, ciprofloxacin and sulfamethoxazole on photosynthetic apparatus in Selenastrum capricornutum.

The effects of three antibiotics (erythromycin, ciprofloxacin and sulfamethoxazole) on photosynthesis process of Selenastrum capricornutum were investigated by determining a battery of parameters including photosynthetic rate, chlorophyll fluorescence, Hill reaction, and ribulose-1.5-bisphosphate carboxylase activity, etc. The results indicated that three antibiotics could significantly inhibit the physiological progress including primary photochemistry, electron transport, photophosphorylation and carbon assimilation. Erythromycin could induce acute toxic effects at the concentration of 0.06 mg L(-1), while the same results were exhibited for ciprofloxacin and sulfamethoxazole at higher than 1.0 mg L(-1). Erythromycin was considerably more toxic than ciprofloxacin and sulfamethoxazole and may pose a higher potential risk to the aquatic ecosystem. Some indices like chlorophyll fluorescence, Mg(2+)-ATPase activity and RuBPCase activity showed a high specificity and sensitivity to the exposure of erythromycin, and may be potentially used as candidate biomarkers for the exposure of the macrolide antibiotics.

Effect of cerium on photosynthetic pigments and photochemical reaction activity in soybean seedling under ultraviolet-B radiation stress.

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.

Effects of lead on activities of photochemical reaction and key enzymes of carbon assimilation in spinach chloroplast.

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.

Influences of calcium deficiency and cerium on the conversion efficiency of light energy of spinach.

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+).

Effects of Nanoanatase TiO2 on photosynthesis of spinach chloroplasts under different light illumination.

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.

Inhibition of photophosphorylation and electron transport chain in thylakoids by lasiodiplodin, a natural product from Botryosphaeria rhodina.

Four natural products were isolated from the fungus Botryosphaeria rhodina, and their effects on photosynthesis were tested. Only lasiodiplodin (1) inhibited ATP synthesis and electron flow from water to methylviologen; therefore, it acts as a Hill reaction inhibitor in freshly lysed spinach thylakoids. Photosystem I and II and partial reactions as well as ATPase were measured in the presence of 1. Three new different sites of 1 interaction and inhibition were found: one at CF1, the second in the water-splitting enzyme, and the third at the electron-transfer path between P680 and QA; these targets are different from that of the synthetic herbicides present. Electron transport chain inhibition by 1 was corroborated by fluorescence induction kinetics studies.

Natural diterpenes from Croton ciliatoglanduliferus as photosystem II and photosystem I inhibitors in spinach chloroplasts.

In our search for new natural photosynthetic inhibitors that could lead to the development of "green herbicides" less toxic to environment, the diterpene labdane-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.

Effect of cerium on photosynthetic characteristics of soybean seedling exposed to supplementary ultraviolet-B radiation.

Effects of cerium (Ce3+) on photosynthetic characteristics were investigated by hydroponics under laboratory conditions when soybean seedlings were exposed to two levels of supplementary UV-B radiation. UV-B radiation badly inhibited the photosynthesis in soybean seedling, leading to a reduction in net photosynthetic rate (Pn), Hill reaction activity, light saturated photosynthetic rate (Ps) and apparent quanta yield (AQY), as well as the CO2 and light saturated photosynthetic rate (Pm) and carboxulation efficiency (CE). On the contrary, Ce obviously promoted the photosynthesis of plants by increasing Hill reaction activity, accelerating electron transport and photophosphorylation, and enhancing carboxylation efficiency. For Ce+UV-B treatments, the values of photosynthetic parameters were still lower than those of the control, but obviously higher than those of UV-B treatment. The results indicated that Ce alleviated the inhibition of UV-B radiation on the photosynthesis in soybean seedling to a certain extent. In correlating of Pn with Hill activity, AQY and CE, we found that the changes of photosynthetic rate were mainly influenced by the regulating effect of Ce on Hill activity and AQY at low level (0.15 W/m(2)) of UV-B radiation, but were dominated by the regulating effect of Ce on CE at high level (0.45 W/m(2)). Thus, Ce could regulate many aspects in photosynthesis of soybean seedling under UV-B stress. The regulating mechanism was close related with the dosage of UV-B radiation.

Reversible inhibition of photophosphorylation in chloroplasts by nitric oxide.

Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. Here we demonstrate that NO is capable of regulating the activity of photophosphorylation in chloroplasts. The electron transport activity in photosystem II determined from chlorophyll a fluorescence was inhibited by NO. NO also inhibited light-induced DeltapH formation across the thylakoid membrane. High concentrations of nitrite and nitrate did not show such inhibitory effects, suggesting that the inhibition is not due to uncoupling effects of the oxidized products of NO. ATP synthesis activity upon illumination was severely inhibited by NO (IC(50)=0.7 microM). The inhibition was found to be temporary and the activity was completely recovered by removing NO. Bovine hemoglobin and bicarbonate were effective in preventing NO-dependent inhibition of photophosphorylation. These results indicate that NO is a reversible inhibitor of photosynthetic ATP synthesis.

Phytotoxic and photosynthetic activities of maduramicin and maduramicin methyl ester.

The polyether antibiotic maduramicin and its methyl ester derivative inhibited photophosphorylation and proton uptake in isolated spinach chloroplasts. Both compounds also enhanced basal and phosphorylating electron transport and stimulated Mg(2+)-dependent ATPase activity, therefore, they behave as uncouplers of photophosphorylation being the methyl ester derivative more potent than the parent compound. On the other hand, maduramicin inhibited germination and radicle elongation of several crop and weed species. In addition, the antibiotic caused phytotoxic injury and fresh weight reduction to 4-to-6 week old seedlings of two weed and two crop species when applied at 10(-4) M by foliar application in the greenhouse.

Effect of nitrite on primary photosynthetic processes in isolated chloroplasts of wheat grown under various conditions of nitrogen supply.

In wheat chloroplasts, NO2- (5 mM) inhibited non-cyclic phosphorylation coupled to the linear electron flow through both photosystems but stimulated cyclic phosphorylation with phenazine methosulfate (plus diuron) and electron flux from an artificial electron donor through PSI and methylviologen to oxygen. During light energization of chloroplasts, NO2- increased the F740/F695 ratio in the low-temperature fluorescence spectra, thus suggesting that the energy of absorbed quanta is redistributed in favor of PSI. Nitrite also stimulated the activity of Mg2+-dependent H+-ATPase. Changes in the slow component of the induction curve of delayed fluorescence also suggest that NO2- affects energy transformation processes that are coupled to electron transport in the chloroplasts. Nitrite had no effect on these functional characteristics of thylakoids and chloroplasts isolated from plants grown under nitrogen deficiency in the medium.

Synthesis, structure, and properties of MeSer1-tentoxin, a new cyclic tetrapeptide which interacts specifically with chloroplast F1 H(+)-ATPase differentiation of inhibitory and stimulating effects.

A new tentoxin analogue, in which the L-methyl alanine residue is substituted by L-methylserine, has been prepared following the synthetic pathway recently described for the synthesis of tentoxin [Cavelier, F., & Verducci, J. (1995) Tetrahedron Lett. 36, 4425-4428]. Using two-dimensional homonuclear proton nuclear magnetic resonance and structural analysis, we observed that MeSer1-tentoxin, like tentoxin, adopts several conformations in aqueous solution and presents self-aggregative properties. This analogue was found to be conformationally similar to the natural toxin. It showed the same efficiency as tentoxin in inhibition of ATPase activity of the isolated chloroplast F1 proton ATPase (CF1) as well as in inhibition of the ATP synthase activity of the membrane-bound enzyme (CF0CF1) in thylakoids and proteoliposomes. At concentrations above 10 microM, MeSer1-tentoxin did not reactivate CF1 to a high extent, contrary to tentoxin. It appeared, however, to bind in the same way, since the reactivating effect of tentoxin was inhibited by MeSer1-tentoxin. These results show that it is possible, using tentoxin analogues, to separate inhibitory and activating effects on the chloroplast ATPase, despite the limited chemical difference between the two toxins.

Mechanism of inhibitory action of the local anaesthetic trimecaine on the growth of algae (Chlorella vulgaris).

Using the model compound trimecaine, it was found that algicidal effects exhibited by the local anaesthetics of the acetanilide type were caused by two different mechanisms. The first inhibitory mechanism occurring at low concentrations of the anaesthetic is connected with the uncoupling of the photophosphorylations in algal chloroplasts and is accompanied by the enhancement of the oxygen evolving rate in algal photosynthesis. The second mechanism of inhibition of the photosynthesis in algae, taking place at higher concentrations of the anaesthetic, is connected with the damaging of the manganese containing protein on the donor side of photosystem 2 and is accompanied by a decrease of the oxygen evolving rate in algal photosynthesis.

Coupling of ATP hydrolysis to phosphate uptake in Rhodospirillum rubrum chromatophores under the influence of Ca2+ and Mg2+.

The Pi-ATP exchange and ATP hydrolytic reactions, by the F0F1 complex, were studied in Rhodospirillum rubrum chromatophores in the dark. An optimal pH between 7.0 and 8.5 was determined for the hydrolytic and exchange reactions. Under these conditions, the hydrolysis/exchange ratio was approximately 2. The kinetic analysis of the hydrolytic and exchange reactions using Mg-ATP as substrate showed a change in the hydrolysis/exchange ratio that varied between 2.0 and 2.8 as the substrate concentration was increased. With Ca-ATP, hydrolysis was not saturated up to a substrate concentration of 5.0 mM, and the hydrolysis/exchange ratios changed from 2 to 240 as the substrate concentration was increased from 0.06 to 5.0 mM. Free Mg2+ inhibited hydrolysis and phosphate uptake without altering the hydrolysis/exchange ratio. Nigericin induced an increase in the hydrolysis/exchange ratio from 2.7 to 130, whereas in the presence of valinomycin, this ratio increased from 2.7 to 21. From these results, it can be concluded that Ca-ATP hydrolysis is loosely coupled to phosphate uptake given that Pi-ATP exchange activity is extremely low, even at high rates of ATP hydrolysis.

The effect of equisetin on energy-linked reactions in Rhodospirillum rubrum chromatophores.

Light-induced proton uptake, light-induced carotenoid absorbance shift, photophosphorylation, and hydrolysis of Mg-ATP, Ca-ATP, and PPi in Rhodospirillum rubrum chromatophores are shown to be inhibited by the antibiotic equisetin. The Mg- and Ca-ATPase activities of purified F0F1-ATPase are inhibited by equisetin. In contrast, only the Ca-ATPase activity of purified F1-ATPase is decreased by equisetin, whereas the Mg-ATPase is stimulated. Both equisetin and N,N'-dicyclohexylcarbodiimide (DCCD) inhibit the hydrolytic activity of the purified H+-PPase but not the hydrolytic activity of soluble PPase from R. rubrum and yeast. The I50 for the PPi hydrolysis is near 20 microM for both equisetin and DCCD. The action of equisetin on membranes is compared to the effect of Triton X-100 and carbonyl cyanide p-trifluoromethoxyhydrazone. On the basis of these new data, equisetin is proposed to act nonspecifically on membranes and hydrophobic domains of proteins.

Some characteristics of cyclic photophosphorylation in maize bundle sheath chloroplasts.

PMS-dependent photophosphorylation in bundle sheath chloroplasts isolated from Zea mays was monitored by using a continuous method. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and venturicidin were shown to inhibit the ATP-synthesis. Venturicidin has been shown to inhibit ATP-formation in both mesophyll and bundle sheath chloroplasts. In contrast to the case in mesophyll chloroplasts, FMN was not able to promote photophosphorylation in bundle sheath chloroplasts. The effects of other cofactors and inhibitors on the ATP-synthesis in bundle sheath chloroplasts are shown. No photoinduced synthesis of inorganic pyrophosphate was seen, neither in bundle sheath chloroplasts, nor in mesophyll chloroplasts.

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D.

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.