Effect of nano-TiO2 on strength of naturally aged seeds and growth of spinach

The effects of nano-TiO₂ (rutile) and non-nano-TiO₂ on the germination and growth of naturally aged spinach seeds were studied by measuring the germination rate and the germination and vigor indexes of aged spinach seeds. An increase of these factors was observed at 0.25–4‰ nano-TiO₂ treatment. During the growth stage, the plant dry weight was increased, as was the chlorophyll formation, the ribulosebisphosphate carboxylase/oxygenase activity, and the photosynthetic rate. The best results were found at 2.5‰ nano-TiO₂. The effects of non-nano-TiO₂ are not significant. It is shown that the physiological effects are related to the nanometer-size particles, but the mechanism by which nano-TiO₂ improves the growth of spinach seeds still needs further study.     

Mechanism of nano-anatase TiO2 on promoting photosynthetic carbon reaction of spinach

Having a photocatalyzed characteristic, our previous research had proved that nano-anatase TiO₂ is closely related to the photosynthesis of spinach. It could not only improve the light absorbance and the transformation from light energy to electron energy and to active chemical energy but also promote carbon dioxide (CO₂) assimilation of spinach. However, the mechanism of carbon reaction promoted by nano-anatase TiO₂ remains largely unclear. By electrophoresis and Western blot methods, the results of the experiments proved that Rubisco from the nano-anatase TiO₂-treated spinach during the extraction procedure of Rubisco was found to consist of Rubisco and a heavier molecular-mass protein (about 1200 kDa) comprising both Rubisco and Rubisco activase. The Rubisco carboxylase activity was 2.67 times that of Rubisco from the control and it could hydrolyze ATP in the same manner as Rubisco activase. The total sulfhydryl groups and available sulfhydryl groups of the Rubisco were 32-SH and 21-SH per mole of enzyme more than those of the Rubisco purified from the control, respectively. The circular dichroism spectra showed that the secondary structure of Rubisco from the nano-anatase TiO₂-treated spinach was very different from Rubisco of the control. It suggested that the mechanism of nano-anatase TiO₂ activating Rubisco of spinach was that the complex of Rubsico and Rubisco activase was induced in spinach, which promoted Rubsico carboxylation and increased the rate of photosynthetic carbon reaction.     

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM.     

A novel hydrogen peroxide sensor based on immobilization of haemoglobin on nano-TiO2/DTAB composite film

Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO₂ and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H₂O₂) biosensor were investigated. On nano-TiO₂/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s^??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H₂O₂. The catalytic reduction current of H₂O₂ was proportional to H₂O₂ concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (K_m^app) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H₂O₂ can potentially be applied in determination of other reactive oxygen species as well.     

Evidence for a lack of phospholipid transfer protein in the stroma of spinach chloroplasts

The possible activity of phospholipid transfer protein in stroma extracts from spinach leaf has been investigated. Stroma, prepared from purified intact chloroplasts, was dialyzed and passed through various chromatography columns. None of the protein fractions eluted was able to stimulate the transfer of phosphatidylglycerol (PG) or phosphatidylcholine (PC) from liposomes to mitochondria, suggesting the lack of phospholipid transfer protein in the stroma from mature spinach chloroplasts.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics     

Influence of State-2 transition on the proton motive force across the thylakoid membrane in spinach chloroplasts

The proton motive force (pmf) across the thylakoid membrane is composed of the proton gradient and the membrane potential, which promotes millisecond-delayed light emission (ms-DLE). In this study, the time courses of LHC II phosphorylation and ms-DLE were investigated in spinach chloroplast during State-2 transition. Red light illumination resulted in an exponential rise in LHC II phosphorylation and a biphasic time course of ms-DLE. The phospho-LHC II appeared upon ∼ 1 min illumination. The phosphorylation level increased exponentially when illumination was elongated to 20 min. The t_&frac; of saturated LHC II phosphorylation was estimated 4–5 min under present illumination. During this process, the amplitudes of ms-DLE increased transiently to a maximal amplitude within 0.5 min illumination, and the reached maximum of the fast phase of ms-DLE was ∼ 140% of the dark control. Then, ms-DLE decreased from the maximum. After ≥3 min illumination, ms-DLE decreased to a lower level than the dark control. In the presence of uncouplers and inhibitors, the transient increase in the biphasic time course of ms-DLE was removed by nigericin and DCMU, and the sequential decrease was delayed by DCCD. The time course was not affected significantly by valinomycin and DBMIB. Moreover, the level of LHC II phosphorylation was enhanced by nigericin, valinomycin and DCCD, and was inhibited completely by DCMU and partially by DBMIB. Taken together, we proposed that the PS II photochemical activity remained unaffected even with a higher level of LHC II phosphorylation, which was reflected by the effect of DCCD on the time course of ms-DLE. Probably, the evidence of LHC II phosphorylation is the rearrangement of LHC II–PS II complex and the thylakoid, a feedback to light-exposure, rather than the redistribution of excitation energy from PS II to PS I.     

Rapid purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts

A rapid procedure for the purification of fructose-1,6-bisphosphate aldolase from spinach chloroplasts is presented which involves two steps; precipitation of bulk protein with polyethylene glycol and partitioning of remaining soluble protein in aqueous two-phase systems. A 94% pure preparation is obtained within 6h with a yield of 19%. A marked difference in the partition behaviour of the aldolase activity from whole leaf tissue suggested that the procedure is less efficient when leaf extract is used as starting material.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - PEG Polyethylene Glycol - SDS Sodium Dodecyl Sulfate     

Activities and multiplicity of phenolase from spinach chloroplasts during leaf ageing

Phenolase activity in spinach leaves homogenates depends on the stage of development of leaves and on the kind of homogenization procedure. Under constant experimental conditions it is low in non-senescent leaves. With the onset of senescence there is a 15–20-fold increase in soluble activity in the supernatants of broken chloroplasts as well as an increase in activation of latent phenolase in fractions containing thylakoids. This rise in activity is due to an increase in particular multiple forms, differing for supernatants and membrane sediments. Phenolase from spinach lacks monophenolase and laccase activities.     

Characteristics of 2-oxoglutarate and glutamate transport in spinach chloroplasts

The transport of glutamate, 2-oxoglutarate and malate in intact spinach chloroplasts was determined using a double-silicone-layer centrifugation technique in which the silicone layers stayed separated at the end of centrifugation. Glutamate was found to be transported via the dicarboxylate but not the 2-oxoglutarate translocator. Hence the kinetic parameters (i.e.K _m,K _i andV _max) determined in glutamate-preloaded chloroplasts represent the kinetic constants of the dicarboxylate translocator. Measurements from malate- or succinate-preloaded chloroplasts represent the aggregate values of both the dicarboxylate and the 2-oxoglutarate translocators. Calculations showed that the 2-oxoglutarate and glutamate transport required to support the high fluxes of photorespiratory NH₃ recycling could be achieved if the transport of these two dicarboxylates occurred on separate translocators. It is proposed that during photorespiration the transport of 2-oxoglutarate into and glutamate out of the chloroplast occurred via the 2-oxoglutarate and the dicarboxylate translocators, respectively. These transports are coupled to malate counter-exchange in a cascade-like manner resulting in a net 2-oxoglutarate/glutamate exchange with no net malate uptake.Abbreviation 2-OG 2-oxoglutarate     

Effect of methanol on spinach thylakoid ATPase

Methanol at 35% ($\text{v}\text{v}$) overcomes the latency of spinach thylakoid ATPase. Activation is immediate and reversible involving changes in the V_max, not the K_m of the enzyme, MgATP is a much better substrate than CaATP; free Mg²⁺ noncompetitively inhibits activity. This inhibition can be overcome by the addition of Na₂SO₃. While both MgATP and MgGTP act as substrates, free ATP and GTP both inhibit activity. ADP and MgADP are also inhibitory. Insensitivity to certain inhibitors indicates that methanol neither induces the same conformational changes in CF₁ as illumination does, nor does it lead to coupling between H⁺ movement through CF₀ and ATP hydrolysis. Methanol activation provides a much improved method for assaying thylakoid ATPase.     

Effect of some heterocyclic herbicides on CO2 assimilation by spinach chloroplasts

We have studied the inhibitory effect of heterocyclic herbicides simazine, paraquat, pyrazon and amitrole on photosynthetic CO₂ fixation and on the level of intermediates of the CO₂ assimilation cycle in isolated spinach (Spinacia oleracea) chloroplasts, as well as their in vitro activities on ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphatase. The half inhibitory concentrations (I₅₀) of CO₂ assimilation were about 1 μM for simazine and paraquat, and 10 μM for pyrazon. Amitrole, with an I₅₀ 100 μM, gave only a weak inhibition. In the presence of simazine or pyrazon the triose-phosphates/phosphoglycerate ratio diminished because of a decrease of the triose-phosphates percentage from 47% to 19%, which means an inhibition of the phosphoglycerate reduction step by a low NADPH synthesis. However, there was not a parallel increase of phosphoglycerate, because of collateral pathways leading to phospho-enolpyruvate, amino acids and other non-identified compounds. Paraquat did not give such a decreased ratio, which could be explained as an inhibition of some step of the Calvin cycle later than triose-phosphates by the H₂O₂ formed in a Mehler reaction. Amitrole did not show any effect on the pattern of intermediates. Simazine and pyrazon at 10 μM concentration promote a 20–30% activation of ribulose-1,5-bisphosphate carboxylase activity, whereas paraquat, pyrazon and simazine showed an I₅₀ about 100 μM for the inhibition of the photosynthetic fructose-1,6-bisphosphatase.     

Influence of nano-anatase TiO2 on the nitrogen metabolism of growing spinach

Previous research showed that nano-TiO₂ could significantly promote photosynthesis and greatly improve growth of spinach, but, we also speculated that an increase of spinach growth by nano-TiO₂ treatment might be closely related to the change of nitrogen metabolism. The effects of nano-anatase TiO₂ on the nitrogen metabolism of growing spinach were studied by treating them with nano-anatase TiO₂. The results showed that, nano-anatase TiO₂ treatment could obviously increase the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic-pyruvic transaminase during the growing stage. Nano-anatase TiO₂ treatment could also promote spinach to absorb nitrate, accelerate, inorganic nitrogen (such as NO ₃ ^t- −N and NH ₄ ⁺ −N) to be translated into organic nitrogen (such as protein and chlorophyll), and enhance the fresh weight and dry weights.     

Photosynthetic CO2 fixation in spinach chloroplasts inhibited by pine chloroplasts or extracts of pine chloroplasts

Chloroplasts isolated from pine needles were found to be inactive with respect to CO₂ fixation. Since it was suspected that pine needles may contain substances inhibitory to photosynthesis, studies were carried out using photosynthetically active isolated spinach chloroplasts and chloroplasts isolated from pine needles. When isolated pine chloroplasts were suspended in buffer and were added to isolated spinach chloroplasts they inhibited photosynthetic CO₂ fixation. When the pine chloroplasts were separated from the medium by centrifugation, the separated pine chloroplasts severely inhibited CO₂ fixation by isolated spinach chloroplasts, but the supernatant solution from the pine chloroplasts was not inhibitory. As little as 5% pine chloroplasts (based on chlorophyll content) produced 50% inhibition of CO₂ fixation by the spinach chloroplasts. Studies of fixation of ¹⁴C-labelled CO₂ by spinach chloroplasts were carried out in which after 5 min photosynthesis the pine chloroplasts were added. It was found that the subsequent inhibition of spinach CO₂ fixation was neither due to any effect on the rate of export of photosynthetic metabolites from the chloroplasts to the medium, nor to a direct effect on the RUBP carboxylase reaction. The principal effect was found to be an inhibition of the conversion of fructose-1,6-bisphosphate and sedoheptulose-1,7-bisphosphate to the respective monophosphates and inorganic phosphate. From this finding it was concluded that a principal effect of the inhibition by pine chloroplasts is probably an inhibition either directly or indirectly of the bisphosphatase enzymes in the spinach chloroplasts. Based on its distribution between organic and aqueous acidic or neutral solutions, the inhibitory factor of the pine chloroplasts must be lipophilic. Most of the factor could be transferred to an aqueous phase in a strongly alkaline solution. Following subsequent acidification of the aqueous phase the activity could be completely transferred back into the organic phase. This procedure allowed for separation of the inhibitory factor from most of the pigments and other lipophilic substances present in the pine chloroplasts and yielded a preparation which could be subsequently fractionated by thin layer chromatography. UV absorption was found in two fast moving spots and at the origin. The fastest running spot from the thin layer chromatography plate was found to be the one containing most of the inhibitory activity.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

Improved rates of CO2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum

Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO₂-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO₂-dependent oxygen evolution of 200–300 mol·mg chlorophyll^-1·hour^-1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO₂ fixation were not superior to those isolated in sorbitol media. The K⁺ content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K⁺ which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO₂ fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - EDTA ethlenediamine tetraacetic acid     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

Control of 35SO 4 2- and 35SO 3 2- incorporation into spinach chloroplasts during photosynthetic CO2 fixation

In addition to membrane translocation, measured in the dark, it was found that pre-illumination of the chloroplasts resulted in an enhancement of sulfate uptake by 25% and of sulfite uptake by 55% as soon as the concentration of the ion in the incubation medium exceeded 2 mmol l^-1. This amount which is additionally taken up after pre-illumination is less readily exchanged for other ions. Kinetics of the uptake in relation to pre-illumination time and to light intensity closely parallel those of titration of SH-groups by 5,5-dithiobis (2-nitrobenzoic acid). As a consequence, 10^-6 mol l^-1 DCMU completely inhibits the light triggered increase of uptake of both ions. Uncoupling with 10^-6 mol l^-1 CCCP increases the light induced ³⁵SO ₃ ^2- binding, but decreases that of ³⁵SO ₄ ^2- , demonstrating the need of ATP formation to initiate sulfate reduction. Rates of uptake, measured at different intensities of pre-illumination under nitrogen or in the presence of bicarbonate, suggest that the presence of a carbon skeleton increases the binding rate for both ions. With respect to ³⁵SO ₄ ^2- , the data further indicate a rate limiting step (ATP sulfurylase or adenosine 5-phosphosulfate sulfotransferase) which is activated by light, thus representing a control step to harmonize the rate of CO₂ fixation and of sulfate incorporation. On the contrary, ³⁵SO ₃ ^2- is directly bound in relation to the amount of SH-groups, which in turn are created by the photosynthetic electron transport, resulting in Car-S-SO ₃ ^- . Since the formation of SH-groups is maximal already at low light intensities, no effective control step for SO ₃ ^2- incorporation is indicated.     

菠菜和蚕豆叶绿体CF_1结构与功能的比较

比较了菠菜和蚕豆叶绿体的光合磷酸化活力以及由不同活化方法活化的叶绿体及可溶CF1的Mg2＋－ATPase和Ca2＋－ATPase的活力,观测到两种叶绿体ATPase的合成和水解ATP的功能有明显差异。从两种叶绿体CF1的SDS－PAGE图谱上可见蚕豆CF1的ε亚基分子量明显小于菠菜的,蚕豆CF1的α和β亚基间分子量的差别也比菠菜的小。