Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts

The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO₂ fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO₂ fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.     

Relationship between the Level of Adenine Nucleotides and the Carboxylation Activity of Illuminated Isolated Spinach Chloroplasts: A Study with Antimycin A

The changes in the levels of intact spinach (Spinacia oleracea L.) chloroplast adenine nucleotides during the time course of light-dependent CO₂ fixation were determined with respect to the effect of antimycin A. This study demonstrated that antimycin A lowered the rate of ATP formation during the induction period of carboxylation. While the steady state levels of ATP and the energy-charge value also decreased in the presence of antimycin, the concomitant increase of the CO₂ fixation activities insured higher ATP turnover rates. Changes in the labeling of CO₂ fixation products during the lag phase suggested a stepwise activation of the Calvin cycle, with fructose 1,6-diphosphate, and ribulose 5-phosphate kinase being activated before ribulose 1,5-diphosphate carboxylase. The possible mechanisms of the enhancement of CO₂ fixation activity by antimycin A in relation to its action on photophosphorylation during the lag phase are discussed.     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

Oxygen Concentration in Isolated Chloroplasts during Photosynthesis

The O(2) concentration in intact and osmotically disrupted isolated spinach (Spinacia oleracea, L.) chloroplasts during photosynthesis was estimated. The chloroplasts were allowed to reduce 3-phosphoglycerate, CO(2), or ferricyanide in light until the rate of O(2) production was linear. When the light was turned off O(2) evolution from the chloroplasts continued for a few seconds. This prolonged O(2) evolution is due to an O(2) surplus inside the chloroplasts which equilibrates with that in the medium. From this surplus the O(2) concentration inside the chloroplasts at the moment when the light had been switched off was calculated. In all experiments the O(2) concentration inside the photosynthesizing chloroplasts was higher than that outside, but was dependent upon the O(2) concentration of the chloroplast medium. At low external O(2) concentration (30 mum) the ratio of the internal to the external O(2) concentration was about 5, whereas at concentrations corresponding to those in airsaturated water this ratio was close to 1. With osmotically broken chloroplasts this ratio was 1.2 at 30 mum O(2) and almost 1 from 150 mum onward. When the O(2) surplus found in broken chloroplasts during photosynthesis was related to the volume of the thylakoids, a ratio of about 2.3 was observed.     

低渗膨胀对菠菜完整叶绿体光合作用的影响

菠菜离体完整叶绿体需要合适的介质渗透压（约０．９ＭＰａ）以保持其较高的光合作用速率。当渗透压因降低介质中山梨醇浓度（从０．３３ｍｏｌ／Ｌ至０．１７ｍｏｌ／Ｌ）而降低时,叶绿体的完整率保持不变。低于临界渗透压（约０．５ＭＰａ）,叶绿体被膜就发生破裂．并丧失ＣＯ２同化能力。在轻度低渗条件下,虽然叶绿体被膜未破,但依赖ＣＯ２的放氧速率已受抑制。渗透压在０．９ＭＰａ与０．５ＭＰａ之间,叶绿体依赖ＰＧＡ的放氧抑制,可由加入山梨醇至正常浓度（０．３３ｍｏｌ／Ｌ）而解除。膨涨叶绿体的ＡＴＰ合成水平与正常叶绿体相同,而ＮＡＤＰＨ形成速率则明显降低。利用能透过被膜的不同电子受体ＮＣ２、ＰＧＡ和ＯＡＡ发现,在膨胀叶绿体中,ＮＯ２的还原不受形响,而ＰＧＡ及ＯＡＡ的还原明显被抑制。我们推测,低渗膨胀叶绿体中光合作用的抑制,至少有一个原因是Ｆｄ－ＮＡＤＰ氧化还原酶作用的受阻。     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts

In isolated barley chloroplasts, the presence of 2 millimolar ZnSO₄ inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, O₂ evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCl₂. These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO₄ is observed. However, with concentrations of ZnSO₄ above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn²⁺-treated chloroplasts partially restores the O₂-evolving activity.     

Transport of Metabolites across Isolated Envelope Membranes of Spinach Chloroplasts

Isolated envelope membranes of spinach chloroplasts (Spinacia oleracea L. var. Viroflay) exhibited selective permeability. Metabolites such as 3-phosphoglycerate, bicarbonate, glyoxylate, and acetate were transported rapidly; 6-phosphogluconate, glycolate, glycine, l-malate, and succinate were intermediate; whereas glucose 6-phosphate, fructose 1,6-diphosphate, and sucrose were hardly transported. Transport rates, metabolite accumulations within the membrane vesicles, and the internal water volume of isolated and in situ envelope membranes were compared and found to show similar trends.     

Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride

Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl₂ (less than 1 μmole Hg²⁺/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl₂ concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl₂ to these sites.     

Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Stimulation of Photoreactions of Isolated Chloroplasts by Serum Albumin

Serum albumin was shown to stimulate markedly various photoreactions in isolated bean and lettuce chloroplasts. The maximal effect was obtained when this compound was present during the homogenization step and continuously in the chloroplast preparation. The "basal" electron transport was enhanced using various acceptors and stimulation was obtained also in the presence of uncouplers. The quantum requirement for ferricyanide reduction was appreciably reduced. Serum albumin increased the rate of cyclic phosphorylation and the ratio of P/e(2) in non-cyclic phosphorylation. The increase in phosphorylation is supposedly due to inhibition of the rate of decay of the high energy non-phosphorylated intermediate, X(E).It is postulated that serum albumin affects chloroplast photoreactions by binding endogenously released unsaturated fatty acids.     

草酰乙酸和苹果酸对菠菜叶片和完整叶绿体光合作用的影响

观测了OAA和MA对菠菜叶片和完整叶绿体光合作用的影响.结果显示,当叶片切块在20μmol/L的OAA存在时,其叶片的光合放氧速率增加了89%,经OAA处理的离体完整叶绿体的光合放氧速率增加了72%;当反应体系中存在有较高浓度的NaHCO3时,OAA的作用不明显.叶片经20 μmol/L的MA处理后,叶片光合放氧速率比对照高127%.用CO2分析仪观测了处理后叶片的净光合速率(Pn),结果显示,OAA和MA处理后的叶片Pn值分别是对照的117%和111%.对在C3植物中建立C4微循环系统来提高光合作用效率的可能性进行了讨论.     

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.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

菠菜叶绿体的光抑制部位

有氧条件下,叶绿体的光抑制部位不是专一的。强光可使PSⅡ氧化侧、PSⅡ反应中心、PSⅡ还原侧,PSⅡ及类囊体膜透性都有不同程度的破坏。这种非专一性可能与类囊体膜蛋白在强光下的降解有关。无氧条件下,叶绿体的光抑制部位只是在PSⅡ反应中心及Q_B蛋白上。