A rapid method for isolation of purified,physiologically active chloroplasts,used to study the intracellular distribution of amino acids in pea leaves

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of ¹⁴CO₂ fixation and CO₂-dependent O₂ evolution (over 100 mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and -(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, -aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.     

Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

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     

Localization of sulfolipid labeling within cells and chloroplasts

Spinach chloroplasts were purified on gradients of Percoll which preserved envelope impermeability and CO₂-dependent oxygen evolution in the light. Application of ³⁵SO₄ to purified chloroplasts resulted in a light-dependent labeling of a lipid component which was indentified as sulfoquinovosyl diacylglycerol. Fractionation of chloroplasts showed that after 5 min of labeling most of the newly synthesized sulfolipid was present in thylakoids. Only a small percentage was recovered from the envelopes. Molecular species from envelopes and thylakoids were identical. The molecular species did not change during incubation times ranging from 5 min up to 4.5 h. Mesophyll protoplasts from ³⁵SO₄-labeled oat primary leaves were gently disrupted and separated into organelles by sucrose gradient centrifugation. Labeled sulfolipid was located almost exclusively in the chloroplasts. This, in combination with the experiments carried out with isolated chloroplasts, indicates that the final assembly steps in the biosynthesis of sulfolipid are confined to the chloroplasts.     

‘Tell me where all past years are’

This is about a young man who wished to go to sea like his father and finished up, instead, in photosynthesis. It describes how he served his apprenticeship in England and the United States and how he was then lucky enough to find himself in the laboratory of Robin Hill, one of the all-time greats in this field. It discusses some of the events that led, via mitochondria in castor beans and carboxylating enzymes in Crassulacean plants, to the isolation of fully functional chloroplasts and the manner in which the first polarographic measurements of CO₂-dependent O₂ evolution contributed to present understanding of the movement of molecules through the chloroplast envelope. It describes some of the problems with materials and apparatus which were commonplace forty years ago and reflects on the advantages of working in foreign places and the pleasures of becoming a member of a truly international community.     

Inhibition of the phosphate transporter during isolation of intact chloroplasts from leaves of sunflower

Chloroplasts isolated from spinach leaves by the mechanical method were intact and exhibited high rates of CO₂-dependent oxygen evolution whereas chloroplasts isolated from sunflower leaves by the same technique were also intact but showed only low rates of oxygen evolution. The rate of uptake of orthophosphate (Pi) from the suspending medium with sunflower chloroplasts was less than 20% of that in spinach chloroplasts. The apparent Km for Pi transport was lower in sunflower chloroplasts but uptake was competitively inhibited by 3-phosphoglycerate in chloroplasts from both species. Uptake of malate (via the dicarboxylate transporter) and of ATP (via the adenine nucleotide transporter) was also reduced in sunflower chloroplasts compared to spinach chloroplasts. The endogenous Pi content and total exchangeable phosphate pool of sunflower chloroplasts were less than half that in spinach chloroplasts.Addition of a number of possible protective agents to the grinding medium failed to prevent the loss of photosynthetic activity during mechanical isolation of sunflower chloroplasts. Grinding mixtures of spinach and sunflower leaves together indicated that spinach chloroplasts were not inhibited by the sunflower leaf extract. Chloroplasts isolated from sunflower leaves via protoplasts had high rates of CO₂-dependent oxygen evolution. The Vmax and Km for Pi uptake, endogenous Pi content and total exchangeable phosphate pool of chloroplasts isolated from sunflower protoplasts were all similar to spinach chloroplasts. It is concluded that inner envelope membrane proteins are damaged during mechanical isolation of sunflower chloroplasts. The decrease in activity of the phosphate transporter and loss of endogenous phosphate may contribute to the low rates of photosynthesis observed in chloroplasts isolated by the mechanical method from leaves of sunflower and possibly other species.Abbreviations PGA 3-phosphoglyceric acid     

PHYLOGENETIC RELATIONSHIPS OF CAULERPA (CHLOROPHYTA) BASED ON COMPARATIVE CHLOROPLAST ULTRASTRUCTURE1,2

The ultrastructure of chloroplasts from 28 of the 73 species of Caulerpa Lamouroux (Chlorophyta, Caulerpales) has been studied to aid in interpreting phylogenetic relationships among the 12 recognized sections. Variations of systematic value include pyrenoid occurrence and fine structure, thylakoid architecture and amount of photosynthate storage. Comparisons of field and culture specimens indicate these characters are consistent. Chloroplast thylakoids are grouped into bands, with the distribution of bands differing among species. In the most common arrangement, bands are evenly distributed throughout the chloroplast. A few species show lateral displacement of bands whereas others have a majority of bands arranged at one end of the chloroplast. Starch is stored cither as one or two large grains (> 1 μm diam.) or numerous small grains (< 0.5 μm diam.). Electron-transparent regions are common in other species in which chloroplasts rarely store starch. Simple, embedded pyrenoids are present in several species of section Sedoideae. An opaque region occurs in chloroplasts of C. elongata which may represent an intermediate stage in the evolutionary loss of the pyrenoid. It is suggested that the chloroplast of Caulerpa evolved, from a large, complex, pyrenoid-containing organelle housing both photosynthetic and amylogenic functions, to a small, structurally simpler one, specialized for photosynthesis alone. A phylogeny of the 12 sections of Caulerpa is constructed, based on chloroplast evolution which agrees with an earlier morphology-based hypothesis on the origin and evolution of Caulerpa.     

Prenyl lipid formation in spinach chloroplasts and in a cell-free system of Synechococcus (Cyanobacteria): polyprenols,chlorophylls, and fatty acid prenyl esters

Isolated chloroplasts from spinach leaf cells, chloroplast subfractions, and a cell-free system of the cyanobacterium Synechococcus CCAP 6312 incorporated [1-¹⁴C]isopentenyl pyrophosphate in high yields into prenyl lipids. Products were polyprenols (C₂₀, C₄₅) chlorophylls, quinoid compounds, and fatty acid prenyl esters; prenyl pyrophosphates occurred in trace amounts, and carotenes were only formed to a limited extent in the Synechococcus system. The formation of fatty acid prenyl esters, which is described here for the first time, was found to occur in two different ways in the chloroplast system; by an acyl-CoA: polyprenol acyltransferase reaction associated with the envelope membranes and by a transesterification reaction from chlorophyll associated with the thylakoids. Endogenous fatty acid prenyl esters made up about 3% by weight of total lipids in spinach chloroplasts and were also found to be natural constituents of the cyanobacterial cells.Abbreviations Chl chlorophyll - Chl_GG chlorophyll a containing a geranylgeranyl side chain - IPP isopentenyl pyrophosphate     

Isolation of Functionally Intact Rhodoplasts from Griffithsia monilis (Ceramiaceae, Rhodophyta)

A procedure is described for isolating photosynthetically active rhodoplasts (“red algal chloroplasts”) from the marine alga Griffithsia monilis. The rhodoplasts exhibited rates of CO₂ fixation and CO₂-dependent O₂ evolution in the order of 200 micromoles per milligram chlorophyll a per hour when illuminated with red or green light and were approximately 80% intact. The response of the rate of photosynthesis to the inorganic phosphate and pyrophosphate concentrations in the medium was qualitatively similar to that previously reported for spinach chloroplasts. Osmotically shocked rhodoplasts evolved O₂ from ferricyanide in red, but not in green, light and were completely uncoupled. Rhodoplast envelope rupture appeared to be accompanied by phycobilisome loss from the thylakoids.     

Transport du sulfate à travers la double membrane limitante, ou enveloppe, des chloroplastes d'épinard

Evidence is presented for low rates of carriermediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [³⁵S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO₂-dependant O₂ evolution rates after addition of sulphate.Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate transport had saturation kinetics of the Michaelis-Menten type (Vmax : 25 μmoles . mg⁻¹ chl . h⁻¹ at 22°C ; Km : 2.5 mM). The rate of transport for sulphate was not influenced either by illumination or pH change in the external medium. Phosphate was a competitive inhibitor of sulphate uptake by chloroplasts (Ki : 0.7 mM, fig. 1). The rate of transport for phosphate appeared to be much higher than for sulphate. When the chloroplasts were pre-loaded with labelled sulphate, radioactivity was rapidly released after addition of phosphate into the external medium. Consequently, the transport of sulphate occurs by a strict counter-exchange : for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa.The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO₂-dependent O₂ evolution.The presence, on the inner membrane of the chloroplast envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.     

Photosynthesis in chloroplasts rapidly isolated from primary leaves of oat (Avena sativa L.): effects of orthophosphate, metal ion and chelators

Abstract A simple mechanical method for the rapid isolation of chloroplasts with high rates of photosynthesis from young leaves of oat (Avena sativa L.) was described. The photosynthetic activity of these chloroplasts was stable for at least 2 h with rates of CO₂-dependent O₂ evolution of 30–40 μmol g ¹ Chl s ¹. The photosynthetic properties of these chloroplasts were similar to those reported for spinach and pea chloroplasts isolated by mechanical disruption. The pH optimum for photosynthetic O₂ evolution was pH 7.6. The induction time was 0.5–2 min. Maximal rates of photosynthetic O₂ evolution in these chloroplast preparations were obtained in the absence of both divalent cations and EDTA. Addition of divilent cations strongly inhibited photosynthesis which could be partially restored by the subsequent addition of EDTA. But when these cations were not present in the assay medium the addition of EDTA greater than 1 mol m ³ decreased photosynthetic activity. The optimal orthophosphate concentration required for photosynthesis in these chloroplast preparations was 0.2–0.3 mol m ³. In contrast, the addition of pyrophosphate either in the light or dark inhibited photosynthesis. In a comparative study, chloroplasts were also isolated from oat and wheat (Triticum aestivum L., cultivar Hybrid C306) protoplasts. These chloroplast preparations were found to have properties similar to those determined for oat chloroplasts isolated by the mechanical method reported above.     

Orthophosphate control of glucose-6-phosphate dehydrogenase light modulation in relation to the induction phase of chloroplast photosynthesis

High concentrations of orthophosphate (Pi) inhibited CO₂-dependent O₂ evolution and prevented the inactivation of glucose-6-P dehydrogenase by light in intact spinach and barley chloroplasts. Addition of glycerate-3-P to chloroplasts inhibited by Pi in the light, induced O₂ evolution and caused rapid inactivation of glucose-6-P dehydrogenase. The activity of phosphofructokinase detected in chloroplast preparations was not affected by light or by Pi.     

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of ¹⁴C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of ¹⁴CO₂, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO₂-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O₂ to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH₃+2-oxoglutarate)-dependent O₂ evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Regulation of chloroplast photosynthetic activity by exogenous magnesium

Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl₂, added in the dark, inhibited CO₂-dependent O₂ evolution by Hordeum vulgare L. and Spinacia oleracea L. (C₃ plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O₂ evolution by Digitaria sanguinalis L. (C₄ plant) mesophyll chloroplasts from 80 to 100%. When Mg²⁺ was added in the light, O₂ evolution was reduced only slightly. O₂ evolution in the presence of phosphoglycerate was less sensitive to Mg²⁺ inhibition than was CO₂-dependent O₂ evolution.

Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg²⁺ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg²⁺ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes.

     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.Abbreviations Rubisco ribulose bisphosphate carboxylase/oxygenase - CAB Chl a/b-binding (proteins) - Chlide chlorophyllide - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - Pchlide protochlorophyllide     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O₂-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.