Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O₂ evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H₂O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O₂ evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H₂O as eventual electron donor via the sequence H₂O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K_m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H₂O₂ reduction by washed intact chloroplasts.     

Ammonia assimilation and oxygen evolution by a reconstituted chloroplast system in the presence of 2-oxoglutarate and glutamate

(Ammonia plus 2-oxoglutarate)-dependent O₂ evolution by intact chloroplasts was enhanced three- to five fold by 2 mM L- and D-malate, attaining rates of 9–15 μmol mg^-1 Chl h^-1. Succinate and fumarate also promoted activity but D-aspartate and, in the presence of aminooxyacetate, L-aspartate inhibited the malate-promoted rate. A reconstituted chloroplast system supported (ammonia plus 2-oxoglutarate)-dependent O₂ evolution at rates of 6-11 μmol mg^-1 Chl h^-1 in the presence of MgCl₂, NADP(H), ADP plus Pi (or ATP), ferredoxin and L-glutamate. The concentrations of L-glutamate and ATP required to support 0.5 V _max were 5 mM and 0.25 mM, respectively. When the reaction was initiated with NH₄Cl, O₂ evolution was preceded by a lag phase before attaining a constant rate. The lag phase was shortened by addition of low concentrations of L-glutamine or by preincubating in the dark in the presence of glutamate, ATP and NH₄Cl. Oxygen evolution was inhibited by 2 mM azaserine and, provided it was added initially, 2 mM methionine sulphoximine. The (ammonia plus 2-oxoglutarate)-dependent O₂ evolution was attributed to the synthesis of glutamine from NH₄Cl and glutamate which reacted with 2-oxoglutarate in a reaction catalysed by ferredoxin-specific glutamate synthase using H₂O as the ultimate electron donor. The lag phase was attributed to the establishment of a steady-state pool of glutamine. L-Malate did not affect the activity of the reconstituted system.     

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

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.     

Characterisation of a H2O2-oxidisable cytochrome b-559 in intact chloroplasts with a new type of LED Array Spectrophotometer

Cytochrome (cyt) b-559 absorbance changes in intact chloroplasts were deconvoluted using a previously described LED-Array-Spectrophotometer (Klughammer et al. (1990), Photosynth Res 25: 317–327). When intact chloroplasts were isolated in the presence of ascorbate, approx. 15% of the total cyt b-559 could be transiently oxidised by 200 M H₂O₂ in the dark. This fraction displays low-potential properties, as it can be also oxidised by menadione in the presence of 5 mM ascorbate. Heat pretreatment increased the size of this fraction by a factor of 3–4. Low concentrations of cyanide (in the M range) prolonged the oxidation time while high concentrations suppressed the oxidation (I₅₀=1.5 mM KCN). The former KCN-effect relates to inhibition of ascorbate dependent H₂O₂-reduction which is catalysed by ascorbate peroxidase, whereas the latter effect reflects competition between H₂O₂ and CN^– for the same binding site at the cytochrome heme. In the light, much lower concentrations of H₂O₂ were required to obtain oxidation, the amplitude depending on light intensity and on the concentration of the added H₂O₂, but never exceeding approx. 15% of the total cyt b-559. In the light, but not in the dark, H₂O₂ also induced the transient oxidation of a cyt f fraction similar in size to the H₂O₂-oxidisable cyt b-559 fraction. In this case, H₂O₂ serves as an acceptor of Photosystem I in conjunction with the ascorbate peroxidase detoxification system. Light can also induce oxidation of a 15% cyt b-559 fraction without H₂O₂-addition, if nitrite is present as electron acceptor and the chloroplasts are depleted of ascorbate. It is concluded that light-induced cyt b-559 oxidation in vivo is likely to be restricted to the H₂O₂-oxidisable cyt b-559 LP fraction and is normally counteracted by ascorbate.Abbreviations APX ascorbate peroxidase - chl chlorophyll - cyt cytochrome - HP high potential - LP low potential - MDA monodehydroascorbate - PQ plastoquinone - PS I and PS II Photosystems I and II     

Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H₂O₂-dependent oxidation of ascorbate and ascorbate-dependent reduction of H₂O₂. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K_m value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN₃, KCN, and 8-hydroxyquinoline but not ZnCl₂ or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.     

Light-dependent incorporation of selenite and sulphite into selenocysteine and cysteine by isolated pea chloroplasts

Illuminated intact pea chloroplasts in the presence of O-acetylserine (OAS) catalysed incorporation of SeO₃^2- and SO₃^2- into selenocysteine and cysteine at rates of ca 0.36 and 6 μmol/mg Chl per hr respectively. Sonicated chloroplasts catalysed SeO₃^2- and SO₃^2- incorporation at ca 3.9 and 32% respectively of the rates of intact chloroplasts. Addition of GSH and NADPH increased the rates to ca 91 and 98% of the intact rates, but SeO₃^2- incorporation under these conditions was essentially light-independent. In the absence of OAS, intact chloroplasts catalysed reduction of SO₃^2- to S^2- at rates of ca 5.8 μmol/mg Chl per hr. In the presence of OAS, S^2- did not accumulate. Glutathione (GSH) reductase was purified from peas and was inhibited by ZnCl₂. This enzyme, in the presence of purified clover cysteine synthase, OAS, GSH and NADPH, catalysed incorporation of SeO₃^2- into selenocysteine (but not SO₃^2- into cysteine). The reaction was inhibited by ZnCl₂. Incorporation of SeO₃^2- into selenocysteine by illuminated intact chloroplasts and sonicated chloroplasts (with NADPH and GSH) was also inhibited by ZnCl₂ but not by KCN. Conversely, incorporation of SO₃^2- into cysteine was inhibited by KCN but not by ZnCl₂. It was concluded that SeO₃^2- and SO₃^2- are reduced in chloroplasts by independent light-requiring mechanisms. It is proposed that SeO₃^2- is reduced by light-coupled GSH reductase and that the Se^2- produced is incorporated into selenocysteine by cysteine synthase.     

Studies on the water-oxidizing system by the effects of different treatments in chloroplasts

Treatments such as trypsinization (50 μg/ml per mg Chl for 1 h), osmotic shock of the chloroplasts or mild heating altered the oxygen evolution in such a way that the properties of the Photosystem II were simplified. After these treatments, the damping of the oscillation pattern of O₂ yields induced by a flash series remained the same, irrespective of the level of inhibition induced by the treatment. This damping did not decrease with increasing flash energy, as observed in untreated chloroplasts. The light saturation curve of the S₂ → S₃ transition of the O₂ evolving system no more exhibited the slow-increasing phase at high flash energy observed under normal conditions. The kinetic properties of the O₂-evolving system were also simplified. After the treatments cited above, deactivation of S₂ and S₃ were identical and accelerated with respect to untreated chloroplasts. Turnover kinetics of the transitions S₁ → S₂ and S₂ → S₃ were also similar and simpler without a lag for S₂ → S₃. These results indicate that the treatments mentioned above disconnect one donor from the O₂-evolving complex. This donor, under normal conditions, contributes to the increase of the quantum yield of the transition S₂ → S₃ at high flash energy. This donor is here denoted by D. Our results are in agreement with the following working hypothesis: the large miss, observed on the S₂ → S₃ transition without any contribution of the donor D, may be due to the fact that the system needs a conformation change of the O₂-evolving complex in the S₂ state, so that the main donor Y can oxidize the second H₂O molecule in the water-splitting complex. In the inactive state corresponding to the absence of a conformation change, the donor D, being different in configuration, is likely to oxidize the S₂ state into an S₃ state at high light intensity.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

Chloroplast cysteine synthases of Trifolium repens and Pisum sativum

About 68–86% of the cysteine synthase activity in leaf tissue of white clover (Trifolium repens) and peas (Pisum sativum cultivar Massey Gem) was associated with chloroplasts. The enzymes from white clover and peas were purified ca 66 and 12-fold respectively. For clover, the K_m values determined by calorimetric and S^2? ion electrode methods were: S^2? 0.51 and 0.13 mM; O-acetylserine (OAS), 3.5 and 2.O mM respectively. The analogous values for the pea enzyme were: S^2?, 0.24 and 0.06 mM; OAS, 3.1 and 0.24 mM. Both enzymes were inhibited by cystathionine and cysteine. Pretreatment with cysteine inactivated the enzyme, but addition of pyridoxal phosphate caused partial reactivation. Isolated pea chloroplasts (70–75 % intact) catalysed OAS-dependent assimilation of sulphide at a mean rate of 88 μmol/mg Chl/hr. About 85 % of the OAS-dependent sulphide assimilated was recovered as cysteine. The rates were unaffected by light and 2 μM DCMU. Sonicating the chloroplasts enhanced the rate by 1.3–2 fold. Cysteine synthase activity was associated with the chloroplast stroma. Similar results were obtained for clover chloroplasts except that both the intactness and the rates were lower.     

Iodination of chloroplasts. I. Properties of iodinated chloroplasts

Spinach chloroplasts exposed to iodide can be washed free of the bulk of the iodide. In the presence of lactoperoxidase and H₂O₂, iodide can be introduced into chloroplasts in high amounts and in non diffusible forms. The resultant particles, which have been named iodochloroplasts, extrude their iodide upon stimulation by light. The form and the amount of extruded iodide bears a definite relationship to the amount of incident light. A flash of marginally effective light is additive to the next such flash even after a lapse of 10 min of darkness. These and other properties of iodochloroplasts may make them of great use in the study of intermediate reactions of photosynthesis.     

Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Hydrogen evolution by co-immobilized chloroplasts and Clostridium butyricum

Spinach chloroplasts and Clostridium butyricum cells were immobilized in 2% agar gel. Crude ferredoxin isolated from spinach and benzyl viologen were used as electron carriers. The optimum pH for both NADP reduction by immobilized chloroplasts and for hydrogen evolution by immobilized Cl. butyricum was 8.0. The optimum temperature was between 25 and 30°C for NADP reduction by immobilized chloroplasts, and 37°C for hydrogen evolution by immobilized cells. The total amount of hydrogen evolved in 6 h was 41 μmol/mg Chl for the immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system, and 11 μmol/mg Chl for the immobilized chloroplast-ferredoxin-Cl. butyricum system. The systems evolved only a trace amount of hydrogen when dichlorophenyldimethylurea was added. The immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system evolved hydrogen continuously for 6 h, and immobilized Cl. butyricum retained the initial hydrogenase activity. However, the photoreduction activity of chloroplasts decreased to 30% of the initial activity after 6 h of reaction.     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

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     

Photosynthesis in cell suspension cultures of the CAM plant Chamaecereus sylvestrii (Cactaceae)

Abstract Photosynthetic properties of cell suspension cultures derived from the callus proliferation of cladophyll explants of Chamaecereus sylvestrii Spegazzini were studied. High content of chlorophyll (105–120 μg/g fresh weight), cyanide sensitive O₂ uptake and maximal rates of O₂ evolution (100–115 μmol/mg Chl x h) and CO₂ fixation (130–150 μmol/mg Chl x h) were some of the properties of the exponential phase cells. Determination of the component reactions, viz. photosystems I and II and photophosphorylation of the chloroplasts isolated from the cells, indicated normal development and functioning of the photosynthetic machinery. Studies on the enzymatic reactions as well as the determination of the early products of ¹⁴CO₂ fixation in light in these cells implicated the operation of both autotrophic and non-auto-trophic pathways, the latter being less pronounced. The diurnal oscillation of titratable acidity and malate content found in the intact cladophyll tissues was absent in the cultured cells. Evidences for a rapid and continuous drain of carbon from malate into the citrate and isocitrate components of the TCA cycle via pyruvate after decarboxylation, and then into the amino acid pool are presented. The absence of large vacuoles and the rapid turnover of malate are considered to account for the lack of diurnal fluctuation of organic acide in the cell cultures.     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

ANTIOXIDATIVE PROTECTION OF PERIDINIUM GATUNENSE IN LAKE KINNERET: SEASONAL AND DAILY VARIATION1

A comprehensive antioxidative mechanism was found in the freshwater dinoflagellate Peridinium gatunense Lemm. during the spring bloom in Lake Kinneret. Ascorbate was present throughout the bloom period and was responsible, together with catalase, for the elimination of photosynthetically produced H₂O₂. As glutathione concentrations and ascorbate regenerative enzymes were negligible during mid-spring, ascorbate was presumably biosynthesized during the photosynthetically active period. Antioxidative activity increased overall at the end of the spring in conjunction with elevated ambient stress conditions, for example high light. Under such circumstances, ascorbate was regenerated. Ascorbate levels doubled when cells were exposed to an increase in irradiance from 60 to 600 μmol photons·m^?2·s^?1, and on addition of H₂O₂, concentrations increased a further 20-fold. Significant antioxidative activity was also noted in the dark, although this was dependent on the presence of H₂O₂. Diurnal changes in antioxidants and their regenerative enzymes were observed. The activities of mono-dehydroascorbate reductase, glutathione reductase, and ascorbate concentrations showed ultraradian periodicity and were completely in phase throughout the day/night period. Dehydroascorbate reductase activity and glutathione concentrations were also in phase but showed aperiodic variation, as did ascorbate peroxidase activity. Superoxide dismutase and catalase activities were generally out of phase during the 24-h period but did show ultraradian periodicity. Lake samples entrained under constant light revealed an inate 12-h rhythm for catalase activity, during at least 36 h.     

Relationship between chloroplast structure and O2 evolution rate of leaf discs in plants from different biotopes in South Finland

Abstract The chloroplast ultrastructure, especially the thylakoid organization, the polypeptide composition of the thylakoid membranes and photosynthetic O₂ evolution rate, chlorophyll (Chl) content and Chi a/b ratio were studied in leaves of nine plants growing in contrasting biotopes in the wild in South Finland. All the measurements were made at the beginning of the period of main growth on leaves approaching full expansion, when the CO₂-saturated O₂ evolution rate (measured at 20°C and 1500 μmol photons m^?2s^?1) was at a maximum, ranging from 19.2 to 6.9 μmol O₂ cm^?2 h^?1. Among the species, the Chi a/b ratio varied between 3.75 and 2.71. In the mesophyll chloroplasts, the ratio of the total length of appressed to non-appressed thylakoid membranes varied between 1.07 and 1.79, the number of partitions per granum varied between 2.8 and 12.0 and the grana area between 21 and 42% of the chloroplast area. There was a significant relationship between the rate of O₂ evolution of the leaf discs and the thylakoid organization in the mesophyll chloroplasts. The higher the O₂ evolution rate, the lower was the ratio of the total length of appressed to non-appressed thylakoid membranes and also the lower the grana area. Although the relationship of the photosynthetic rate with the Chi content and the Chi a/b ratio of the leaves was not as clear, a significant negative correlation existed between the Chi a/b ratio and the ratio of appressed to non-appressed thylakoid membranes, indicating lateral heterogeneity in the distribution of different Chl- protein complexes.