Effect of Disalicylidenepropanediamine on the Light-dependent Reduction of Carbon Dioxide and Glycerate 3-Phosphate in Intact Spinach Chloroplasts

Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light-dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3-phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+)-linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane-mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248).     

Ferricyanide Reduction in Photosystem II of Spinach Chloroplasts

Ferricyanide can be reduced in Photosystem II of spinach chloroplasts at 2 separate sites, both of which are sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but only one of which is sensitive to dibromothymoquinone. Data presented in this paper emphasize ferricyanide site II of Photosystem II, which is sensitive to thiol inhibition and may reflect a cyclic pathway around Photosystem II. Ferricyanide reduction sites 1 and 2 also differ from each other in fractions isolated from discontinuous sucrose gradients, from fragmented chloroplasts, and upon trypsin treatment. Sucrose density gradient centrifugation shows that ferricyanide reduction site 1 activity at pH 6 decreases from 30 to 50% in various isolated fractions, while the dibromothymoquinone-insensitive activity at pH 8 (site 2) is stimulated from 15 to 35%.     

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K_m for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.     

Kinetics of NADPH-Induced Non-Photochemical Reduction of the Plastoquinone Pool in Spinach Chloroplasts

Kinetics of non-photochemical reduction of the photosynthetic intersystem electron transport chain by exogenous NADPH was examined in osmotically lysed spinach chloroplasts by chlorophyll (Chl) fluorescence measurements under anaerobic condition. Upon the addition of NADPH, the apparent F₀ increased sigmoidally, and the value of the maximal slope was calculated to give the reduction rate of plastoquinone (PQ) pool. Application of 5 µM antimycin A lowered significantly both the ceiling and the rate of the NADPH-induced Chl fluorescence increase, while the suppressive effect of 10 µM rotenone was slighter. This indicated that dark reduction of the PQ pool by NADPH in spinach chloroplasts under O₂-limitation condition could be attributed mainly to the pathway catalysed sequentially by ferredoxin-NADP⁺ oxidoreductase (FNR) and ferredoxin-plastoquinone reductase (FQR), rather than that mediated by NAD(P)H dehydro- genase (NDH).     

Influence of pH upon the Warburg Effect in Isolated Intact Spinach Chloroplasts: I. Carbon Dioxide Photoassimilation and Glycolate Synthesis

The influence of pH upon the O₂ inhibition of ¹⁴CO₂ photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO₂ and 100% O₂, O₂ inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced ¹⁴C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The ¹⁴C-labeling of starch was also more inhibited by O₂ at higher pH. The enhanced synthesis of glycolate during ¹⁴CO₂ assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.     

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。     

Effect of Elevated Temperature on Nitrite and Nitrate Reduction in Leaves and Intact Chloroplasts

Barley (Hordeum vulgare L.) leaves and intact spinach (Spinacia oleracea L.) chloroplasts were exposed to short-term heating, and the aftereffects of heat treatment on in vitro andin vivo activities of nitrate reductase and noncyclic electron transport associated with nitrite reduction were studied. Heating of leaves at temperatures above 40°C led to a monotonic decrease in nitrate reductase in vitro activity. On the contrary, the in vivo enzyme activity, assayed in intact leaf tissues after 5-min heat treatment, increased 1.5 times upon elevating the pretreatment temperature from 37 to 40°C and gradually decreased at higher temperatures. Noncyclic electron transport related to CO₂ fixation in intact chloroplasts decreased gradually after heat exposures above 39°C, unlike the electron transport to nitrite as a terminal acceptor, which was stimulated by heating of intact chloroplast suspensions in the temperature range from 33 to 40°C. The heating at higher temperatures inhibited nitrite photoreduction. It is concluded that the heating of phototrophic cells at sublethal temperatures stimulates the mobilization of inorganic nitrogen and thereby facilitates the repair of thermally induced injuries of proteinaceous cell structures. The stimulation of nitrate reductase activity in vivo at the temperature range 37–40°C provides an evidence for the increase in the availability of reductants in the cytosolic compartment of the leaf cell.     

Photophosphorylation and Carbon Dioxide Fixation by Chloroplasts Isolated from Populus deltoides

A system has been developed for the isolation of photosynthetically active chloroplasts from leaves of Populus deltoides. A high proportion of the chloroplasts appeared intact. The maximum rates of different photosynthetic processes were as follows: CO₂ fixation 3.5 micromoles per milligram chlorophyll per hour, noncyclic ATP synthesis 10 micromoles per milligram chlorophyll per hour, and cyclic ATP synthesis 300 micromoles per milligram chlorophyll per hour.     

beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form β-carotene from NaH¹⁴CO₃ or [U-¹⁴C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-¹⁴C]-3-PGA or 3 to 6 millimolar NaH¹⁴CO₃ was fully sufficient to supply β-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles ¹⁴C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the β-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO₂ to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute β-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.     

Palisade Tissue Chloroplasts and Spongy Tissue Chloroplasts in Spinach: Biochemical and Ultrastructural Differences

Palisade tissue chloroplasts (P-Chlts) and spongy tissue chloroplasts(S-Chlts) were separately isolated from spinach leaves, andtheir photosynthetic properties were compared. The followingresults were obtained: (1) At saturating light, the activities of overall electrontransport and CO₂ fixation in P-Chlts were respectively 1.6–2.0and 2.5–3.0 times higher than those in S-Chlts on a Chlbasis. (2) The contents of PS I and PS II reaction centers (P700 and47 kDa polypeptide, respectively) were slightly higher in P-Chltsthan in S-Chlts, while the contents of plastoquinone, Cyt f,plastocyanin, ferredoxin, ferredoxin-NADP⁺ reductase, couplingfactor and ribulose-bisphosphate carboxylase were 1.6–2.2times higher in P-Chlts than in S-Chlts on a Chl basis. (3) Electron microscopic examination of chloroplast ultrastructureshowed that S-Chlts have highly stacked grana accompanied byhigher proportion of appressed thylakoids relative to non-appressedthylakoids, while P-Chlts have poorly stacked grana. The volumeratio of thylakoids to stroma was higher in S-Chlts than inP-Chlts. These results indicate that mesophyll chloroplasts adapt tothe light environment within a leaf in a similar way that thesun and shade plant chloroplasts adapt to the light environmentwithin a canopy. (Received July 19, 1984; Accepted October 13, 1984)     

Two Forms of Division Profile in Spinach Chloroplasts

CHLOROPLAST division is generally thought to take place by constriction (fission)^1–7. Division may also occasionally occur by the growth of a central baffle before separation^8–11. Usually only one type has been observed for a given tissue although searches have been made for the alternative form. The present communication, forming part of a study on the effect of light and dark treatments on cultured leaf disks, describes the ultrastructure of spinach chloroplasts which exhibit both dumb-bell and central baffle profiles suggestive of division.     

Induction of Hexose-Phosphate Translocator Activity in Spinach Chloroplasts

Many environmental and experimental conditions lead to accumulation of carbohydrates in photosynthetic tissues. This situation is typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly to leaves. In this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for several days. These chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability to perform starch synthesis from exogenous glucose-6-phosphate (Glc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Glucose-1-phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Glc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a novel Glc-6-P-translocator capacity is present in the envelope membrane, exhibiting an apparent Km of 0.54 mM and a Vmax of 2.9 [mu]mol Glc-6-P mg-1 chlorophyll h-1. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. The generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates is not supported by these results. A physiological role for a Glc-6-P translocator in green plastids is presented with reference to the source/sink function of the leaf.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.

Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.

Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any `feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

     

Characteristics of Prompt and Delayed Fluorescence from Spinach Chloroplasts

Effects of ferricyanide, dichlorophenyldimethylurea (DCMU), and uncouplers of phosphorylation on the prompt and delayed fluorescences from spinach chloroplasts are described. Any factor that affects the yield of prompt fluorescence will similarly influence the intensity of delayed fluorescence. This idea, recently investigated by Lavorel, should be expressed in terms of a “live” component of fluorescence; that is, the component from chlorophyll associated with the photochemical traps of System II. Some of the effects of ferricyanide and DCMU on delayed fluorescence can then be explained in terms of effects on the yield of prompt fluorescence. From the internal consistency of the explanation, applied to various observations, a judgment can be made that most of the prompt fluorescence observed initially when dark-adapted chloroplasts are first illuminated is “dead,” coming from chlorophyll not associated with trap II. The live fluorescence is represented almost entirely by the time-varying component that develops during illumination. The observed intensity of delayed fluorescence can be divided by the yield of live prompt fluorescence to give an intrinsic delayed fluorescence. This intrinsic delayed fluorescence is proportional to the square root of exciting light intensity (as long as the excitation is not saturating) and decays with second order kinetics. This behavior may reflect the photochemical formation and second order dissipation of an oxidized product of Photosystem II.