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PubMed1976

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive gamma-Irradiation of the Seed.

Spinach seeds (Spinacia oleracea L.) given massive doses of gamma-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis.

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PubMed1979

Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L.

Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg(2+) and thiamine pyrophosphate.Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants.

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PubMed1981

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II.

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO(2) photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.

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PubMed1981

Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE.

Addition of exogenous Mg(2+) (2 millimolar) to illuminated intact spinach (Spinacia oleracea L.) chloroplasts caused acidification of the stroma and a 20% decrease in stromal K(+). Addition of K(+) (10-50 millimolar) reversed both stromal acidification and K(+) efflux from the chloroplast caused by Mg(2+). These data suggested that Mg(2+) induced reversible H(+)/K(+) fluxes across the chloroplast envelope. Ca(2+) and Mn(2+) (2 millimolar) were as effective as 4 millimolar Mg(2+) in causing K(+) efflux from chloroplasts and inhibition of O(2) evolution. In contrast, 10 millimolar Ba(2+) induced only a small amount of inhibition. The lack of strong inhibition by Ba(2+) indicated that the effects of divalent cations such as Mg(2+) cannot be attributed to generalized electrostatic interactions of the cation with the chloroplast envelope. With the chloroplasts used in this study, stromal acidification caused by 2 millimolar Mg(2+) was small (0.07 to 0.15 pH units), but sufficient to account for the inhibition of O(2) evolution (43%) induced by Mg(2+).A variety of ATPase inhibitors were tested for effects on Mg(2+)-induced H(+)/K(+) fluxes. Oligomycin was the only ATPase inhibitor which specifically inhibited photosynthesis in the presence of Mg(2+) + K(+), but had little or no effect in the absence of these cations. In the presence of oligomycin, much higher concentrations (50 millimolar) of exogenous K(+) were required to reverse Mg(2+)-induced acidification and inhibition of O(2) evolution than in its absence. Oligomycin (in the absence of divalent cations) increased the inhibition of photosynthesis caused by sodium acetate, which acts by causing stromal acidification. In addition, the chloroplast envelope ATPase was inhibited partially (45%) by oligomycin. These results suggested that H(+) fluxes across the chloroplast envelope are regulated by two mechanisms: (a) an active, oligomycin-sensitive H(+) efflux and (b) a reversible, Mg(2+)-dependent, oligomycin-insensitive H(+)/K(+) exchange.

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PubMed1984

Hydrogen peroxide-dependent oxidation of flavonols by intact spinach chloroplasts.

Externally added quercetin (100 micromolar) was oxidized by intact spinach chloroplasts at a rate of 30 micromoles per mg chlorophyll per hour in the presence of 100 micromolar H(2)O(2). The oxidation rate was increased by about 20% in a hypotonic reaction mixture. The thylakoid fraction also oxidized the flavonol in the presence of H(2)O(2), and the rate was about 25% of that by intact chloroplasts. The oxidation of quercetin was inhibited by KCN and NaN(3). Ascorbate, which permeates slowly across chloroplast envelope, only slightly suppressed the initial rate of quercetin oxidation by intact chloroplasts, while the oxidation by ruptured chloroplasts was suppressed by ascorbate by about 60%. Quercetin glycosides, quercitrin and rutin, were also oxidized by chloroplasts in the presence of H(2)O(2). These results suggest that flavonols are oxidized by peroxidase-like activity in chloroplasts and that externally added flavonols can permeate into the stroma through the envelope of intact chloroplasts.

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PubMed1984

Rates and products of long-chain Fatty Acid synthesis from [1-C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants.

Chloroplasts highly active in the synthesis of long-chain fatty acids from [1-(14)C]acetate were prepared from leaves of Solanum nodiflorum, Chenopodium quinoa, Carthamus tinctorius, and Pisum sativum. These preparations were used to test whether the various additions to incubation media found to stimulate the synthesis of particular lipid classes in vitro by Spinacia oleracea chloroplasts were applicable generally. Chloroplasts from 18:3 plants incorporated a greater proportion of radioactivity into unesterified fatty acids under control conditions than did those from 16:3 plants. Supplying exogenous sn-glycerol 3-phosphate or Triton X-100 to chloroplasts increased the synthesis of glycerolipids in all cases and accentuated the capacity of chloroplasts from 18:3 plants to accumulate phosphatidic acid rather than the diacylglycerol accumulated by chloroplasts from 16:3 plants. The UDP-galactose-dependent synthesis of labeled diacylgalactosylglycerol was much less active in incubations of chloroplasts from 18:3 plants also containing sn-glycerol 3-phosphate and Triton X-100 compared with similar incubations from 16:3 plants. Exogenous CoA stimulated total fatty acid synthesis in all chloroplast preparations and the further addition of ATP diverted radioactivity from the unesterified fatty acid to acyl-CoA. The results have been discussed in terms of the two pathway hypothesis for lipid synthesis in leaves.

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PubMed1986

The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays.

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K(m) (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K(i) of about 6 millimolar. For comparison the K(i) for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K(a) values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and beta-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.

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PubMed1986

Biosynthetic cause of in vivo acquired thermotolerance of photosynthetic light reactions and metabolic responses of chloroplasts to heat stress.

Thermotolerance of photosynthetic light reactions in vivo is correlated with a decrease in the ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol and an increased incorporation into thylakoid membranes of saturated digalactosyl diacylglycerol species. Although electron transport remains virtually intact in thermotolerant chloroplasts, thylakoid protein phosphorylation is strongly inhibited. The opposite is shown for thermosensitive chloroplasts in vivo. Heat stress causes reversible and irreversible inactivation of chloroplast protein synthesis in heat-adapted and nonadapted plants, respectively, but doe not greatly affect formation of rapidly turned-over 32 kilodalton proteins of photosystem II. The formation on cytoplasmic ribosomes and import by chloroplasts of thylakoid and stroma proteins remain preserved, although decreased in rate, at supraoptimal temperatures. Thermotolerant chloroplasts accumulate heat shock proteins in the stroma among which 22 kilodalton polypeptides predominate. We suggest that interactions of heat shock proteins with the outer chloroplast envelope membrane might enhance formation of digalactosyl diacylglycerol species. Furthermore, a heat-induced recompartmentalization of the chloroplast matrix that ensures effective transport of ATP from thylakoid membranes towards those sites inside the chloroplast and the cytoplasm where photosynthetically indispensable components and heat shock proteins are being formed is proposed as a metabolic strategy of plant cells to survive and recover from heat stress.

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PubMed1986

Microspectrofluorometric Measurement of Chloroplast DNA in Dividing and Expanding Leaf Cells of Spinacia oleracea.

Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.

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PubMed1987

CO(2) Photoassimilation by the Spinach Chloroplast at Low Temperature.

Spinach chloroplasts were used to study the relationship between photosynthetic CO(2) fixation and temperature from 30 to -15 degrees C. In saturating light and high concentrations of CO(2), the temperature coefficients (Q(10)) above 20 degrees C were less than 2 in the intact chloroplast. Below 15 degrees C, the Q(10) values were greater than 2 and gradually increased with decreasing (down to 0 degrees C) temperature to approximately 4.4. Photosynthesis responded similarly to temperature in a reconstituted chloroplast preparation fortified with ribose 5-phosphate. In the intact chloroplast, temperature did not alter the Q(10) value in low light and high CO(2). Elevating the temperature to 25 degrees C after photosynthesizing at -15 degrees C (46 minutes) or 0 degrees C (17 minutes) restored the temperature-depressed photosynthetic rate without a lag in the intact chloroplast to the rate of a chloroplast continually at 25 degrees C. At 0 degrees C, the intact chloroplast photosynthetic rate responded slightly to the inorganic phosphate concentration (0.1-1.0 millimolar) and to pH (7.0-8.6). Relative to 25 degrees C, the levels of ribulose 1,5-bisphosphate and glycerate 3-phosphate were increased 1300 and 200%, respectively, whereas glycolate decreased 57% during intact chloroplast photosynthesis at 0 degrees C. Chilling temperature impeded the transport of photosynthetic intermediates from the stromal compartment to the external medium. Ethylene glycol was shown to be an appropriate additive to prevent freezing of the reaction mixture down to -15 degrees C for photosynthetic CO(2) assimilation.

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PubMed1987

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide.

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var "Turkish Samsun" control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by "Samsun" chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.

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PubMed1987

Two immunological approaches to the detection of ribulose-1,5-bisphosphate carboxylase in guard cell chloroplasts.

Two immunological approaches were used to determine if ribulose bisphosphate carboxylase oxygenase (RuBisCo) is present in guard cell chloroplasts. Immunocytochemistry on thin plastic sections using tissue samples that were processed using traditional glutaraldehyde/osmium fixation and then restored to antigenicity with metaperiodate treatment, resulted in labeling over wild-type mesophyll and guard cell plastids of several green and white variegated Pelargonium chimeras. The density of immunogold labeling in guard cell chloroplasts was only about one-seventh of that noted in mesophyll chloroplasts on a square micron basis. Because guard cell chloroplasts are much smaller than mesophyll chloroplasts, and occur at lower quantities/cell, the relative differences in RuBisCo concentration between the cell types indicate that guard cells have only 0.48% of the RuBisCo of mesophyll cells. No reaction was noted over 70S ribosomeless plastids of these chimeras even though adjacent green chloroplasts were heavily stained, indicating the high specificity of the reaction for RuBisCo. Spurr's resin gave the most successful colloidal gold labeling in terms of low background staining and structural detail but L. R. White's resin appeared to be superior for antigen retention. In the white leaf edges of the white and green Pelargonium chimeras, the only green, functional chloroplasts are in the guard cells. When either whole tissue or plastid enriched extracts from this white tissue were electrophoresed, blotted, and probed with anti-RuBisCo a large subunit band was detected, identical to that in the green tissue. These data indicate that a low, but detectable, level of RuBisCo is present in guard cell chloroplasts.

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PubMed1988

Chloroplast osmotic adjustment and water stress effects on photosynthesis.

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var ;Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Psi(w)) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Psi(w) reached -1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Psi(w) may involve stromal volume maintenance.

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PubMed1989

Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg.

Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg(2+) bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg(2+) was altered by pretreatment with Mg(2+) and divalent cation chelating agents and by additions of Mg(2+) to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg(2+) can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with (28)Mg(2+) confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg(2+). It was concluded that changes in Mg(2+) associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg(2+) may be exclusively due to increased envelope-associated Mg(2+). The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg(2+) inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K(+) in the photosynthetic reaction media.

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PubMed1989

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella.

Neither Dunaliella cells grown with 5% CO(2) nor their isolated chloroplasts had a CO(2) concentrating mechanism. These cells primarily utilized CO(2) from the medium because the K((0.5)) (HCO(3) (-)) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K((0.5)) CO(2) was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO(2) concentrating mechanism was present that decreased the K(0.5) (CO(2)) to about 0.5 micromolar and K(0.5) (HCO(3) (-)) to 11 micromolar at pH 8. These K(0.5) values suggest that air-adapted cells preferentially concentrated CO(2) but could also use HCO(3) (-) from the medium. Chloroplasts isolated from air-adapted cells had a K((0.5)) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO(2). Chloroplasts from air-adapted cells, but not CO(2)-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO(2) concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO(2) concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO(2) concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.

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PubMed1990

Variations in the Levels of Chloroplast tRNAs and Aminoacyl-tRNA Synthetases in Senescing Leaves of Phaseolus vulgaris.

The relative amounts of chloroplast tRNAs(Leu), tRNA(Glu), tRNA(Phe), tRNAs(Thr), and tRNA(Tyr) and of chloroplastic and cytoplasmic aminoacyl-tRNA synthetases were compared in green leaves, yellowing senescing leaves, and N(6)-benzyladenine-treated senescing leaves from bean (Phaseolus vulgaris, var Contender). Aminoacylation of the tRNAs using Escherichia coli aminoacyl-tRNA synthetases indicated that in senescing leaves the relative amount of chloroplast tRNA(Phe) was significantly lower than in green leaves. Senescing leaves treated with N(6)-benzyladenine contained higher levels of this tRNA than untreated senescing leaves. No significant change in the relative amounts of chloroplast tRNAs(Leu), tRNAs(Thr), and tRNA(Tyr) was detected in green, yellow senescing, or N(6)-benzyladine-treated senescing leaves. Relative levels of chloroplast tRNAs were also estimated by hybridization of tRNAs to DNA blots of gene specific probes. These experiments confirmed the results obtained by aminoacylation and revealed in addition that the relative level of chloroplast tRNA(Glu) is higher in senescing leaves than in green leaves. Transcription run-on assays indicated that these changes in tRNA levels are likely to be due to a differential rate of degradation rather than to a differential rate of transcription of the tRNA genes. Chloroplastic and cytoplasmic leucyl-, phenylalanyl-, and tyrosyl-tRNA synthetase activities were greatly reduced in senescing leaves as compared to green leaves, whereas N(6)-benzyladenine-treated senescing leaves contained higher enzyme activities than untreated senescing leaves. These results suggest that during senescence, as well as during senescence-retardation by cytokinins, changes in enzyme activities, such as aminoacyl-tRNA synthetases, rather than reduced levels of tRNAs, affect the translational capacity of chloroplasts.

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PubMed1990

Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport.

An artificial Na(+) gradient across the envelope (Na(+) jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C(4) plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, (22)Na(+) and pyruvate uptake were examined in mesophyll chloroplasts of several species of C(4) plants. Enhancement of pyruvate uptake by a Na(+) jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na(+) uptake in the dark when added together with Na(+). When flux of endogenous Na(+) was measured in these mesophyll chloroplasts preincubated with (22)Na(+), pyruvate addition induced Na(+) influx, and the extent of the pyruvate-induced Na(+) influx positively correlated with that of pyruvate uptake. A Na(+)/H(+) exchange ionophore, monensin, nullified all the above mutual effects of Na(+) and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na(+) uptake and increased equilibrium level of chloroplast (22)Na(+). Measurements of initial uptake rates of pyruvate and Na(+) gave a stoichiometry close to 1:1. These results point to Na(+)/pyruvate cotransport into mesophyll chloroplasts of some C(4) plants.

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PubMed1991

Dissipation of the proton electrochemical potential in intact and lysed chloroplasts : I. The electrical potential.

Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts.

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