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M Stitt

Publications and source records attributed to M Stitt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis.

Diurnal changes in carbohydrates and nitrate reductase (NR) activity were compared in tobacco (Nicotiana tabacum. L.cv. Gatersleben) plants growing in a long (18 h light/6 h dark) and a short (6 h light/18 h dark) day growth regime, or after short-term changes in the light regime. In long-day-grown plants, source leaves contained high levels of sugars throughout the light and dark periods. In short-day-grown plants, levels of sucrose and reducing sugars were very low at the end of the night and, although they rose during the light period, remained much lower than in long days and declined to very low levels again by the middle of the night. Starch accumulated more rapidly in short-day-than long-day-grown plants. Starch was completely remobilised during the night in short days, but not in long days. A single short day/long night cycle sufficed to stimulate starch accumulation during the following light period. In long-day-grown plants, the Nia transcript level was high at the end of the night, decreased during the day, and recovered gradually during the night. In short-day-grown plants, the Nia transcript level was relatively low at the end of the night, decreased to very low levels at the end of the light period, increased to a marked maximum in the middle of the night, and decreased during the last 5 h of the dark period. In long-day-grown plants, NR activity in source leaves rose by 2- to 3-fold in the first part of the light period and decreased in the second part of the light period. In short-day-grown plants, NR activity was low at the end of the night, and only increased slightly after illumination. Dark inactivation of source-leaf NR was partially reversed in long-day-grown plants, but not in short day-grown plants. In both growth regimes, mutants with one instead of four functional copies of the Nia gene had a 60% reduction in maximum NR activity in the source leaves, compared to wild-type plants. The diurnal changes in NR activity were almost completely suppressed in the mutants in long days, whereas the mutants showed similar or slightly larger diurnal changes than wild-type plants in short days. When short-day-grown plants were transferred to long-day conditions for 3 d, NR activity and the diurnal changes in NR activity resembled those in long-day-grown plants. Phloem export from source leaves of short-day-grown plants was partially inhibited by applying a cold-girdle for one light and dark cycle. The resulting increase in leaf sugar was accompanied by an marked increase in the Nia transcript level and a 2-fold increase in NR activity at the end of the dark period. When wild-type plants were subjected to a single short day/long night cycle of increasing severity, NR activity in source leaves at the end of the night decreased when the endogenous sugars declined below about 3 mumol hexose (g FW)-1. In sink leaves in short-day conditions, sugars were higher and the light-induced rise in NR activity was much larger than in source leaves on the same plants. The source leaves of wild-type plants in short-day conditions contained very high levels of nitrate, very low levels of glutamine, low levels of total amino acids, and lower protein and chlorophyll, compared to long-day-grown plants. Plants grown in short days had relatively high levels of glutamate and aspartate, and extremely low levels of most of the minor amino acids in their source leaves at the end of the night. Illumination led to a decrease in glutamate and an increase in the minor amino acids. A single short day/long night cycle led to an increase in glutamate, and a large decrease in the minor acids at the end of the dark period, and reillumination led to a decrease in glutamate and an increase in the minor amino acids. It is proposed that sugar-mediated control of Nia expression and NR activity overrides regulation by nitrogenous compounds when sugars are in short supply, resulting in a severe inhibition of nitrate assimilation. It is also proposed that su

Amino Acids↗
PubMed1998

A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants.

Antisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several 'antisense' potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32-43%, 56-71%, 79-83% and 91-97% in A-70, A-3, A-51 and A-2. Separation on a Q-Sepharose-FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose-1,7-biphosphatase and plastid fructose-1,6-bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A-70, A-3, A-51 and A-2. The transformants contained increased triose phosphates, and very low ribulose-1,5-bisphosphate and glycerate-3-phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A-70 and A-3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A-70, was decreased by 41% in A-3, and was severely inhibited in plants with under 20% of wild-type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.

Carbohydrate Metabolism↗
PubMed1998

High-temperature perturbation of starch synthesis is attributable to inhibition of ADP-glucose pyrophosphorylase by decreased levels of glycerate-3-phosphate in growing potato tubers

To investigate the short-term effect of elevated temperatures on carbon metabolism in growing potato (Solanum tuberosum L.) tubers, developing tubers were exposed to a range of temperatures between 19 degreesC and 37 degreesC. Incorporation of [14C]glucose (Glc) into starch showed a temperature optimum at 25 degreesC. Increasing the temperature from 23 degreesC or 25 degreesC up to 37 degreesC led to decreased labeling of starch, increased labeling of sucrose (Suc) and intermediates of the respiratory pathway, and increased respiration rates. At elevated temperatures, hexose-phosphate levels were increased, whereas the levels of glycerate-3-phosphate (3PGA) and phosphoenolpyruvate were decreased. There was an increase in pyruvate and malate, and a decrease in isocitrate. The amount of adenine diphosphoglucose (ADPGlc) decreased when tubers were exposed to elevated temperatures. There was a strong correlation between the in vivo levels of 3PGA and ADPGlc in tubers incubated at different temperatures, and the decrease in ADPGlc correlated very well with the decrease in the labeling of starch. In tubers incubated at temperatures above 30 degreesC, the overall activities of Suc synthase and ADPGlc pyrophosphorylase declined slightly, whereas soluble starch synthase and pyruvate kinase remained unchanged. Elevated temperatures led to an activation of Suc phosphate synthase involving a change in its kinetic properties. There was a strong correlation between Suc phosphate synthase activation and the in vivo level of Glc-6-phosphate. It is proposed that elevated temperatures lead to increased rates of respiration, and the resulting decline of 3PGA then inhibits ADPGlc pyrophosphorylase and starch synthesis.

Journal Article↗
PubMed1997

Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase.

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Circadian Rhythm↗
PubMed1995

Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants.

Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants. The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem. To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene. Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth. From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning. To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells. To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed. Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.

Base Sequence↗
PubMed1993

Sucrose-phosphate synthase phosphatase, a type 2A protein phosphatase, changes its sensitivity towards inhibition by inorganic phosphate in spinach leaves.

The activity of a type 2A protein phosphatase from spinach leaves was monitored using phosphorylated sucrose-phosphate synthase (SPS) as a substrate. After partial purification the overall activities of sucrose-phosphate synthase phosphatase (SPS-P) recovered from leaves harvested in the dark and in the light did not vary. However, SPS-P preparations from darkened leaves were more strongly inhibited by inorganic phosphate and certain phosphorylated compounds than preparations from illuminated or mannose fed leaves. We conclude, that activation of SPS involves an interconversion of multiple forms of SPS-P activity.

Catalysis↗
PubMed1993

Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli.

Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylene glycol and chromatography over diethylaminoethylcellulose, omega-aminohexyl-agarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat.(mg protein)-1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121-1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.

Amino Acid Sequence↗
PubMed1991

A Quantification of the Significance of Assimilatory Starch for Growth of Arabidopsis thaliana L. Heynh.

These studies use starch synthesis mutants to quantify the contribution of assimilatory starch to whole plant growth and form. Arabidopsis thaliana (L.) Heynh plants were used with null plastid phosphoglucomutase (T Caspar, SC Huber, CR Sommerville, [1986] Plant Physiol 79; 1-7) or 7% of wild-type ADP-glucose pyrophosphorylase (T-P Lin, T Caspar, CR Sommerville, J Preiss [1988] Plant Physiol 88; 1175-1179). The daily turnover of starch and the rate of biomass increase in the mutants and the wild type were investigated during growth in a 14 hour light/10 hour dark cycle in high irradiance (600 micromoles per square meter per second) and nitrogen (6 millimolar NH(4)NO(3)), in high irradiance and low nitrogen (0.1 millimolar NH(4)NO(3)) or in low irradiance (80 micromoles per square meter per second) and high nitrogen. There is some variability in the data, but the following conclusions can be drawn. Growth was slow in the absence of starch turnover. In high nitrogen conditions, about 1 mole of carbon per gram dry weight per day was incorporated additionally into structural biomass for every one mole of carbon turned over as starch per gram dry weight per day. In low nitrogen, the gain was much lower. This indicates that temporary storage of photosynthate is important for rapid growth in high nitrogen, but not in low nitrogen when carbohydrate is in excess. Starch-deficient plants showed the usual decrease of the shoot/root ratio in low nitrogen and increase of the ratio in low light. This shows that adjustment of plant form to nitrogen nutrition and irradiance is not mediated via regulation of photosynthate partitioning in the leaf. Starch deficient plants had lower shoot/root ratios than the wild type and the nitrogen concentration in their leaves was increased. It is discussed how interactions between carbohydrate allocation, respiration and growth at the organ and whole plant level generate these changes. We conclude that mutants with a decreased capacity to carry out a particular partial process provide a powerful tool to disect complex mutually interacting systems, and define and quantify causal interactions at the level of whole plant growth.

Journal Article↗
PubMed1991

Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.

In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis.

Base Sequence↗
PubMed1990

Sucrose-phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves. Evidence from the effects of okadaic acid and microcystin.

Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PP2A). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PPI and PP2A), while inhibitor-2 (a PP1 inhibitor) or Mg2+ (essential for PP2C) were ineffective. In vivo, okadaic acid and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO2 fixation. It is concluded that PP2A is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.

Animals↗
PubMed1990

Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N-terminal portions of the potato-derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly-A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50- to 500-fold higher invertase activity compared to non-transformed control plants. This invertase is N-glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.

Amino Acid Sequence↗
PubMed1990

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves.

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.

Journal Article↗
PubMed1989

Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana. Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of Flux Control Coefficients and Elasticity Coefficients.

1. Subcellular-compartment-specific decreased-activity mutants of phosphoglucose isomerase in Clarkia xantiana were used to analyse the control of sucrose and starch synthesis during photosynthesis. Mutants were available in which the plastid phosphoglucose isomerase complement is decreased to 75% or 50% of the wild-type level, and the cytosol complement to 64%, 36% or 18% of the wild-type level. 2. The effects on the [product]/[substrate] ratio and on fluxes to sucrose or starch and the rate of photosynthesis were studied with the use of saturating or limiting light intensity to impose a high or low flux through these pathways. 3. Removal of a small fraction of either phosphoglucose isomerase leads to a significant shift of the [product]/[substrate] ratio away, from equilibrium. We conclude that there is no 'excess' of enzyme over that needed to maintain its reactants reasonably close to equilibrium. 4. Decreased phosphoglucose isomerase activity can also alter the fluxes to starch or sucrose. However, the effect on flux does not correlate with the extent of disequilibrium, and also varies depending on the subcellular compartment and on the conditions. 5. The results were used to estimate Flux Control Coefficients for the chloroplast and cytosolic phosphoglucose isomerases. The chloroplast isoenzyme exerts control on the rate of starch synthesis and on photosynthesis in saturating light intensity and CO2, but not at low light intensity. The cytosolic enzyme only exerts significant control when its complement is decreased 3-5-fold, and differs from the plastid isoenzyme in exerting more control in low light intensity. It has a positive Control Coefficient for sucrose synthesis, and a negative Control Coefficient for starch synthesis. 6. The Elasticity Coefficients in vivo of the cytosolic phosphoglucose isomerase were estimated to lie between 5 and 8 in the wild-type. They decrease in mutants with a lowered complement of cytosolic phosphoglucose isomerase. 7. The implications of these results for regulation and for evolution are discussed.

Chloroplasts↗
PubMed1989

Purification and characterization of fructose-2,6-bisphosphatase, a substrate-specific cytosolic enzyme from leaves.

Fructose-2,6-bisphosphatase (EC 3.1.3.46), which hydrolyzes fructose 2,6-bisphosphate to fructose 6-phosphate and Pi, has been purified to apparent homogeneity from spinach leaves and found to be devoid of fructose-6-phosphate,2-kinase activity. The isolated enzyme is a dimer (76 kDa determined by gel filtration) composed of two 33-kDa subunits. The enzyme is highly specific and displays hyperbolic kinetics with its fructose 2,6-bisphosphate substrate (Km = 32 microM). The products of the reaction, fructose 6-phosphate and Pi, along with AMP and Mg2+ are inhibitors of the enzyme. Nonaqueous cell fractionation revealed that, like the fructose 2,6-bisphosphate substrate, fructose-2,6-bisphosphatase as well as fructose-6-phosphate,2-kinase occur in the cytosol of spinach leaves.

Chromatography, DEAE-Cellulose↗
PubMed1989

Product inhibition of potato tuber pyrophosphate:fructose-6-phosphate phosphotransferase by phosphate and pyrophosphate.

The product inhibition of potato (Solanum tuberosum) tuber pyrophosphate:fructose-6-phosphate phosphotransferase by inorganic pyrophosphate and inorganic phosphate has been studied. The binding of substrates for the forward (glycolytic) and the reverse (gluconeogenic) reaction is random order, and occurs with only weak competition between the substrate pair fructose-6-phosphate and pyrophosphate, and between the substrate pair fructose-1,6-bisphosphate and phosphate. Pyrophosphate is a powerful inhibitor of the reverse reaction, acting competitively to fructose-1,6-biphosphate and noncompetitively to phosphate. At the concentrations needed for catalysis of the reverse reaction, phosphate inhibits the forward reaction in a largely noncompetitive mode with respect to both fructose-6-phosphate and pyrophosphate. At higher concentrations, phosphate inhibits both the forward and the reverse reaction by decreasing the affinity for fructose-2,6-bisphosphate and thus, for the other three substrates. These results allow a model to be proposed, which describes the interactions between the substrates at the catalytic site. They also suggest the enzyme may be regulated in vivo by changes of the relation between metabolites and phosphate and could act as a means of controlling the cytosolic pyrophosphate concentration.

Journal Article↗
PubMed1989

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts.

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [(14)C]5,5-dimethyl-2,4-oxazolidinedione and [(14)C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO(2) fixation by superoptimal medium Pi or by adding glyceraldehyde-an inhibitor of the Calvin cycle-leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific ;low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP(+) and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N'-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N'-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific ;low phosphate' proton leak in the thylakoids, and its origin is discussed.

Journal Article↗