Control of photosynthetic sucrose synthesis by fructose-2,6-bisphosphate

The metabolite levels in the mesophyll of leaves of Zea mays L. have been compared with the regulatory properties of the cytosolic fructose-1,6-bisphosphatase from the mesophyll to show how withdrawal of triose phosphate for sucrose synthesis is reconciled with generation of the high concentrations of triose phosphate which are needed to allow intercellular diffusion of carbon during photosynthesis. i) A new technique is presented for measuring the intercellular distribution of metabolites in maize. The bundle-sheath and mesophyll tissues are partially separated by differential homogenization and filtration through nylon nets under liquid nitrogen. ii) considerable gradients of 3-phosphoglycerate, triose phosphate, malate and phosphoenolpyruvate exist between the mesophyll and bundle sheath which would allow intercellular shuttles to be driven by diffusion. These gradients could result from the distribution of electron transport and the Calvin cycle in maize leaves. iii) consequently, the mesophyll contains high concentrations of triose phosphate and fructose-1,6-bisphosphate. iv) Most of the regulator metabolite fructose-2,6-bisphosphate, is present in the mesophyll. v) The cytosolic fructose-1,6-bisphosphatase has a lower substrate affinity than that found for the enzyme from C₃ species, especially in the presence of inhibitors like fructose-2,6-bisphosphate. vi) This lowered affinity for substrate makes it possible to reconcile use of triose phosphate for sucrose synthesis with the maintenance of the high concentration of triose phosphate in the mesophyll needed for operation of photosynthesis in this species.Abbreviations DHAP Dihydroxyacetonephosphate - Fru1,6-bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - PEP(Case) phosphoenolpyruvate (carboxylase) - PGA 3-phosphoglycerate - Rubisco ribulose-1,5-bisphosphate carboxylase     

Metabolic integration in locust flight: the effect of octopamine on fructose 2,6-bisphosphate content of flight muscle in vivo

The biogenic amine octopamine was injected into the haemolymph of 20-days old male locusts,Locusta migratoria, and the content of fructose 2,6-bisphosphate, a potent activator of glycolysis, was measured in the flight muscle after various time. Octopamine brought about a transient increase in fructose 2,6-bisphosphate. After the injection of 10 l of 10 mmol·l^-1 d, l-octopamine fructose 2,6-bisphosphate was increased by 61% within 2 min. Ten minutes after the injection fructose 2,6-bisphosphate was increased to 6.71±0.89 nmol·g^-1 flight muscle, almost 300% over the control value. Flight caused fructose 2,6-bisphosphate in flight muscle to decrease, but this decrease was counteracted by octopamine injected into the haemolymph of flying locusts. Octopamine and fructose 2,6-bisphosphate may act as signals to stimulate the oxidation of carbohydrate and to integrate muscle performance and metabolism. This mechanism appears particularly significant in the initial stage of flight when carbohydrates are the main fuel.Abbreviations F2,6P₂ fructose 2,6-bisphosphate - F6P fructose 6-phosphate - PFK1 6-phosphofructokinase (EC 2.7.1.11) - P _i inorganic phosphate - PP _i -PFK pyrophosphate dependent fructose 6-phosphate phosphotransferase (EC 2.7.1.90)     

Reduced-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana

(i) We have studied the influence of reduced phosphoglucose-isomerase (PGI) activity on photosynthetic carbon metabolism in mutants of Clarkia xantiana Gray (Onagraceae). The mutants had reduced plastid (75% or 50% of wildtype) or reduced cytosolic (64%, 36% or 18% of wildtype) PGI activity. (ii) Reduced plastid PGI had no significant effect on metabolism in low light. In high light, starch synthesis decreased by 50%. There was no corresponding increase of sucrose synthesis. Instead glycerate-3-phosphate, ribulose-1,5-bisphosphate, reduction of Q_A (the acceptor for photosystem II) and energy-dependent chlorophyll-fluorescence quenching increased, and O₂ evolution was inhibited by 25%. (iii) Decreased cytosolic PGI led to lower rates of sucrose synthesis, increased fructose-2,6-bisphosphate, glycerate-3-phosphate and ribulose-1,5-bisphosphate, and a stimulation of starch synthesis, but without a significant inhibition of O₂ evolution. Partitioning was most affected in low light, while the metabolite levels changed more at saturating irradiances. (iv) These results provide decisive evidence that fructose-2,6-bisphosphate can mediate a feedback inhibition of sucrose synthesis in response to accumulating hexose phosphates. They also provide evidence that the ensuing stimulation of starch synthesis is due to activation of ADP-glucose pyrophosphorylase by a rising glycerate-3-phosphate: inorganic phosphate ratio, and that this can occur without any loss of photosynthetic rate. However the effectiveness of these mechanisms varies, depending on the conditions. (v) These results are analysed using the approach of Kacser and Burns (1973, Trends Biochem. Sci. 7, 1149–1161) to provide estimates for the elasticities and flux-control coefficient of the cytosolic fructose-1,6-bisphosphatase, and to estimate the gain in the fructose-2,6-bisphosphate regulator cycle during feedback inhibition of sucrose synthesis.Abbreviations and symbols Chl chlorophyll - Fru6P fructose-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - Fru-1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - Fru2,6Pase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - PGI phosphoglucose isomerase - Pi inorganic phosphate - Q_A acceptor for photosystem II - Ru1,5bisP ributose-1,5-bisphosphate - SPS sucrose-phosphate synthase     

Control of photosynthate partitioning in spinach leaves

Experiments were carried out to estimate the elasticity coefficients and thence the distribution of control of sucrose synthesis and photosynthate partitioning between cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase (SPS), by applying the dualmodulation method of Kacser and Burns (1979, Biochem. Soc. Trans. 7, 1149–1161). Leaf discs of spinach (Spinacia oleracea L.) were harvested at the beginning and end of the photoperiod and illuminated at five different irradiances to alter (i) the extent of feedback inhibition and (ii) the rate of photosynthesis. The rate of CO₂ fixation, sucrose synthesis and starch synthesis were measured and compared with the activation of SPS, and the levels of fructose-2,6-bisphosphate (Fru2,6bisP) and metabolites. Sucrose synthesis increased progressively with increasing irradiance, accompanied by relatively large changes of SPS activity and Fru2,6bisP, and relatively small changes of metabolites. At each irradiance, leaf discs harvested at the end of the photoperiod had (compared with leaf discs harvested at the beginning of the photoperiod) a decreased rate of sucrose synthesis, increased starch synthesis, decreased SPS activity, increased Fru2,6bisP, a relatively small (20%) increase of most metabolites, no change of the glycerate-3-phosphate: triose-phosphate ratio, a small increase of NADPmalate dehydrogenase activation, but no inhibition of photosynthesis. The changes of sucrose and starch synthesis were largest in low light, while the changes of SPS and Fru2,6bisP were as large, or even larger, in high light. It is discussed how these results provide evidence that the control of sucrose synthesis is shared between SPS and fructose-1,6-bisphosphatase, and provide information about the in-vivo response of these enzymes to changes in the levels of their substrates and effectors. At low fluxes, feedback regulation is very effective at altering partitioning. In high light, changes of SPS activation and Fru2,6bisP can be readily overriden by increasing levels of metabolites.     

Coarse control of sucrose-phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose

It has been investigated whether diurnal rhythms of sucrose-phosphate synthase (SPS) are involved in controlling the rate of photosynthetic sucrose synthesis. Extracts were prepared from spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) leaves and assayed for enzyme activity. The activity of SPS increased in parallel with a rising rate of photosynthesis, and was increased by feeding mannose and decreased by supplying inorganic phosphate. In leaf material where sucrose had accumulated during the photoperiod or when sucrose was supplied exogenously, SPS activity decreased. During a diurnal rhythm, SPS activity increased after illumination, declined gradually during the light period, decreased further after darkening and then recovered gradually during the night. These changes did not involve an alteration of the maximal activity, but were caused by changes in the kinetic properties, revealed as a change in sensitivity to inhibition by inorganic phosphate. In experiments which modelled the response of SPS to changing metabolite concentrations, it was shown that these alterations of kinetic properties would strongly modify the activity of SPS in vivo. It is proposed that SPS can exist in kinetically distinct forms in vivo, and that the distribution between these forms can be rapidly altered. As the rate of photosynthesis increases there is an activation of SPS, which may be directly or indirectly linked to changes in the availability of P_i. This activation can be modified by factors related to the accumulation of sucrose. Under normal conditions there is a balance between these factors, and the leaf contains a mixture of the different forms of SPS.Abbreviations Chl chlorophyll - Frul,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Fru1,6bisPase fructose-1,6-bisphosphatase - Fru6P 2kinase fructose-6-phosphate, 2kinase - Fru2,6bisPase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - P_j inorganic phosphate - SPS sucrose-phosphate synthase - UDPGLc uridine 5-diphosphate glucose     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea

Images of chlorophyll-a-fluorescence oscillations were recorded using a camera-based fluorescence imaging system. Oscillations with frequencies around 1 per min were initiated by a transient decrease in light intensity during assimilation at an elevated CO₂-concentration. The oscillation was inhomogenously distributed over the leaf. In cells adjacent to minor veins, frequency and damping rate was high, if there was any oscillation. In contrast, the amplitude was highest in cells most distant from phloem elements (maximal distance about 300 m). The appearance of minor veins in oscillation images is explained by a gradient in the metabolic control in the mesophyll between minor veins and by transport of sugar from distant cells to phloem elements. The potential of fluorescence imaging to visualize microscopic source-sink interactions and metabolic domains in the mesophyll is discussed.Abbreviations P_i inorganic phosphate - Fru2,6BP fructose-2,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - SPS sucrose-phosphate synthetase - HP hexosephosphate     

Ribulose-1,5-bisphosphate carboxylase-oxygenase,other Calvin-cycle enzymes,and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream

The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the sink regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 mol photons · m^–2 · s^–1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO₂, less marked in saturating irradiance and saturating CO₂, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.Abbreviations and Symbols C_i CO₂ concentration in the air space within the leaf - F_m fluorescence yield with a saturating pulse in dark-adapted material - F_o ground level of fluorescence using a weak non-actinic modulated beam in the dark - Fru1,6bisP fructose-1,6-bisphosphate - Fru1,6Pase fructose-1,6-bisphosphatase - Fru2,6bisP fructose-2,6-bisphosphate - IRGA infrared gas analyser - NAD-MDH NAD-dependent malate dehydrogenase - NADP-MDH NADP-dependent malate dehydrogenase - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PFK phospho-fructokinase - PFP pyrophospate: fructose-6-phosphate-phosphotransferase - 3-PGA glycerate-3-phospate - P_i inorganic phosphate - Ru1,5bisP ribulose 1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - triose-phosphates sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate This research was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells

Fructose 2,6-bisphosphate (Fru2,6P₂) appears to function as a regulator metabolite in glycolysis and gluconeogenesis in animal tissues, yeast, and the photosynthetic cells of leaves. We have investigated the role of Fru2,6P₂ in guard-cell protoplasts from Vicia faba L. and Pisum sativum L. (Argenteum mutant), and in epidermal strips purified by sonication from all cells except for the guard cells. Guard-cell protoplasts were separated into fractions enriched in cytosol and in chloroplasts by passing them through a nylon net, followed by silicone oil centrifugation. The cytosol contained a pyrophosphate: fructose 6-phosphate phosphotransferase (involved in glycolysis) which was strongly stimulated by Fru2,6P₂. A cytosolic fructose 1,6-bisphosphatase (a catalyst of gluconeogenesis) was inhibited by Fru2,6P₂. There was virtually no fructose 1,6-bisphosphatase activity in guard-cell chloroplasts of V. faba. It is therefore unlikely that the starch formed in these chloroplasts originates from imported triose phosphates or phosphoglycerate.

The level of Fru2,6P₂ in guard-cell protoplasts and epidermal strips was about 0.1 to 1 attomole per guard cell in the dark (corresponding to 0.05 to 0.5 nanomole per milligram chlorophyll) and increased three- to tenfold within 15 minutes in the light. Within the same time span, hexose phosphate levels in guard-cell protoplasts declined to approximately one-half, indicating that acceleration of glycolysis involved stimulation of reactions using hexose phosphates. The level of Fru2,6P₂ in guard cells appears to determine the direction in which carbohydrate metabolism proceeds.

     

Short-term water stress leads to a stimulation of sucrose synthesis by activating sucrose-phosphate synthase

The aim of this work was to identify which aspects of photosynthetic metabolism respond most sensitively to leaf water deficit. Spinach (Spinacia oleracea L.) leaf discs were floated on sorbitol concentrations of increasing molarity and changes of the protoplast volume were estimated using [¹⁴C]sorbitol and ³H₂O penetration. Detached leaves were also wilted until 10% of their fresh weight was lost. Photosynthesis was studied at very high external CO₂ concentrations, to eliminate the effect of closing stomata. There was no large inhibition of CO₂ fixation after wilting leaves, or until the external water deficit was greater than-1.2 MPa. However, partitioning changed markedly at these moderate water deficits: more sucrose and less starch was made. When an inhibition of CO₂-saturated photosynthesis did appear at a water deficit of-2.0 MPa and above, measurements of chlorophyll-fluorescence quenching and metabolite levels showed the thylakoid reactions were not especially susceptible to short-term water stress. The inhibition was accompanied by a small increase of the triose phosphate: ribulose-1,5-bisphosphate ratio, showing regeneration of ribulose-1,5-bisphosphate was affected. However, there was also a general increase of the estimated concentrations of most metabolites, indicating that there is no specific site for the inhibition of photosynthesis. Increasing water deficit led to a large increase of fructose-2,6-bisphosphate. This is explained in terms of a simultaneous increase of fructose-6-phosphate and inorganic phosphate as the cell shrinks. The high fructose-2,6-bisphosphate led to the accumulation of triose phosphates, and the potential significance of this for protection against photoinhibition is discussed. There was an increase in the extractable activity of sucrose-phosphate synthase. This was only detected when the enzyme was assayed in conditions which distinguish between different kinetic forms which have previously been identified in spinach leaves. It is proposed that activation of sucrose-phosphate synthase is one of the first sites at which spinach leaves respond to a rising water deficit. This could be of importance for osmoregulation.Abbreviations Chl chlorophyll - Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA glycerate-3-phosphate - Pi inorgamic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - SPS sucrose-phosphate synthase - triose-P sum of glyceraldehyde-3-phosphate and dehydroxyacetone phosphate - UDPGlc uridine diphosphoglucose     

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with antisense constructs to the genes encoding the -and -subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-¹⁴C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U¹⁴-C] sucrose. (vii) Metabolism (cycling) of [U-¹⁴C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-¹⁴C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-¹³C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.Abbreviations Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NMR nuclear magnetic resonance - 3PGA glycerate-3-phosphate - PEP phosphoenolpyruvate - PEP pyrophosphate: fructose-6-phosphate phosphotransferase - PFK phosphofructokinase - UDPGlc UDP glucose - WT wild type This research was supported by the Bundesministerium for Forschung and Technology (M.S., U.S.), the Canadian Research Council (S.C., D.D.), the Agricultural and Food Research Council (R.V.) and Sandoz Agro Ltd. (M.H., M.S.).     

Perturbation of photosynthesis in spinach leaf discs by low concentrations of methyl viologen

Spinach leaf discs were floated on methyl-viologen solutions (5–200 nmol·l^-1) and the effect on photosynthetic metabolism was then investigated under conditions of saturating CO₂. Methyl viologen led to increased non-photochemical quenching, and the ATP/ADP ratio increased from <2 to >10. Comparison of the apparent quantum yield and non-photochemical quenching indicated that these concentrations of methyl viologen were only catalysing a marginal electron flux, and that the decrease in quantum yield was mainly the result of pH-triggered energy dissipation. Similar changes were also obtained after supplying tentoxin to inhibit the chloroplast ATP synthase and increase the energisation of the thylakoids. The photosystem-II acceptor, Q_A, was monitored by photochemical fluorescence quenching, and became more reduced. In contrast, the activation of NADP-malate dehydrogenase decreased, showing that the acceptor side of photosystem I becomes more oxidised. Similar changes were observed after supplying tentoxin. It is concluded that increased thylakoid energisation can lead to a substantial restriction of linear electron transport. Analysis of metabolite levels showed that glycerate-3-phosphate reduction was imporved, but that there was a large accumulation of triose phosphates and fructose-1,6-bisphosphate. This is the consequence of an inhibition of the regeneration of ribulose-1,5-bisphosphate, caused by inactivation of the stromal fructose-1,6-bisphosphatase and, to a lesser extent, phosphoribulokinase. Methyl viologen also led to inactivation of sucrose-phosphate synthase, and abolished the response of fructose-2,6-bisphosphate to rising rates of photosynthesis. This provides evidence for a primary role of glycerate-3-phosphate in controlling the activity of fructose-6-phosphate, 2-kinase and, thence, the fructose-2,6-bisphosphate concentration as the rate of photosynthesis increases. It is concluded that the very moderate ATP/ADP ratios found in chloroplasts are the results of constraints on the operation of ATP synthase. They can be increased if the thylakoid energisation is increased. However, the increased energisation acts directly or indirectly to disrupt many other aspects of photosynthetic metabolism including linear electron transport, activation of the Calvin cycle, and the control of sucrose and starch synthesis.Abbreviations and symbols Frul,6P₂ (Fru1,6Pase) fructose-1,6-bisphosphate(ase) - Fru2,6P, (Fru2,6Pase) fructose-2,6-bisphosphate(-ase) - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PSI and PSII photosystems I and II - qE high energy' quenching of chlorophyll fluorescence - PGA glycerate-3-phosphate - Q_A primary stable acceptor of PSII - Ru5P (Ru1,5P₂) ribulose-5-phosphate (-1,5-bisphosphate) - SPS sucrose-phosphate synthase - triose P dihydroxyacetone phosphate plus glyceraldehyde-3-phosphate - _s apparent quantum yield Dedicated to Professor E. Latzko on the occasion of his 65th birthday     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

Subcellular compartmentation of pyrophosphate and dark/light kinetics in comparison to fructose 2,6-bisphosphate

Subcellular compartmentation of pyrophosphate (PP₁) was determined by rapid membrane filtration of evaeuolated oat mesophyll protoplasts. By improving both the extraction procedure and its assay via bioluminescence, PP₁ recovery from samples was quantitative and linear down to below 200 fmol. Based on the content of the different fractions obtained after membrane filtration and compared to the respective pools of marker metabolites [cytosol, fructose 2,6-bisphosphate (F26BP); chloroplast stroma, ribulose bisphosphate] rather than enzymes, we found ca 2/3 of the total cellular content to be chloroplast-assotiated. Referred to compartmental volumes, cytosolic and stromal concentrations of PP₁ were nearly equal (70–100 μ M ). PP₁ was higher in evacuolated compared to racuotated protoplasts which indicates a possible role of the tonoplast-located H⁺ pumping PP₁ase in regulating the cellular pool size of PP₁. During dark-light-transition the pool sizes of PP₁ changed only marginally in both vacuolated and evacuolated protoplasts, while there were pronounced changes in those of F26BP, starch and sucrose. Thus our findings support the notion that the cellular pool size of PP₁ is kept rather constant. They are, however, in contrast to the assumption that appreciable PP₁ levels only exist in the cytosol.     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP at the base of the leaf of transformants implies that PFP plays a more important role in metabolism at the leaf base than in mature leaf tissue. Nevertheless, there were no major changes in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large changes in expression can be compensated by changes in Fru2,6-bisP, even in growing tissues.     

Effects of temperature on the regulation of photosynthetic carbon assimilation in leaves of maize and barley

The aim of this work was to examine the effect of temperature in the range 5 to 30 ° C upon the regulation of photosynthetic carbon assimilation in leaves of the C₄ plant maize (Zea mays L.) and the C₃ plant barley (Hordeum vulgare L.). Measurements of the CO₂-assimilation rate in relation to the temperature were made at high (735 bar) and low (143 bar) intercellular CO₂ pressure in barley and in air in maize. The results show that, as the temperature was decreased, (i) in barley, pools of phosphorylated metabolites, particularly hexose-phosphate, ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate, increased in high and low CO₂; (ii) in maize, pools of glycerate 3-phosphate, triose-phosphate, pyruvate and phosphoenolpyruvate decreased, reflecting their role in, and dependence on, intercellular transport processes, while pools of hexose-phosphate, ribulose 1,5-bis phosphate and fructose 1,6-bisphosphate remained approximately constant; (iii) the redox state of the primary electron acceptor of photosystem II (Q_A) increased slightly in barley, but rose abruptly below 12° C in maize. Non-photochemical quenching of chlorophyll fluorescence increased slightly in barley and increased to high values below 20 ° C in maize. The data from barley are consistent with the development of a limitation by phosphate status at low temperatures in high CO₂, and indicate an increasing regulatory importance for regeneration of ribulose 1,5-bisphosphate within the Calvin cycle at low temperatures in low CO₂. The data from maize do not show that any steps of the C₄ cycle are particularly cold-sensitive, but do indicate that a restriction in electron transport occurs at low temperature. In both plants the data indicate that regulation of product synthesis results in the maintenance of pools of Calvin-cycle intermediates at low temperatures.Abbreviations Glc6P glucose-6-phosphate - Fru6P fructase-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - PGA glycerate-3-phosphate - p _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate We thank the Agricultural and Food Research Council, UK (Research grant PG50/67) and the Science and Engineering Research Council, UK for financial support. C.A.L. was supported by the British Council, by the Conselho Nacional de Desenvolvimento Cientiflco e Tecnologico (CNPq), Brazil and by an Overseas Research Student Award. We also thank Mark Stitt (Bayreuth, FRG) and Debbie Rees for helpful discussions.