Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : II. Partitioning between Sucrose and Starch

The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6-bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6-bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6-bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6-bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch.     

Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate

Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P₂) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6-P₂ were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P₂, suggesting that fru-2,6-P₂ is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru-2,6-P₂ changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P₂ are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P₂ did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state.     

Relationship between Fructose 2,6-Bisphosphate and Carbohydrate Metabolism in Darkened Barley Primary Leaves

Initial dark fructose 2,6-bisphosphate levels in 10-day-old barley (Hordeum vulgare L.) leaves increased when the photosynthetic period was lengthened, when the temperature during the prior photosynthetic period was reduced, and following leaf excision. These treatments also increased the leaf sucrose concentration. Conversely, a decrease in dark fructose 2,6,-bisphosphate occurred during extended darkness, with increasing leaf age and when photosynthate in the leaf was reduced by earlier low light treatments. These variations in fructose 2,6-bisphosphate content correlate with known changes in dark respiration. These findings suggest, but do not conclusively prove, a causal relationship between dark fructose 2,6-bisphosphate levels and dark respiration rates.     

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO₂ concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.     

A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves

The subcellular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level of fructose 2,6-bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf.     

In Vitro Inhibition of the Plastid and Cytosolic Isozymess of 6-Phosphogluconate Dehydrogenase from Developing Endosperm of Ricinus communis by Fructose 2,6-bisphosphate

Activities of the cytosolic and plastid isozymes of 6-phosphogluconate dehydrogenase from developing endosperm of Ricinus communis L. seeds were inhibited in vitro by hexosebisphosphates. Inhibition constants for glucose 1,6-bisphosphate were 221 and 209 micromolar for the cytosolic and plastid isozymes, respectively, and corresponding values for fructose 2,6-bisphosphate were 10.5 and 8.6 micromolar. In each case inhibition was of a mixed noncompetitive nature relative to 6-phosphogluconate. While the levels and distribution of fructose 2,6-bisphosphate in castor oil seed endosperm cells are not yet known, the levels reported to occur in leaf cytosol would be high enough to significantly inhibit carbon flux through the pentosephosphate pathway due to inhibition of 6-phosphogluconate dehydrogenase activity.     

Photosynthetic carbohydrate metabolism in wheat (Triticum aestivum L.) leaves: optimization of methods for determination of fructose 2, 6-bisphosphate

The accurate measurement of fructose 2,6-bisphosphate from plants such as wheat is fraught with difficulty. Extraction and assay methods for fructose 2,6-bisphosphate that give near 100% recovery of the metabolite, and a linear response with volume have therefore been developed for extracts prepared from wheat leaves of different ages. Amounts of fructose 2,6-bisphosphate in different regions of leaves generally showed a positive correlation with chlorophyll content. Measurements of sucrose and starch in third leaves harvested at different times of the diurnal cycle demonstrated that sucrose is the major form in which photosynthate is stored in the leaf, but starch can account for up to about 30% of the stored carbohydrate. Virtually all of the carbohydrate accumulated as starch and sucrose during the day was degraded at night. Amounts of fructose 2,6-bisphosphate were generally lower in extracts prepared from leaves harvested in the light than in the dark. Additionally, there was no change in either the amount of fructose 2, 6-bisphosphate or the ratio of sucrose to starch in samples prepared from leaves harvested at different times of the day. These results are broadly consistent with a role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis and the partitioning of carbohydrate between sucrose and starch in wheat leaves.     

Leaf Phosphate Status, Photosynthesis, and Carbon Partitioning in Sugar Beet: III. Diurnal Changes in Carbon Partitioning and Carbon Export

The effect of low phosphate supply (low P) was determined on the diurnal changes in the rate of carbon export, and on the contents of starch, sucrose, glucose, and fructose 2,6-bisphosphate (F2,6BP) in leaves. Low-P effects on the activities of a number of enzymes involved in starch and sucrose metabolism were also measured. Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers and the low-P treatment induced nutritionally. Low-P treatment decreased carbon export from the leaf much more than it decreased photosynthesis. At growth chamber photon flux density, low P decreased carbon export by 34% in light; in darkness, export rates fell but more so in the control so that the average rate in darkness was higher in low-P leaves. Low P increased starch, sucrose, and glucose contents per leaf area, and decreased F2, 6BP. The total extractable activities of enzymes involved in starch and sucrose synthesis were increased markedly by low P, e.g. adenosine 5-diphosphoglucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, uridine 5-diphosphoglucose pyrophosphorylase, and sucrose-phosphate synthase. The activities of some enzymes involved in starch and sucrose breakdown were also increased by low P. We propose that plants adapt to low-P environments by increasing the total activities of several phosphatases and by increasing the concentrations of phosphate-free carbon compounds at the expense of sugar phosphates, thereby conserving Pi. The partitioning of carbon among the various carbon pools in low-P adapted leaves appears to be determined in part by the relative capacities of the enzymes for starch and sucrose metabolism.     

Kinetic properties of pyrophosphate:fructose-6-phosphate phosphotransferase from germinating castor bean endosperm

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was purified over 500-cold from endosperm of germinating castor bean (Ricinus commiunis L. var. Hale). The kinetic properties of the purified enzyme were studied. PFP was specific for pyrophosphate and had a requirement for a divalent metal ion. The pH optimum for activity was 7.3 to 7.7. The enzyme had similar activities in the forward and reverse directions and exhibited hyperbolic kinetics with all substrates. Kinetic constants were determined in the presence of fructose 2,6-bisphosphate, which stimulated activity about 20-fold and increased the affinity of the enzyme for fructose 6-phosphate, fructose 1,6-bisphosphate, and pyrophosphate up to 10-fold. Half-maximum activation of PFP by fructose 2,6-bisphosphate was obtained at 10 nanomolar. The affinity of PFP for this activator was reduced by decreasing the concentration of fructose 6-phosphate or increasing that of phosphate. Phosphate inhibited PFP when the reaction was measured in the reverse direction, i.e. fructose 6-phosphate production. In the presence of fructose 2,6-bisphosphate, phosphate was a mixed inhibitor with respect to both fructose 6-phosphate and pyrophosphate when the reaction was measured in the forward direction, i.e. fructose 1,6-bisphosphate production. The possible roles of fructose 2,6-bisphosphate, fructose 6-phosphate, and phosphate in the control of PFP are discussed.     

Regulation of photosynthetic carbon assimilation at the cellular level: a review

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.Paper presented at the FESP meeting (Strasbourg, 1984)     

Differential inhibition and activation of two leaf dihydroxyacetone phosphate reductases : role of fructose 2,6-bisphosphate

The chloroplastic and cytosolic forms of spinach (Spinacia oleracea cv Long Standing Bloomsdale) leaf NADH:dihydroxyacetone phosphate (DHAP) reductase were separated and partially purified. The chloroplastic form was stimulated by dithiothreitol, reduced thioredoxin, dihydrolipoic acid, 6-phosphogluconate, and phosphate; the cytosolic isozyme was stimulated by fructose 2,6-bisphosphate but not by reduced thioredoxin. End product components that severely inhibited both forms of the reductase included lipids and free fatty acids, membranes, and glycerol phosphate. In addition, two groups of inhibitory peptides were obtained from the fraction precipitated by 70 to 90% saturation with (NH₄)₂SO₄. Chromatography of this fraction on Sephadex G-50 revealed a peptide peak of about 5 kilodaltons which inhibited the chloroplastic DHAP reductase and a second peak containing peptides of about 2 kilodaltons which inhibited the cytosolic form of the enzyme. Regulation of the reduction of dihydroxyacetone phosphate from the C₃ photosynthetic carbon cycle or from glycolysis is a complex process involving activators such as thioredoxin or fructose 2,6-bisphosphate, peptide and lipid inhibitors, and intermediary metabolites. It is possible that fructose 2,6-bisphosphate increases lipid production by stimulating DHAP reductase for glycerol phosphate production as well as inhibiting fructose 1,6-bisphosphatase to stimulate glycolysis.     

Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum)

Here we show that fructose 2,6-bisphosphate cannot be reliably measured in mature leaves of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), or stinging nettle (Urtica dioica L.) using conventional extraction techniques, since the recoveries of fructose 2,6-bisphosphate added during extraction are poor. However, fructose 2,6-bisphosphate could be extracted by boiling leaves in ethanol and aqueous buffer. Evidence for the reliability of this technique is provided by high recovery measurements of fructose 2,6-bisphosphate added to the leaves before extraction. This extraction method was used to measure changes in the level of fructose 2,6-bisphosphate throughout the photoperiod in tobacco and potato leaves. These changes are compared with the rate of accumulation of sucrose and starch in the leaf samples. Variations in the levels of fructose 2,6-bisphosphate, and the relationship between this metabolite and sucrose and starch accumulation in these leaves during the photoperiod are similar to the pattern observed in leaves of other plant species.Abbreviations BSA bovine serum albumin - Fru-2,6-P₂ fructose 2,6-bisphosphate This research was supported by the Agricultural and Food Research Council (Grant no. PG43/531), and the Royal Society.     

Fructose 2,6-bisphosphate in relation with the resumption of metabolic activity in slices of Jerusalem artichoke tubers

When slices of Jerusalem artichoke tubers were incubated at 25°C, their concentration in fructose 2,6-bisphosphate increased up to 250-fold within 2 h. Fructose 2,6-bisphosphate was also formed, although at a slower rate, in slices incubated at 0°C. Its formation could not be explained by an increase in the concentration of fructose 6-phosphate or of ATP either by an activation of phosphofructo-2-kinase. Pyrophosphate—fructose-6-phosphate 1-phosphotransferase was the only enzyme present in a tuber extract which was found to be sensitive to fructose 2,6-bisphosphate. An improved procedure for the assay of fructose 2,6-bisphosphate is also reported.     

Effect of fructose 2,6-bisphosphate on the kinetic properties of cytoplasmic fructose 1,6-bisphosphatase from germinating castor bean endosperm

The cytoplasmic form of fructose 1,6-bisphosphatase (FBPase) was purified over 60-fold from germinating castor bean endosperm (Ricinus communis). The kinetic properties of the purified enzyme were studied. The preparation was specific for fructose 1,6-bisphosphate and exhibited optimum activity at pH 7.5. The affinity of the enzyme for fructose 1,6-bisphosphate was reduced by AMP, which was a mixed linear inhibitor. Fructose 2,6-bisphosphate also inhibited FBPase and induced a sigmoid response to fructose 1,6-bisphosphate. The effects of fructose 2,6-bisphosphate were enhanced by low levels of AMP. The latter two compounds interacted synergistically in inhibiting FBPase, and their interaction was enhanced by phosphate which, by itself, had little effect. The enzyme was also inhibited by ADP, ATP, UDP and, to a lesser extent, phosphoenolpyruvate. There was no apparent synergism between UDP, a mixed inhibitor, and fructose 2,6-bisphosphate. Similarly ADP, a predominantly competitive inhibitor, did not interact with fructose 2,6-bisphosphate. Possible roles for fructose 2,6-bisphosphate and the other effectors in regulating FBPase are discussed.     

Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Fructose 2,6-bisphosphate hydrolyzing enzymes in higher plants

The phosphatases that hydrolyze fructose 2,6-bisphosphate in a crude spinach (Spinacia oleracea L.) leaf extract were separated by chromatography on blue Sepharose, into three fractions, referred to as phosphatases I, II, and III, which were further purified by various means. Phosphatase I hydrolyzed fructose 2,6-bisphosphate, with a K_m value of 30 micromolar, to a mixture of fructose 2-phosphate (90%) and fructose 6-phosphate (10%). It acted on a wide range of substrates and had a maximal activity at acidic pH. Phosphatase II specifically recognized the osyl-link of phosphoric derivatives and had more affinity for the β-anomeric form. Its apparent K_m for fructose 2,6-bisphosphate was 30 micromolar. It most likely corresponded to the fructose-2,6-bisphosphatase described by F. D. Macdonald, Q. Chou, and B. B. Buchanan ([1987] Plant Physiol 85: 13-16). Phosphatase III copurified with phosphofructokinase 2 and corresponded to the specific, low-K_m (24 nanomolar) fructose-2,6-bisphosphatase purified and characterized by Y. Larondelle, E. Mertens, E. Van Schaftingen, and H. G. Hers ([1986] Eur J Biochem 161: 351-357). Three similar types of phosphatases were present in a crude extract of Jerusalem artichoke (Helianthus tuberosus) tuber. The concentration of fructose 2,6-bisphosphate decreased at a maximal rate of 30 picomoles per minute and per gram of fresh tissue in slices of Jerusalem artichoke tuber, upon incubation in 50 millimolar mannose. This rate could be accounted for by the maximal extractable activity of the low-K_m fructose-2,6-bisphosphatase. A new enzymic method for the synthesis of β-glucose 1,6-bisphosphate from β-glucose 1-phosphate and ATP is described.     

Fructose 2,6-bisphosphate and the regulation of pyrophosphate-dependent phosphofructokinase activity in germinating pea seeds

The activity of pyrophosphate: d-fructose-6-phosphate-1-phosphotransferase (EC 2.7.1.90, PPi-PFK) in cotyledons and sprouts of germinating pea seeds (Pisum sativum cv Alaska or Green Arrow) increases rapidly during the first 2 to 3 days after imbibition and then declines to a lower activity. The reaction toward fructose 1,6-bisphosphate formation is activated greatly by fructose 2,6-bisphosphate (fru 2,6-P₂); however, the sensitivity of the enzyme's activity to fru 2,6-P₂ activation changes during germination.     

Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.     

Fructose 2,6-Bisphosphate Changes in Rat Brain During Ischemia

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.