Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K_a) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.     

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).     

Immunological Characterization of the Pyrophosphate Dependent Fructose-6-Phosphate Phosphotransferase

The pyrophosphate dependent phosphofructokinase (PFP, EC 2.7.1.90 [EC] )was purified from potato tubers, bean seeds and cucumber seeds.The PFP of all three species appears to contain two subunitswith a molecular weight of approximately 60,000 and 66,000 dalton.The purified proteins were used as the antigens to produce polyclonalantibodies in rabbits. Two of the obtained sera (anti-potatoPFP and anti-cucumber PFP) proved to be monospecific for thePFP polypeptides on protein blots. The antipotato serum crossreacts with the PFP from all the tested higher plant specieson protein blots, but no cross reaction with the PFP of Propionibacteriumsharmanii was found. This shows that the PFP subunits from thehigher plant species have similar antigenic determinants inthe primary structure but differes largely from that of thePropionibacterium. The differences observed in the efficiencyof the sera to inactivate the PFP from the different species,however, indicate that the surface antigenic determinants onthe native PFP enzymes differ between the higher plant speciesand even within the Cucurbitaceae. (Received June 15, 1987; Accepted November 20, 1987)     

果糖-2,6-二磷酸对香蕉成熟过程中焦磷酸果糖-6-磷酸转移酶活性的调节作用

从成熟香蕉果实中部分纯化了焦磷酸:果糖—6—磷酸磷酸转移酶(PFP)。研究了酶的果糖—2,6—二磷酸的活化动力学特性.果糖—2,6—二磷酸通过降低酶的K_m(F6P)值和增进最大反应速度(V_(max))促进酶的果糖—6—磷酸磷酸化活性。底物(F6P)浓度和温度影响果糖—2,6—二磷酸对酶的活化作用。 本工作中还观察了香蕉成熟过程中PFP和依赖ATP的磷酸果糖激酶(PFK)活性的变化,并对PFP在果实成熟中的生理意义和调节特性进行了讨论。     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Wound-Induced Metabolism in Potato (Solanum tuberosum) Tubers: Biosynthesis of Aliphatic Domain Monomers

Suberin, a cell specific, wall-associated biopolymer, is formed during normal plant growth and development as well as in response to stress conditions such as wounding. It is characterized by the deposition of both a poly(phenolic) domain (SPPD) in the cell wall and a poly(aliphatic) domain (SPAD) thought to be deposited between the cell wall and plasma membrane. Although the monomeric components that comprise the SPPD and SPAD are well known, the biosynthesis and deposition of suberin is poorly understood. Using wound healing potato tubers as a model system, we have tracked the flux of carbon into the aliphatic monomers of the SPAD in a time course fashion. From these analyses, we demonstrate that newly formed fatty acids undergo one of two main metabolic fates during wound-induced suberization: (1) desaturation followed by oxidation to form the 18:1 ω-hydroxy and dioic acids characteristic of potato suberin, and (2) elongation to very long chain fatty acids (C20 to C28), associated with reduction to 1-alkanols, decarboxylation to n-alkanes and minor amounts of hydroxylation. The partitioning of carbon between these two metabolic fates illustrates metabolic regulation during wound healing, and provides insight into the organization of fatty acid metabolism.Key Words: suberin, potato, Solanum tuberosum, carbon flux analysis, abiotic stress     

Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje

Summary The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, -glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.     

Salt stress induced biometric and physiological changes in Solanum tuberosum L. cv. Bintje grown in vitro

Potato plants grown in vitro were subjected to different salt stresses by providing the salts NaCl, Na₂SO₄, MgCl₂ and MgSO₄ in different concentrations up to 300 mM. Salinity greatly affected the survival and the rooting of the plants. Shoot and root growth decreased with increasing salt concentrations. Under mild stress conditions, i.e. in conditions where the plant is able to adapt to the stress, the observed decrease was dependent upon the salt used. Under severe stress conditions, however, the decrease of the shoot and root growth was independent of the nature of the ions.     

Lipoxygenase Pathway and Membrane Permeability and Composition during Storage of Potato Tubers (Solanum tuberosum L. cv Bintje and Désirée) in Different Conditions

Abstract: Potato tubers ( Solanum tuberosum L. cv Bintje and Désirée) were stored for 12 months under three different storage conditions: 4 °C, 20 °C with sprout inhibitor and 20 °C without sprout inhibitor. Independent of the storage conditions, our results show that the increase of membrane permeability, as revealed by electrolyte leakage, is not correlated with the lipid saturation status. Moreover, there is no simple correlation between cold sweetening and membrane permeability or lipid saturation status. During storage at 20 °C without sprout inhibitor, the increase in membrane permeability is inversely correlated to sucrose accumulation, but this is not the case when tubers were stored with sprout inhibitors. Lipoxygenase (LOX) is often proposed as responsible for peroxidative damage to membrane lipids. The gradual peroxidation resulting in double bond index decrease is regarded as a cause of senescence sweetening. Our results revealed that the role of LOX in aging and senescence of potato tubers is far from clear. LOX activity and gene expression are not correlated with the fatty acids composition of the membrane. Moreover, LOX activity and fatty acid hydroperoxide content are low in older tubers, whatever the storage conditions or the varieties. On the basis of our results, the correlation between sugar accumulation (low temperature and senescence sweetening) and peroxidative damage occurring during storage of potato tubers is discussed.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Properties of Pyrophosphate:Fructose-6-Phosphate Phosphotransferase from Endosperm of Developing Wheat (Triticum aestivum L.) Grains

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90) from endosperm of developing wheat (Triticum aestivum L.) grains was purified to apparent homogeneity with about 52% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose and gel filtration through Sepharose-CL-6B. The purified enzyme, having a molecular weight of about 170,000, was a dimer with subunit molecular weights of 90,000 and 80,000, respectively. The enzyme exhibited maximum activity at pH 7.5 and was highly specific for pyrophosphate (PPi). None of the nucleoside mono-, di- or triphosphate could replace PPi as a source of energy and inorganic phosphate (Pi). Similarly, the enzyme was highly specific for fructose-6-phosphate. It had a requirement for Mg²⁺ and exhibited hyperbolic kinetics with all substrates including Mg²⁺. K_m values as determined by Lineweaver-Burk plots were 322, 31, 139, and 129 micromolar, respectively, for fructose-6-phosphate, PPi, fructose-1,6-bisphosphate and Pi. 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 its substrates. Initial velocity studies indicated kinetic mechanism to be sequential. At saturating concentrations of fructose-2,6-bisphosphate (1 micromolar), Pi strongly inhibited PFP; the inhibition being mixed with respect to both fructose-6-phosphate and PPi, with K_i values of 0.78 and 1.2 millimolar, respectively. The inhibition pattern further confirmed the mechanism to be sequential with random binding of the substrates. Probable role of PFP in endosperm of developing wheat grains (sink tissues) is discussed.     

PFP的研究进展

焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP)可催化果糖-6-磷酸与果糖-1,6-二磷酸间的可逆转变.该酶广泛存在于各种高等植物及一些微生物体内.文章综述了90年代以来有关PFP的一些研究进展.包括:PFP的种类与亚基构成、活性中心、底物特异性、酶活性的调节及功能等.     

Complete Sucrose Metabolism Requires Fructose Phosphotransferase Activity in Corynebacterium glutamicum To Ensure Phosphorylation of Liberated Fructose

Sucrose uptake by Corynebacterium glutamicum involves a phosphoenolpyruvate-dependent sucrose phosphotransferase (PTS), but in the absence of fructokinase, further metabolism of the liberated fructose requires efflux of the fructose and reassimilation via the fructose PTS. Mutant strains lacking detectable fructose-transporting PTS activity accumulated fructose extracellularly but consumed sucrose at rates comparable to those of the wild-type strain.     

Sucrose Metabolism in Tubers of Potato (Solanum tuberosum L.): Effects of Sink Removal and Sucrose Flux on Sucrose-Degrading Enzymes

Excision of developing potato (Solanum tuberosum L.) tubers from the mother plant, followed by storage at 10°C, resulted in a rapid, substantial decrease in sucrose synthase activity and considerable increases in hexose content and acid invertase activity. A comparison of the response of three genotypes, known to accumulate different quantities of hexoses in storage, showed that both sucrose synthase activity and the extent to which activity declined following excision were similar in all cases. However, there was significant genotypic variation in the extent to which acid invertase activity developed, with tubers accumulating the highest hexose content also developing the highest extractable activity of invertase. Similar effects were found in nondetached tubers when growing plants were maintained in total darkness for a prolonged period. Furthermore, supplying sucrose to detached tubers through the cut stolon surface prevented the decline in sucrose synthase activity. Maltose proved to be ineffective. Western blots using antibodies raised against maize sucrose synthase showed that the decline in sucrose synthase activity was associated with the loss of protein rather than the effect of endogenous inhibitors. Although there were indications that maintaining a flux of sucrose into isolated tubers could prevent the increase in acid invertase activity, the results were not conclusive.     

AFM Investigations of Cellulose Fibers in Bintje Potato (Solanum tuberosum L.) Cell Wall Fragments

Atomic force microscopy (AFM) has been used to image the cellulose networks in moist fragments of the cell walls of Bintje potato (Solanum tuberosum L.). The interfiber spacing in hydrated native cell wall fragments was found to be 26.2 nm. This value is consistent with published estimates of the contour length of xyloglucan cross-links determined by transmission electron microscopy (TEM) studies of cell walls. Sequential extraction of the pectin using CDTA and Na₂CO₃ led to shrinkage of the cell wall fragment and a reduction in interfiber spacing to 20.2 nm. Partial extraction of xyloglucan using 1 M KOH caused a small decrease in interfiber spacing to 19.5 nm. Finally, the almost complete removal of xyloglucan with 4 M KOH substantially reduced the interfiber spacing to 11 nm. The results are consistent with a model for the cell wall in which the cellulose–xyloglucan network is immersed in a swollen, hydrated pectin network.     

Cytokinin Activity in Stolons and Tubers of Solanum tuberosum during the Period of Tuberization

In water-culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), changes in cytokinin activity in the stolon tips and newly formed tubers during tuberization were studied. Tuberization was induced by withdrawing nitrogen from the nutrient solution. — The cytokinin activity was low in the stolon tips prior to tuberization, but increased considerably in both stolon tips and young tubers during tuberization. At the same time qualitative changes in the cytokinin spectrum occurred. These qualitative changes are reversible if ‘regrowth’ of young tubers is brought about by a sudden high supply of nitrogen. — Despite the close correlation between tuberization and cytokinin activity, it is assumed that cytokinins are not directly responsible for the onset of tuberization, although they play an important role in tuber growth.     

Differential Activities of Chorismate Mutase Isozymes in Tubers and Leaves of Solanum tuberosum L

Chromatography on DEAE cellulose equilibrated with Pipes buffer resolved three forms of chorismate mutase (CM) from tubers and leaves of Solanum tuberosum: CM-1A and CM-1B were activated by tryptophan and inhibited by phenylalanine and tyrosine; CM-2 was unaffected by these aromatic amino acids. When compared to freshly excised discs, 3 day old tuber discs demonstrated a 4.5-fold increase in CM-1 activity following wounding. By contrast, CM-2 activity levels were not affected by this treatment. In aged tuber discs the CM-1:CM-2 activity ratio was 9:1. However, in green leaves the CM-1:CM-2 activity ratio was 1:4 suggesting organ specific regulation for the expression of these isozymes. The CM-1 isozymes isolated from both tubers and leaves shared similar native molecular weight values of 55,000, K_m values of 40 to 56 micromolar, and inhibition by phenylalanine (110-145 micromolar concentrations required for 50% inhibition) and tyrosine (50-70 micromolar concentrations required for 50% inhibition). The resolution of CM-1 into two forms occurred only in the presence of Pipes buffer. When this buffer was replaced with Aces, Bes, imidazole or Tris, only a single peak of CM-1 activity was observed. In these buffers CM-2 eluted as a shoulder on the CM-1 peak. Analytical isoelectric focusing of the CM-1 fraction followed by assay of the gel yielded only one form of CM-1 with an isoelectric point of 5.0. Gel filtration studies with Pipes buffer yielded molecular weights of 60,000 for both CM-1A and CM-1B indicating these forms are not the result of aggregation. The two forms of CM-1 may be artifacts generated by Pipes buffer.     

NAD Malic Enzyme and the Control of Carbohydrate Metabolism in Potato Tubers

Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations.