Showing papers on "Sucrose published in 1993"

An Improved Colorimetric Method to Quantify Sugar Content of Plant Tissue

Abstract: A modification of the phenol-sulphuric acid method to determine the bulk of soluble or insoluble sugars in plant tissue is proposed. The method is based on optimizing the phenol concentration to obtain equal colour yields for glucose, fructose and sucrose. The optimal phenol concentration is found to depend on the ethanol concentration of the measuring solution. The method is quick, easy and its costs are low, permitting its use in routine analysis

Rapid Extraction and Analysis of Nonstructural Carbohydrates in Plant Tissues

Abstract: Considerable time is necessary to determine the nonstructural carbohydrates (i.e., glucose, fructose, sucrose, starch) in plant tissues and some methods used for this purpose lack specificity. Two steps in such assays typically occupy much of the assay time: sample drying and homogenization/centrifugation. A laboratory method was therefore developed to carry out such assays without either of these stepsThe new method involved separation of the ethanol-soluble carbohydrates from disks or slices of plant tissue with hot aqueous ethanol and the in situ conversion of starch to glucose by enzymes. The amount of ethanol-soluble sugars and the glucose released from the tissue during the starch digestion were then determined in a microplate assay using an enzyme-coupled colorimetric reaction which was highly specific for glucose

Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues

Abstract: Experiments were carried out to investigate whether sucrose synthase (Susy) catalyses a readily reversible reaction in vivo in potato (Solanum tuberosum L.) tubers, Ricinus communis L. cotyledons, and heterotrophic Chenopodium rubrum L. cell-suspension cultures. (i) The contents of sucrose, fructose, UDP and UDP-glucose were measured and the mass-action ratio compared with the theoretical equilibrium constant. In all three tissues the values were similar. (ii) Evidence for rapid turnover of label in the sucrose pool was obtained in pulse-chase experiments with potato discs and with intact tubers attached to the plant. The unidirectional rates of sucrose synthesis and degradation were considerably higher than the net flux through the sucrose pool in the tubers. (iii) Labelling of the glucosyl and fructosyl moieties of sucrose from [14C]glucose in the presence of unlabelled fructose provided evidence that Susy contributes to the movement of label into sucrose. Methods for estimating the contribution of sucrose-phosphate synthase and Susy are presented and it is shown that their relative contribution varies. For example, the contribution of Susy is high in developing tubers and is negligible in harvested tubers which contain low Susy activity. (iv) The absolute values of the forward (v+1) and backward (v−1) reaction direction of Susy are calculated from the kinetic labelling data. The estimated values of v+1 and v−1 are comparable, and much higher than the net flux through the sucrose pool. (v) The estimated concentrations of the substrates and products of Susy in tubers are comparable to the published K m values for potato-tuber Susy. (vi) It is concluded that Susy catalyses a readily reversible reaction in vivo and the relevance of this conclusion is discussed with respect to the regulation of sucrose breakdown and the role of Susy in phloem unloading.

Sucrose Synthase, Starch Accumulation, and Tomato Fruit Sink Strength.

Abstract: Contrasting evidence has accumulated regarding the role of acid invertase and sucrose synthase in tomato fruit sink establishment and maintenance. In this work the relationships among the activities of sucrose synthase and acid invertase, Lycopersicon esculentum Mill cv UC-82B fruit growth, and starch accumulation were analyzed in fruit at 0 to 39 d after anthesis. Sucrose synthase, but not acid invertase, was found to be positively correlated with tomato fruit relative growth rate and with starch content in the pericarp tissue. A similar association between sucrose synthase activity and starch accumulation was also evident in the basal portion of the stem. Heat-shock treatments, which inhibited the increase in sucrose synthase activity at the beginning of the light period and had no effect on acid invertase activity, were used to examine the importance of sucrose synthase in relation to sucrose metabolism and starch synthesis. After the heat-shock treatment, concomitantly with the suppressed sucrose synthase activity relative to the controls, there was a reduction in sucrose cleavage and starch accumulation. These data substantiate the conclusion that, during the early phases of tomato fruit development, sucrose synthase rather than acid invertase is the dominant enzyme in metabolizing imported sucrose, which in turn plays a part in regulating the import of sucrose into the fruit.

Potato sucrose transporter expression in minor veins indicates a role in phloem loading.

Abstract: The major transport form of assimilates in most plants is sucrose. Translocation from the mesophyll into the phloem for long-distance transport is assumed to be carrier mediated in many species. A sucrose transporter cDNA was isolated from potato by complementation of a yeast strain that is unable to grow on sucrose because of the absence of an endogenous sucrose uptake system and the lack of a secreted invertase. The deduced amino acid sequence of the potato sucrose transporter gene StSUT1 is highly hydrophobic and is 68% identical to the spinach sucrose transporter SoSUT1 (pS21). In yeast, the sensitivity of sucrose transport to protonophores and to an increase in pH is consistent with an active proton cotransport mechanism. Substrate specificity and inhibition by protein modifiers are similar to results obtained for sucrose transport into protoplasts and plasma membrane vesicles and for the spinach transporter, with the exception of a reduction in maltose affinity. RNA gel blot analysis shows that the StSUT1 gene is highly expressed in mature leaves, whereas stem and sink tissues, such as developing leaves, show only low expression. RNA in situ hybridization studies show that the transporter gene is expressed specifically in the phloem. Both the properties and the expression pattern are consistent with a function of the sucrose transporter protein in phloem loading.

Expression of an Arabidopsis Sucrose Synthase Gene Indicates a Role in Metabolization of Sucrose Both during Phloem Loading and in Sink Organs

Abstract: Sucrose synthase, an important enzyme in carbohydrate metabolism, catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose in vitro. To investigate the in vivo function of sucrose synthase, both the gene (Asus1) and a corresponding cDNA from roots of Arabidopsis were isolated. The Asus1 gene has homologies of 67-72% to sucrose synthase genes from other species. Histochemical GUS analysis of Arabidopsis and tobacco plants transformed with a 1.5 kb Asus1 promoter fragment transcriptionally fused to the beta-glucuronidase reporter gene showed that the Asus1 gene is expressed in the phloem of leaves, and in roots. Induction is found under conditions of limited ATP supply and increased demand for translocation of carbohydrates such as anaerobic or cold treatment. During anaerobiosis the increase in RNA level leads to increased sucrose synthase activity in roots. The expression pattern and regulation of the gene suggest that sucrose synthase is involved in the supply of energy for phloem loading in source tissues, and in metabolization of sucrose in sink tissues after unloading.

The unusual sugar composition in leaves of the resurrection plant Myrothamnus flabellifolia

Abstract: To understand mechanisms of osmoprotection, the composition of sugars and related compounds were analyzed in extracts of fully hydrated and desiccated leaves of the desiccation-tolerant resurrection plant Myrothamnus flabellifolia. During the dehydration process the concentrations of fructose and glucose decrease, whereas sucrose, arbutin and glucopyranosyl-β-glycerol increase. The substances were identified by GC-MS and NMR-analyses. This is the first report of large amounts of glucopyranosyl-β-glycerol in higher plants which may act as an osmoprotectant. Significant levels of the nonreducing sugar trehalose were present in all samples tested.

Reduction of Blood Glucose Levels by Tea Catechin

Abstract: The influence of tea catechins on the absorption of starch or sucrose was investigated in vivo. Tea catechins were administered orally to rats before soluble starch or sucrose administration. Saccharide-dosed rats were killed and the blood and the contents of the intestine were collected at intervals over two hours. Catechins of certain concentrations suppressed the increase of plasma glucose levels, thus concurrently suppressing insulin activity. Increased activity of intestinal α-amylase by starch dosing was inhibited markedly in the catechin-administered rats. Sucrase on the brush border membrane was also inhibited by prior catechin administration. From these results it was assumed that orally administered catechins will inhibit intestinal α-amylase or sucrase, thereby deterring the digestion of certain amounts of starch or sucrose and eventually reducing the plasma glucose levels.

Baroprotective Effects of High Solute Concentrations Against Inactivation of Rhodotorula rubra

Abstract: High hydrostatic pressure (200 to 400 MPa) treatment of Rhodotorula rubra suspended in solutions of sucrose, glucose, fructose or sodium chloride resulted in water activity (aw) dependent barotolerance. The protective effect started at values below aw = 0.92 to 0.88. Increase in time of treatment over 15 min had a small influence. In sucrose, the effect was independent of pH between pH 3.0 and 8.0. Inactivation of the yeast at low aw could be achieved by pressure treatment at increased temperature (45°C). At atmospheric pressure, processing temperatures of 70 to 80°C were required to accomplish the same effects.

Carbohydrate Metabolism in Drought-Stressed Leaves of Pigeonpea (Cajanus cajan)

Abstract: Pigeonpea is a tropical grain-legume, which is highly dehydration tolerant. The effect of drought stress on the carbohydrate metabolism in mature pigeonpea leaves was investigated by withholding water from plants grown in very large pots (50 kg of soil). The most striking feature of drought-stressed plants was the pronounced accumulation of D-pinitol (lD-3-methyl-c/nVo inositol), which increased from 14 to 85 mg g"1 dry weight during a 27 d stress period. Concomitantly, the levels of starch, sucrose and the pinitol precursors myoinositol and ononitol all decreased rapidly to zero or near-zero in response to drought. The levels of glucose and fructose increased moderately. Drought stress induced a pronounced increase of the activities of enzymes hydrolysing soluble starch (amylases) and sucrose (invertase and sucrose synthase). The two anabolic enzymes sucrose phosphate synthase (sucrose synthetic pathway) and myoinositol methyl transferase (pinitol synthetic pathway) also showed an increase of activity during stress. These results indicate that pinitol accumulated in pigeonpea leaves, because the carbon flux was diverted from starch and sucrose into polyols.

Patterns in floral nectar characteristics of some bird-visited plant species from Costa Rica

Abstract: Nectar samples from a wide range of flowers (120 samples, 112 species, 22 families) visited by hummingbirds (subfamilies Phaethorninae and Trochilinae) were collected over a wide range of elevations and environments in Costa Rica. Some species visited are believed to be pollinated principally by perching birds, lepidopterans, and bees, however. We measured sugar composition (% fructose, glucose, and sucrose), daily secretion rates, and sugar concentration. In general, sugar compositions of all hummingbird nectars were found to be highly clustered toward the high-sucrose end of the composition spectrum and not significantly different from sugar compositions of other hummingbird nectar assemblages from northwestern Mexico and the southwestern United States. Significant correlations were detected between elevation and the percentage of fructose and of sucrose in the nectar, with the fructose percent positive and the sucrose negative. These correlations were found to be due to both elevation and hummingbirdgroup effects. Daily secretion rate and sugar concentration were both negatively correlated with elevation. Discriminant analysis indicated that nectar sugar concentration and daily secretion rates together could usually predict whether a species was pollinated by hermit (Phaethorninae) or nonhermit (Trochilinae) hummingbirds and could often predict which eco-morphological group of nonhermits.

Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers

Abstract: The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose Km=64, 90 and 100 (μM) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (Km=26, 25 and 240 μM) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (Ki=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the Ki (ADP) was about threefold below the Km (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (Ki=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (Km=41, 130 and 35 μM) and mannose but a poor affinity for fructose (Km=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (Km=90, 280 and 560 μM) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (Ki=40 and 108 μM) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (Ki=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.

Expression of Acid Invertase Gene Controls Sugar Composition in Tomato (Lycopersicon) Fruit

Abstract: A wild tomato species, Lycopersicon chmielewskii, accumulates high levels of soluble sugar in mature fruit and, unlike the domesticated tomato species, Lycopersicon esculentum, accumulates sucrose rather than glucose and fructose. Genetic and biochemical analyses of progeny resulting from a cross of L. chmielewskii with L. esculentum have previously indicated that the trait of sucrose accumulation is controlled by a single recessive gene and is associated with low levels of acid invertase protein in the developing fruit. Analysis of progeny from the BC2F3 generation from the L. esculentum x L. chmielewskii cross revealed that sucrose-accumulating fruit accumulate sugar in two phases corresponding to fruit expansion and fruit maturation and that the majority of the sucrose was stored in the latter phase after the fruit had reached maximum size. The only significant enzymic difference between the sucrose-accumulating and hexose-accumulating fruit was the lack of acid invertase activity in sucrose-accumulating fruit. Sucrose phosphate synthase activity did not increase in the sucrose-accumulating fruit during late development when the rate of sucrose accumulation increased. The lack of acid invertase activity in sucrose-accumulating fruit was correlated with inheritance of the L. chmielewskii acid invertase gene and the absence of acid invertase mRNA in developing fruit. This suggests that the L.chmielewskii invertase gene is transcriptionally silent in fruit and that this is the basis for sucrose accumulation in progeny derived from the interspecific cross of L. esculentum and L. chmielewskii.

Carbohydrates and water status in wheat plants under water stress

Abstract: SUMMARY Changes in sugar content during water stress and recovery were examined in leaves of two varieties of durum wheat (Triticum durum). The drought-resistant Mohamed Ben Bachir (MBB) from Algeria and the droughtsensitive European variety Capdur differed in the time and type of sugar increase during water stress. Glucose accumulated at a rate closely corresponding with decreasing water potential but more rapidly and to a higher concentration in MBB than in Capdur. Sucrose content correlated less well than that of monosaccharides with changes in water potential. Glucose and, to a lesser extent, fructose appeared to play an important role during water stress and to be more sensitive indicators of the degree of stress and of potential tolerance than proline which increased later and to the same extent in both varieties. After rewatering, the amounts of accumulated solutes in leaves of both types fell quickly to normal, coincident with relatively rapid growth.

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

Abstract: Potato (Solanum tuberosum L.) plants were transformed with “antisense” constructs to the genes encoding the α-and β-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.

Effects of nitrogen and phosphorus deficiencies on levels of carbohydrates, respiratory enzymes and metabolites in seedlings of tobacco and their response to exogenous sucrose

Abstract: A simple method of growing plants in agar was exploited to investigate the effect of long-term nitrogen (N) and phosphorus (P) deficiencies on respiratory metabolism and growth in shoots and roots of Nicotiana tabacum seedlings, and their interaction with exogenously supplied sucrose. Levels of hexose phosphates and 3-phosphoglyceric acid (3-PGA) were low in P-deficient shoots and roots and high in N-deficient shoots and roots. The ratio of hexose phosphates to 3-PGA and levels of fructose-2,6-bisphosphate were high in P-deficient plants and low in N-deficient plants. These data reflect differences in the way metabolism was perturbed, yet both deficiencies were associated with increased root growth relative to shoot growth, starch accumulation in the shoots, and soluble carbohydrate accumulation, especially hexoses, in the roots. Enzymes for sucrose degradation (sucrose synthase, acid and alkaline invertase) and glycolysis (phosphofructokinase, pyrophosphate-dependent phospho-fructokinase and pyruvate kinase) remained unaltered or declined in the shoots and roots. The accumulation of hexoses in roots of N- and P-deficient plants may result from maintenance of high invertase activities relative to sucrose synthase and glycolytic enzymes in the roots. The possibility that hexose accumulation may drive preferential root growth osmotically in N and P deficiencies is discussed. The addition of sucrose to roots to further investigate the interaction of carbohydrates with growth and allocation in low N and low P produced clear effects even though endogenous levels of soluble carbohydrate were already high in the nutrient-deficient plants. In complete nutrition, growth was stimulated, protein content particularly of the roots was increased and there was a preferential increase in activity of sucrose synthase in roots. At low P, enzyme activities in roots were increased, including sucrose synthase, and protein content increased, particularly in the roots, but there was no increase in growth. In N-deficient plants, exogenous sucrose led to decreased protein, Rubisco and chlorophyll content in shoots, in contrast to the other conditions, and a higher protein content and a general increase of catabolic enzyme activities and growth in the roots.

Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin

Abstract: The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocytes have been preincubated. In hepatocytes pre-cultured with 5 mM glucose the release of glucokinase was much slower than that of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 microM-1 mM) markedly increased glucokinase release. This suggests that, in cells maintained in 5 mM glucose, glucokinase is present predominantly in a bound state and this binding is dependent on the presence of Mg2+. The enzyme can be released or translocated from its bound state by an increase in [glucose] (A50 15 mM) or by fructose (A50 50 microM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insulin and counteracted by glucagon. They were inhibited by cyanide, but not by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-like effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D-glucose and 2-deoxyglucose were ineffective. When hepatocytes were incubated with [2-3H, U-14C]glucose, the incorporation of 3H/14C label into glycogen correlated with the extent of glucokinase release. Since 2-3H is lost during conversion of glucose 6-phosphate into fructose 6-phosphate, substrate-induced translocation of glucokinase from a Mg(2+)-dependent binding site to an alternative site might favour the partitioning of glucose 6-phosphate towards glycogen, as opposed to phosphoglucoisomerase.

Sugars versus starch as supplements to grass silage: Effects on ruminal fermentation and the supply of microbial protein to the small intestine, estimated from the urinary excretion of purine derivatives, in sheep

Abstract: The effects of various purified carbohydrates, given as supplements to a basal diet of grass silage, on ruminal fermentation and on the urinary excretion of purine derivatives (PD) were investigated using six sheep in a 6 × 6 Latin square experiment with period lengths of 14 days. The six experimental treatments were (i) the basal diet of 4 kg day−1 of grass silage, supplemented with (ii) sucrose, (iii) lactose, (iv) xylose, (v) wheat starch, and (vi) fructose. All supplements were given at 200 g day−1. The basal diet and the supplements were given in two equal meals each day. Relative to the basal diet, xylose, starch and fructose all reduced (P 0·05) whilst lactose produced virtually no depression of pH. All supplements reduced (at least P P > 0·05). Starch did not alter the volatile fatty acid (VFA) pattern from the basal diet but the sugars produced very marked changes in the molar proportions of VFA. All sugars reduced the proportions of acetic acid (for xylose, 0·10 > P > 0·05, for the others, at least P 0·05). The calculated supply of microbial nitrogen to the small intestine was 10·2, 14·8, 14·3, 13·1, 11·9 and 13·7 g day−1 for the basal, sucrose, lactose, xylose, starch and fructose treatments respectively; the value for sucrose was significantly (P 0·05). It is concluded that sugars, particularly sucrose, are clearly superior to starch as an energy source for the microbial fixation of nitrogen in the rumen.

Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity

Abstract: A combination of chromatography on DE-52 cellulose, Cibacron Blue agarose, Mono Q anion exchanger and gel filtration was used to resolve different hexose-phosphorylating enzymes from growing “sink” potato tubers (Solanum tuberosum L.). Three enzymes (fructokinases: FK1, FK2 and FK3) are active with fructose and inactive with glucose, and three (hexokinases: HK1, HK2 and HK3) are active with glucose but not with fructose. Elution from DE-52 columns showed that the relative abundance of the six activities changes, depending on the organ and on the developmental stage. FK1 and FK2 were present at high activities in tubers but at very low activity in leaves; conversely FK3 was present at very low activity in tubers but at high activity in leaves. During storage of potato tuber, and also during sprouting, there was a decrease of FK1 and FK2. In contrast, glucose-phosphorylating activity was very low in growing tubers. During storage and sprouting the activity of the glucose-phosphorylating enzymes rose, until they exceeded FK1 and FK2. This was due particularly to an increase of HK1, whereas HK2 declined relative to HK1, and HK3 was always negligible. These changes in the pattern of hexose-phosphorylating enzyme forms are compared with the changing metabolic fluxes and pools of hexose sugars in potato tubers. It is concluded that organ- and development-specific changes in the abundance of the various enzyme forms contribute to the regulation of hexose metabolism in the potato.

Sucrose is metabolised by sucrose synthase and glycolysis within the phloem complex of Ricinus communis L. seedlings

Abstract: Metabolites and enzyme activities were measured in the phloem sap exuding from a cut hypocotyl of germinating castor-bean (Ricinus communis L.) seedlings. The sap contained considerable quantities of adenine nucleotides, uridine nucleotides, uridine diphosphoglucose (UDPGlc), all the major phosphorylated metabolites required for glycolysis, fructose-2,6-bisphosphate and pyrophosphate. Supplying 200 mM glucose instead of sucrose to the cotyledons resulted in high concentrations of glucose in the sap, but did not modify the metabolite levels. In contrast, when 200 mM fructose was supplied we found only a low level of fructose but a raised sucrose concentration in the sap, which was accompanied by a three-to fourfold decrease of UDPGlc, and an increase of pyrophosphate, UDP and UTP. The measured levels of metabolites are used to estimate the molar mass action ratios and in-vivo free-energy change associated with the various reactions of sucrose breakdown and glycolysis in the phloem. It is concluded that the reactions catalysed by ATP-dependent phosphofructokinase and pyruvate kinase are removed from equilibrium in the phloem, whereas the reactions catalysed by sucrose synthase, UDPGlc-pyrophosphorylase, phosphoglucose mutase, phosphoglucose isomerase, aldolase, triose-phosphate isomerase, phosphoglycerate mutase and enolase are close to equilibrium within the conducting elements of the phloem. Since the exuded sap contained negligible or undetectable activities of the enzymes, it is concluded, that the responsible proteins are bound, or sequesterd behind plasmodesmata, possibly in the companion cells. It is argued that sucrose mobilisation via a reversible reaction catalysed by sucrose synthase is particularily well suited to allow the rate of sucrose breakdown in the phloem to respond to changes in the metabolic requirement for ATP, and for UDPGlc during callose production. It is also calculated that the transport of nucleotides in the phloem sap implies that there must be a very considerable uptake or de-novo biosynthesis of these cofactors in the phloem.

Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: Insights into growth limitation and metabolism

Abstract: Growth, nutrient consumption, and end-product accumulation were quantitated in shake-flask cultures of two insect cell lines, Sf-9 and BTI-EAA, in three different serum-supplemented media. Per cell consumption or production rates were calculated for most medium components analyzed. Glucose was growth-limiting in TNM-FH medium and was the most important single source of organic-C for the cells in all cultures. Cells utilized fructose and maltose but not sucrose. alpha-Ketoglutarate and malate contributed significantly to the carbon budget of cells in TNM-FH. Lactate generally did not accumulate during growth. Most of the amino acids were consumed by the cells, with the exception of alanine which was produced. Most of the amino acids appeared to be present in adequate supply in the cultures. Glutamate was generally the most rapidly consumed of the amino acids, followed closely by glutamine. Alanine accumulation was correlated with glucose consumption. In Sf-9 cultures, ammonia accumulated only slightly or not at all as long as glucose was present in the medium, and uric acid was detectable at the end of growth and in the stationary phase. Added ammonia up to a concentration of 10 mM did not affect the growth of either cell line. Ammonia and lactate may be of less importance in limiting growth in insect cell cultures than in mammalian cell cultures. A hypothetical outline of the major metabolic pathways of the cultured insect cells is presented on the basis of information obtained here and in the literature.

Increase in the content of low-molecular carbohydrates at lumber surfaces during drying and correlations with nitrogen content, yellowing and mould growth

Abstract: In this study a quantitative analysis of the low molecular carbohydrates (predominantly sucrose, fructose and glucose) in a series of lumber samples of Pinus sylvestris and Picea abies taken at various distances from the surface has been made. The increase of nitrogenous compounds towards the surface had been shown in a previous study. Several of the lumber samples showed a marked sugar accumulation at the surface, which correlated quite well with a corresponding nitrogen accumulation. In one case, the total amount of the three sugars was as high as 4.9% of the dry matter content in the 0–1 mm layer. It was of special interest to note that samples with high nitrogen and sugar contents also had a yellow surface colour, which probably formed during the drying process by the well-known Maillard reaction — a complex of reactions occurring when sugars and amino acids, peptides and proteins are heat-treated together. Growth of the mould fungus Penicillium brevicompactum was well correlated with the content of nitrogen and low molecular carbohydrates in adjacent samples. The initial colonization was somewhat delayed in material from the outermost sapwood zone despite high nutrient contents indicating effects of antifungal compounds from the bark or toxic Maillard reaction products effective against germination. Growth of Aspergillus versicolor was likewise most elaborate on samples with the highest nitrogen and soluble carbohydrate content but the results also indicate a sensitivity to antifungal compounds present.

Apoplastic Sugar Concentration and pH in Barley Leaves Infected with Brown Rust

Abstract: Soluble sugars were extracted by low speed centrifugation from the apoplast of leaves of barley (Hordeum distichum L.) infiltrated with water. Infection of the leaf with the brown rust fungus (Puccinia hordeii) resulted in a reduction in the concentration of sucrose, glucose and fructose in the apoplast. Sugars were present in an apoplastic space occupying 12 and 17 cm 3 m -2 of leaf area in healthy and infected tissue, respectively. Uptake of hexoses by intercellular hyphae is suggested as a cause of this reduction. The pH of apoplastic sap extracted from rust-infected leaves was increased to pH 7.3 from pH 6.6 in controls. The effect of a reduced apoplastic sugar pool and increased pH on export from infected leaves is discussed

Seasonal variations in soluble sugars and starch within woody stems of Cornus sericea L.

Abstract: Carbohydrate composition changed seasonally in red osier dogwood (Cornus sericea L.) stem tissues. Starch concentration was highest in fall and decreased to a minimum in midwinter. Coincident with the breakdown of starch in fall, there was an increase in the concentrations of soluble sugars. Soluble sugars were present in highest concentrations in midwinter. Glucose, fructose, sucrose, and raffinose were the predominant soluble sugars present in both bark and wood tissues. In early spring, the soluble sugar concentration decreased and the concentration of starch increased. The seasonal interchange between sugars and starch did not simply reflect a general quantitative shift in the balance between sugars and starch because qualitative changes in soluble sugars were also noted. The most striking changes involved the trisaccharide raffinose. Raffinose was barely detectable in summer and early fall, but increased to one fifth and one third of the total soluble sugars in January samples of bark and wood tissues, respectively. The potential physiological role of raffinose in overwintering red osier dogwood tissue is discussed.

Fluxes of carbohydrate metabolism in ripening bananas

Abstract: The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO2 and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-14C]-, [1-14C]-, [3,4-14C]-and [6-14C]glucose, [U-14C]glycerol, 14CO2. The data obtained were used to estimate the following fluxes, values given as μmol hexose · (g FW)−1 · h−1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO2 fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.

Fructan Synthesis in Excised Barley Leaves (Identification of Two Sucrose-Sucrose Fructosyltransferases Induced by Light and Their Separation from Constitutive Invertases).

Abstract: Excised leaves of barley (Hordeum vulgare L.) exposed to continuous light accumulate large amounts of soluble carbohydrates. Carbohydrates were analyzed in deionized extracts by high-pressure liquid chromatography on an anion exchange column coupled with pulsed amperometric detection. During the first few hours of illumination, the main sugar to accumulate was sucrose. The levels of glucose and fructans (oligofructosylsucroses) increased later. The trisaccharide 1-kestose (1-kestotriose) predominated initially among the fructans. Later, 6-kestose (6-kestotriose) and tetra- and pentasaccharides accumulated also. Total extracts from barley leaves were chromatographed on a MonoQ column, and each fraction was assayed for enzymes of interest by incubation with 200 mM sucrose for 3 h, followed by carbohydrate analysis. Freshly excised leaves yielded two peaks of invertase, characterized by formation of fructose and glucose, but had almost no trisaccharide-forming activities. In leaves exposed to continuous light, two new enzyme activities appeared that generated fructan-related trisaccharides and glucose from sucrose. One of them was a sucrose-sucrose fructosyl-1-transferase (1-SST), producing 1-kestose exclusively: the peak fractions of this activity contained almost no invertase. The other was a sucrose-sucrose fructosyl-6-transferase (6-SST), producing 6-kestose. It comigrated with one of the constitutive invertases on MonoQ but was separated from it by subsequent chromatography on alkyl Superose. Nevertheless, the preparation retained invertase activity, suggesting that this enzyme may act both as fructosidase and fructosyltransferase. When incubated with 1-kestose in addition to sucrose, this enzyme formed less 6-kestose but instead produced large amounts of the tetrasaccharide bifurcose (1&6-kestotetraose), the main fructan tetrasaccharide accumulating in vivo. These results suggest that two inducible enzymes, 1-SST and 6-SST, act in concert to initiate fructan accumulation in barley leaves.

The production of high-content fructo-oligosaccharides from sucrose by the mixed-enzyme system of fructosyltransferase and glucose oxidase

Abstract: A technique to produce high-content fructo-oligosaccharides by the mixed- enzyme system of fructosyltransferase and glucose oxidase was investigated. The mixed-enzyme reaction was carried out in a stirred tank reactor containing 40 %(w/v) sucrose with 10 unit of fructosyltransferase and 10 unit of glucose oxidase per gram sucrose for 25 h, at 40 °C and pH 5.5. Highly concentrated fructo-oligosaccharides up to 90 % was obtained by the mixed-enzyme system.