Fructansucrase enzymes and sucrose analogues: A new approach for the synthesis of unique fructo-oligosaccharides

Fructansucrase enzymes of lactic acid bacteria use the cheap compound sucrose (Glc-Fru) to synthesize a variety of poly- and oligosaccharide products. Recently, it has been shown that a variety of sucrose analogues (e.g. Gal-Fru, Man-Fru) can be efficiently synthesized. This has exciting potential for the development of novel (fructo) oligosaccharide derivatives.     

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.     

The effect of sulphite on the regulation of photosynthetic sucrose synthesis in poplar leaves

Sulphite at concentrations from 0.05 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bart. ex Marsh) leaves via the transpiration stream. The rate of CO2 fixation and partitioning of newly fixed carbon between sucrose and starch were measured and compared with the contents of selected phosphorylated intermediates, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and the activation of sucrose-phosphate synthase (SPS). Supplying leaves with 0.5 mM sulphite led to an increase in the sucrose/starch partitioning ratio without altering the rate of ¹⁴CO2 fixation. The increase in sucrose synthesis compared to starch synthesis was accompanied by relatively small changes of 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate (Fru1,6BP), hexose phosphates (hexose-)), uridine 5'-diphosphoglucose (UDPGlc), an accumulation of triose phosphates (triose-P), an activation of SPS, and decreased Fru2,6BP contents. Supplying leaves with 1.0 mM sulphite decreased ¹⁴CO2 assimilation and increased partitioning of fixed carbon into starch. A selective inhibition of sucrose synthesis was accompanied by an accumulation of triose-P, Fru1,6BP, hexose-P, and a decrease of PGA contents. There was also a large increase of Fru2,6BP contents and a decline in the activation of SPS. It could be argued that sulphite affects the allocation of photosynthetic carbon to sucrose and that sulphite can inhibit photosynthesis via a selective inhibition of sucrose synthesis.     

Carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing elevated fructose 2,6-bisphosphate levels

The aim of this work was to investigate the role of fructose 2,6-bisphosphate (Fru 2,6-P₂) during photosynthesis. The level of Fru 2,6-P₂ in tobacco plants was elevated by the introduction of a modified mammalian gene encoding 6-phosphofructo-2-kinase (6-PF-2-K). Estimates of the metabolite control coefficient (C) for Fru 2,6-P₂ levels in response to increased 6-PF-2-K activity, suggest that small increases in 6-PF-2-K activity have little effect upon steady-state Fru 2,6-P₂ levels (_C = +0.08 for a 0–58% increase in 6-PF-2-K activity). However, larger changes resulted in dramatic rises in Fru 2,6-P₂ levels (C = +3.35 for 206–268% increase in 6-PF-2-K activity). Transgenic plants contained Fru 2,6-P₂ levels in the dark that ranged from 104 to 230% of the level in wild-type tobacco. Plants with altered levels of Fru 2,6-P₂ were used to determine the effects of this signal metabolite upon carbohydrate metabolism during the initial phase of the light period. Here we provide direct evidence that Fru 2,6-P₂ contributes to the regulation of carbon partitioning in tobacco leaves by inhibiting sucrose synthesis.     

Optimization of simultaneously enzymatic fructo- and inulo-oligosaccharide production using co-substrates of sucrose and inulin from Jerusalem artichoke

Prebiotic substances are extracted from various plant materials or enzymatic hydrolysis of different substrates. The production of fructo-oligosaccharide (FOS) and inulo-oligosaccharide (IOS) was performed by applying two substrates, sucrose and inulin; oligosaccharide yields were maximized using central composite design to evaluate the parameters influencing oligosaccharide production. Inulin from Jerusalem artichoke (5–15% w/v), sucrose (50–70% w/v), and inulinase from Aspergillus niger (2–7 U/g) were used as variable parameters for optimization. Based on our results, the application of sucrose and inulin as co-substrates for oligosaccharide production through inulinase hydrolysis and synthesis is viable in comparative to a method using a single substrate. Maximum yields (674.82?mg/g substrate) were obtained with 5.95% of inulin, 59.87% of sucrose, and 5.68 U/g of inulinase, with an incubation period of 9?hr. The use of sucrose and inulin as co-substrates in the reaction simultaneously produced FOS and IOS from sucrose and inulin. Total conversion yield was approximately 67%. Our results support the high value-added production of oligosaccharides using Jerusalem artichoke, which is generally used as a substrate in prebiotics and/or bioethanol production.     

The determination time of the carpel whorl is differentially sensitive to carbohydrate supply in Pharbitis nil

A shoot apical meristem is florally determined if, following its removal from an induced plant, it flowers when cultured in non-inductive conditions. Determination times were measured in the short-day plant Pharbitis nil to examine whether floral whorls are determined simultaneously or sequentially. Shoot apices were excised at daily intervals following a 48-h dark-inductive treatment, cultured in non-inductive conditions for 4 weeks in continuous light, and the number of floral organs scored. The culture medium was White's supplemented with sucrose, glucose (Glc), fructose (Fru), or 1:1 Glc:Fru at 2% (w/v), 4% (w/v), or 6% (w/v) or sugar-mannitol combinations of osmotic potentials equivalent to 4% (w/v) or 6% (w/v). The minimum whorl determination time was 1 d for sepals, petals, and stamens regardless of carbon supply. However, for carpels it varied remarkably from 5 d on sucrose, to 2 to 3 d on Fru or Glc:Fru, to 1 d for 2% (w/v) and 6% (w/v) Glc. Therefore, depending on the carbon supply, the carpel whorl was determined at the same time or after the outer whorls. Generally, these effects could not be reproduced on the sugar-mannitol treatments.     

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     

Exopolysaccharide and kestose production by Lactobacillus sanfranciscensis LTH2590

The effect was investigated of sucrose concentration on sucrose metabolism and on the formation of exopolysaccharide (EPS) by Lactobacillus sanfranciscensis LTH2590 in pH-controlled fermentations with sucrose concentrations ranging from 20 to 160 g liter(-1). The EPS production increased and the relative sucrose hydrolysis activity decreased by increasing the sucrose concentration in the medium. The carbon recovery decreased from 95% at a sucrose concentration of 30 g liter(-1) to 58% at a sucrose concentration of 160 g liter(-1) because of the production of an unknown metabolite by L. sanfranciscensis. This metabolite was characterized as a fructo-oligosaccharide. The oligosaccharide produced by L. sanfranciscensis was purified and characterized as a trisaccharide with a glucose/fructose ratio of 1:2. The comparison of the retention time of this oligosaccharide and that of pure oligosaccharide standards using two different chromatography methods revealed that the oligosaccharide produced by L. sanfranciscensis LTH2590 is 1-kestose. Kestose production increased concomitantly with the initial sucrose concentration in the medium.     

Mapping of putative quantitative trait loci controlling the total oligosaccharide and sucrose content of Glycine max seeds

Although oligosaccharides and sucrose are very important nutritional components of soybean seeds, little information is available about inheritance of oligosaccharide and sucrose content. The objective of this study was to identify quantitative trait loci (QTLs) that determine the oligosaccharide and sucrose content of soybean. The 117 F_2:10 recombinant inbred lines developed from a cross of “Keunolkong” and “Shinpaldalkong” were used. Narrow-sense heritability estimates, on a plot mean basis, of oligosaccharide and sucrose content were 79.07 and 74.84%, respectively. Four QTLs for oligosaccharide content were located on linkage groups (LG) C2, H, J, and L. Sucrose content was related with two QTLs located on LG H and J. Total oligosaccharide and sucrose content have two common QTLs on LG H and J.     

Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-(13C)Fru]sucrose, [1-(13C)Glc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Lack of fructose 2,6‐bisphosphate compromises photosynthesis and growth in Arabidopsis in fluctuating environments

The balance between carbon assimilation, storage and utilisation during photosynthesis is dependent on partitioning of photoassimilate between starch and sucrose, and varies in response to changes in the environment. However, the extent to which the capacity to modulate carbon partitioning rapidly through short‐term allosteric regulation may contribute to plant performance is unknown. Here we examine the physiological role of fructose 2,6‐bisphosphate (Fru‐2,6‐P₂) during photosynthesis, growth and reproduction in Arabidopsis thaliana (L.). In leaves this signal metabolite contributes to coordination of carbon assimilation and partitioning during photosynthesis by allosterically modulating the activity of cytosolic fructose‐1,6‐bisphosphatase. Three independent T‐DNA insertional mutant lines deficient in 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (F2KP), the bifunctional enzyme responsible for both the synthesis and degradation of Fru‐2,6‐P₂, lack Fru‐2,6‐P₂. These plants have normal steady‐state rates of photosynthesis, but exhibit increased partitioning of photoassimilate into sucrose and have delayed photosynthetic induction kinetics. The F2KP‐deficient plants grow normally in constant environments, but show reduced growth and seed yields relative to wildtype plants in fluctuating light and/or temperature. We conclude that Fru‐2,6‐P₂ is required for optimum regulation of photosynthetic carbon metabolism under variable growth conditions. These analyses suggest that the capacity of Fru‐2,6‐P₂ to modulate partitioning of photoassimilate is an important determinant of growth and fitness in natural environments.     

Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase <Emphasis Type="Italic">Bacillus subtilis</Emphasis> levansucrase

Background  

Fructans – β-D-fructofuranosyl polymers with a sucrose starter unit – constitute a carbohydrate reservoir synthesised by a considerable number of bacteria and plant species. Biosynthesis of levan (αGlc(1–2)βFru [(2–6)βFru]_n), an abundant form of bacterial fructan, is catalysed by levansucrase (sucrose:2,6-β-D-fructan-6-β-D-fructosyl transferase), utilizing sucrose as the sole substrate. Previously, we described the tertiary structure of Bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, Asp86, Asp247 and Glu342, which are central to the double displacement reaction mechanism of fructosyl transfer. Still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined.     

Carbohydrate status in dormant and afterripened excised wild oat embryos

Germination and carbohydrate concentrations were determined in excised dormant and afterripened wild oat (Avena fatua L. line M73) embryos cultured on N6 medium with and without 88 mM fructose (Fru). Without Fru dormant embryos began to germinate after approximately 2 weeks, and the germination rate was greater at 12 than 16°C. With addition of Fru 80% of dormant embryos germinated in 3 days. More than 80% of afterripened embryos germinated within 1 day on N6 with or without additional sugars. Therefore, relative to afterripened embryos, true embryo dormancy exists in line M73. Concentrations of starch and soluble sugars were initially similar in dormant and afterripened embryos. Culturing dormant and afterripened embryos on medium with Fru resulted in concentrations of glucose (Glu), sucrose (Sue), Fru and maltose (Mal) that were the same or higher than the initial levels. The concentration of starch in embryos initially increased slightly then remained constant or declined, except in dormant embryos on Fru-amended medium, where starch accumulated to 34 μg Glu equivalents (mg fresh weight)^-1 at 52 h. Raffinose (Raf) and stachyose (Stach) concentrations declined over time in all embryos. Carbohydrate concentrations in afterripened embryos on medium without Fru decreased to nearly undetectable levels by 52 h. Soluble sugar concentrations in dormant embryos on medium without Fru also declined by 52 h, but changes were not as extensive as those in afterripened embryos without Fru. In 52 h Raf and Stach were nearly depleted in all afterripened embryos, and in dormant embryos cultured on Fru-containing medium but not in dormant embryos without Fru. The concentration of Stach in dormant embryos without Fru declined 60% at 12 to 18 days coinciding with the potential for germination. The results demonstrate that a decline in Stach concentration is associated with the potential for germination of dormant (D) excised embryos. The mechanism of dormancy-breaking associated with the Raf family oligosaccharides remains to be determined.     

The effect of sulphite on chlorophyll fluorescence and sucrose metabolism in poplar leaves

Sulphite at concentrations from 0.5 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bartr. ex Marsh) leaves via the transpiration stream. Chlorophyll a fluorescence parameters, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and starch, and extractable specific activity of sucrose-phosphate synthase (SPS), sucrose synthase (SuSy), acid invertase (AI), neutral invertase (NI), ATP-dependent fructose-6-phosphate 1-phosphotransferase (PFK) and pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) were measured. Chlorophyll fluorescence parameters appeared to be unaffected by sulphite. Application of ≥ 1.0 mM sulphite led to an increase in the content of Fru2,6BP and starch. There was also a decline in the activity of SPS, NI and PFK. On the other hand, the influence of sulphite on the activity of AI and PFP was negligible. Specific activity of SuSy was inhibited by 1.0 and 2.5 mM but activated by 5.0 mM of sulphite. On the basis of the results obtained in the present study, we postulate that sulphite at concentrations ≥ 1.0 mM inhibits primarily sucrose synthesis, favours starch accumulation and has an indirect effect on the sucrolytic activities in poplar leaves.     

Exopolysaccharide and Kestose Production by Lactobacillus sanfranciscensis LTH2590

The effect was investigated of sucrose concentration on sucrose metabolism and on the formation of exopolysaccharide (EPS) by Lactobacillus sanfranciscensis LTH2590 in pH-controlled fermentations with sucrose concentrations ranging from 20 to 160 g liter⁻¹. The EPS production increased and the relative sucrose hydrolysis activity decreased by increasing the sucrose concentration in the medium. The carbon recovery decreased from 95% at a sucrose concentration of 30 g liter⁻¹ to 58% at a sucrose concentration of 160 g liter⁻¹ because of the production of an unknown metabolite by L. sanfranciscensis. This metabolite was characterized as a fructo-oligosaccharide. The oligosaccharide produced by L. sanfranciscensis was purified and characterized as a trisaccharide with a glucose/fructose ratio of 1:2. The comparison of the retention time of this oligosaccharide and that of pure oligosaccharide standards using two different chromatography methods revealed that the oligosaccharide produced by L. sanfranciscensis LTH2590 is 1-kestose. Kestose production increased concomitantly with the initial sucrose concentration in the medium.     

Crystal structure of the Glu328Gln mutant of Neisseria polysaccharea amylosucrase in complex with sucrose and maltoheptaose

Several enzymes acting on sucrose are found in glycoside hydrolase family 13 (the α–amylase family). They all transfer a glucosyl moiety from sucrose to an acceptor, but the products can be very different. The structure of a variant of one of these, the Glu328Gln mutant of Neisseria polysaccharea amylosucrase, has been determined in a ternary complex with sucrose and an oligosaccharide to 2.16 Å resolution using x-ray crystallography. Sucrose selectively binds in the active site and the oligosaccharide only binds at surface sites. When this structure is compared to structures of other enzymes acting on sucrose from glycoside hydrolase family 13, it is found that the active site residues are very similar around the glucose part of sucrose while much variation is seen around the fructose moiety.     

Rheological characteristics of heat-induced custard pudding gels with high antioxidative activity

Custard pudding gels were prepared from fresh whole egg, milk and sugar. The effects of D-psicose (Psi), a non-calorie rare hexose, on the antioxidative activity and rheological properties of the custard pudding gels were investigated at different temperatures for comparison with those of control sugars (sucrose, Suc; D-fructose, Fru). The rheological behavior of the heat-induced pudding gels was evaluated by using breaking and creep tests. During the heat-induced gel formation, custard pudding containing Psi (15%, wt/wt) demonstrated a stronger breaking strength and higher viscoelasticity than those containing Fru and Suc. The thermodynamic parameters obtained from DSC indicated that the egg white (EW) proteins were made less thermally stable when heated in the presence of Psi than in the presence of Fru and Suc. These findings are consistent with enhanced aggregation of the EW solution in the presence of Psi. Furthermore, the Psi pudding gels possessed higher antioxidative activity than the control sugar pudding gels by using an analysis of the scavenging activity on DPPH radicals and the ferric-reducing antioxidative power. These results suggest that Psi favored cross-linking of Psi-protein molecules through the Maillard reaction which increased the formation of intermediate products to improve functionality. Custard pudding containing Psi could therefore be an effective functional sweet desert with high antioxidative activity and the outstanding gelling characteristics.     

Properties of fructan:fructan 1-fructosyltransferases from chicory and globe thistle,two Asteracean plants storing greatly different types of inulin

Remarkably, within the Asteraceae, a species-specific fructan pattern can be observed. Some species such as artichoke (Cynara scolymus) and globe thistle (Echinops ritro) store fructans with a considerably higher degree of polymerization than the one observed in chicory (Cichorium intybus) and Jerusalem artichoke (Helianthus tuberosus). Fructan:fructan 1-fructosyltransferase (1-FFT) is the enzyme responsible for chain elongation of inulin-type fructans. 1-FFTs were purified from chicory and globe thistle. A comparison revealed that chicory 1-FFT has a high affinity for sucrose (Suc), fructose (Fru), and 1-kestose as acceptor substrate. This makes redistribution of Fru moieties from large to small fructans very likely during the period of active fructan synthesis in the root when import and concentration of Suc can be expected to be high. In globe thistle, this problem is avoided by the very low affinity of 1-FFT for Suc, Fru, and 1-kestose and the higher affinity for inulin as acceptor substrate. Therefore, the 1-kestose formed by Suc:Suc 1-fructosyltransferase is preferentially used for elongation of inulin molecules, explaining why inulins with a much higher degree of polymerization accumulate in roots of globe thistle. Inulin patterns obtained in vitro from 1-kestose and the purified 1-FFTs from both species closely resemble the in vivo inulin patterns. Therefore, we conclude that the species-specific fructan pattern within the Asteraceae can be explained by the different characteristics of their respective 1-FFTs. Although 1-FFT and bacterial levansucrases clearly differ in their ability to use Suc as a donor substrate, a kinetic analysis suggests that 1-FFT also works via a ping-pong mechanism.     

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