A. Van Laere

X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana

Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the alpha1-beta2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15 A. The structure comprises an N-terminal fivefold beta-propeller domain followed by a C-terminal domain formed by two beta-sheets. The active site is positioned in the fivefold beta-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme.

ISOLATION AND STRUCTURAL ANALYSIS OF NEW FRUCTANS PRODUCED BY CHICORY

This report describes a new series of oligosaccharides, which is formed in chicory roots under forcing conditions and during in vitro experiments using purified chicory 1-FFT (fructan:fructan 1-fructosyl transferase). It was demonstrated that the three smallest members of this new series (disaccharide, trisaccharide and tetrasaccharide) contain exclusively β-D-fructosyl residues after hydrolysis. The present data demonstrate that the smallest compound is levanbiose and that the other oligomers of this new series of fructans do not belong to the linear 2→6 linked levan-oligosaccharides nor to the linear 2→1 linked inulo-oligosaccharides. A combination of several chromatographic techniques yielded a fraction that contained only the compound with degree of polymerisation (DP) 2 (levanbiose, β-D-fructofuranosyl-(2→6)-D-fructofuranose), and a mixture of DP 3 of the new series and 1-kestose. Using homonuclear and heteronuclear 2D NMR experiments the complete 1H and 13C NMR assignments of levanbiose and DP 3 were obtained. From High Performance Anion Exchange Chromatography (HPAEC) and NMR experiments of DP 3 of the new series it was concluded that the molecule contains a β-D-fructofuranosyl residue 2→1 linked to the non-reducing moiety of levanbiose, and thus has to be named β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→6)-D-fructofuranose. The simple and regular pattern of the HPAEC retention times of the new oligosaccharides suggests that it is a homologous series of oligomers build by 2→1 elongation with β-D-fructofuranosyl residues at the non-reducing residue of levanbiose.

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-14C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1 mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early develpment. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. This increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.

Water potential, glycerol synthesis, and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.

Glucose Metabolism of Dormant and Heat-activated Spores of Phycomyces blakesleeanus Burgeff

SummaryThe metabolism of [14C]glucose has been studied in Phycomyces spores during dormancy, activation, and the initial stages of germination. Dormant spores are able to take up and metabolize exogenous glucose into different products; the major part of it goes to trehalose synthesis (up to 60% when the external glucose concentration exceeds 10-3 M). During activation itself (i.e. a prolonged treatment at 50°) there is a general increase of glucose uptake and metabolism, without major changes in the relative rates of 14C-label distribution in the different fractions (as compared to the metabolism of dormant spores), except for a drop in material insoluble in 80% ethanol and a still higher percentage (73%) going to trehalose synthesis. In the early hours of germination we find an enhancement of the uptake and metabolism of glucose. Trehalose synthesis is practically switched off within 2 h whereas the major part of glucose (65%) is metabolized to CO2 and ethanol-insoluble proteinaceous material.

Induction of 1-FEH in Mature Chicory Roots Appears to be Related to Low Temperatures Rather than to Leaf Damage

Large-scale inulin production from chicory roots (Cichorium intybus L.) is hampered by the induction of 1-FEH activity (fructan 1-exohydrolase) and concomitant fructose production in autumn, coincident with a period with low night temperatures that cause leaf damage. To understand whether leaf damage per se is sufficient for 1-FEH induction and fructan breakdown, we defoliated mature chicory plants at a preharvest stage (September 10) and investigated the changes in carbohydrate levels and 1-FEH activities. Also, the activities of 1-SST (sucrose:sucrose 1-fructosyl transferase, EC 2.4.1.99), 1-FFT (fructan:fructan 1-fructosyl transferase, EC 2.4.1.100), and acid invertase (EC 3.2.1.26) were determined. Defoliation did not result in a prompt fructan breakdown and increase in 1-FEH activity, but after 10 days fructan breakdown and 1-FEH activities became higher in the defoliated plants. Defoliation resulted in a sharp decrease in 1-SST activity over the first 24 h. Afterwards, root 1-SST activities of defoliated plants remained at a lower level than in control plants. 1-FFT and invertase activities were not affected by defoliation. It can be concluded that defoliation of plants at the preharvest stage by itself did not induce the same rapid changes as observed in naturally induced October roots by low temperature (harvest stage). Taken together with our finding that 1-FEH is not induced in chicory roots when plants are transferred to the greenhouse early autumn (minimal temperature 14°C), we conclude that low temperatures might be essential for 1-FEH induction.

Compartmentation and respiration pathways in Avena coleoptile segments

Summary The distribution of label from glucose-14C into different products has been followed during incubation and chase experiments with Avena coleoptiles. Compartmentation of glucose between a small and a large compartment has been demonstrated. Further compartmentation of glucose within the cytoplasm is shown to be improbable taking into account the high randomisation of label within the glucose molecule, the radioactivity distribution upon incubation in different glucose concentrations and the specific radioactivity data of glucose-6-P and UDPG. Respiratory pathways are computed from data obtained from incubations in glucose-1-, -2- and -6-14C. The specific radioactivity data of glucose-6-P and UDPG allowed an expression of the nanomoles of glucose metabolised along the different pathways. Nearly half of the glucose was utilised for cell wall synthesis. The remaining metabolism was mainly along glycolysis and only minor amounts were metabolised along the pentose phosphate pathway.

The fate of label from glucose-U-14-C during incubation and chase experiments in mung bean tissue. An approach to the problem of dilution and compartmentation

Summary The fate of label from glucose-U- 14 C has been followed during 8 hours in mung bean tissue. Most of the label was recovered in cell wall material, CO 2 and free sugars while only minor amounts are found in organic acids, amino acids, lipids and phosphate esters. The specific activity (S. A.) data of the sugars provide evidence for cellular compartmentation of carbohydrates. Compartmentation of glucose within the cytoplasm is considered improbable, taking into account experimental data of label randomization in cell wall material and incorporation of 14 C in different compounds with varying glucose concentrations. The data of chase experiments suggest a low turnover for cell wall material, fructose, glucose, organic acids and lipids. Only the label in sucrose, amino acids and phosphate esters decreases to a measurable extent during the chase period. We calculated that less than 1 per cent of the cellular glucose is in the active compartment. Our results provide some support for the estimation of respiratory pathways by means of the 14 CO 2 -release, corrected for label randomization, using glucose-1, -2- and -6- 14 C.

Fructan 1-exohydrolase IIa fromCichorium intybusin complex with ligands

Fructan 1-Exohydrolase (1-FEH) is involved in fructan degradation. It can remove a terminal fructose from fructans, but not from sucrose. Together with the invertases and fructosyl transferases, the fructan exohydrolases belong to the glycosyl hydrolase family 32 (GH 32). Earlier, we reported the structure of 1-FEH IIa from Cichorium intybus [1]. Here, the 1-FEH IIa is described in complex with different inhibitors and substrates. All structures are characterized with one single molecule in the active site, positioned in the β-propeller domain. 1-FEH IIa in complex with sucrose, a strong competitive inhibitor, was resolved with a resolution of 2.50 Å. Also complexes with fructose and 2,5 dideoxy-2,5-imino-D-mannitol, two weaker inhibitors, were elucidated at a resolution of 2.65 Å and 3.25 Å, respectively. In order to create a complex with the best substrate of 1-FEH IIa, 1-kestose, an inactive mutant was produced (E201Q). The complex of 1-FEH IIa and 1-kestose was resolved with a resolution of 3.05 Å. The comparison of the complexes can clarify why sucrose acts as a strong inhibitor whereas 1-kestose acts as an ideal substrate.