W. Van den Ende

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.

Water stress drastically reduces root growth and inulin yield in Cichorium intybus (var. sativum) independently of photosynthesis

Root chicory (Cichorium intybus var. sativum) is a cash crop cultivated for inulin production in Western Europe. This plant can be exposed to severe water stress during the last 3 months of its 6-month growing period. The aim of this study was to quantify the effect of a progressive decline in water availability on plant growth, photosynthesis, and sugar metabolism and to determine its impact on inulin production. Water stress drastically decreased fresh and dry root weight, leaf number, total leaf area, and stomatal conductance. Stressed plants, however, increased their water-use efficiency and leaf soluble sugar concentration, decreased the shoot-to-root ratio and lowered their osmotic potential. Despite a decrease in photosynthetic pigments, the photosynthesis light phase remained unaffected under water stress. Water stress increased sucrose phosphate synthase activity in the leaves but not in the roots. Water stress inhibited sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1 fructosyltransferase after 19 weeks of culture and slightly increased fructan 1-exohydrolase activity. The root inulin concentration, expressed on a dry-weight basis, and the mean degree of polymerization of the inulin chain remained unaffected by water stress. Root chicory displayed resistance to water stress, but that resistance was obtained at the expense of growth, which in turn led to a significant decrease in inulin production.

Prebiotics to fight diseases: reality or fiction?

Bacteria living in the gastrointestinal tract are crucial for human health and disease occurrence. Increasing the beneficial intestinal microflora by consumption of prebiotics, which are ‘functional foods’, could be an elegant way to limit the number and incidence of disorders and to recover from dysbiosis or antibiotic treatments. This review focuses on the short‐chain low‐digestible carbohydrates (LDCs) which are metabolized by gut microbiota serving as energy source, immune system enhancers or facilitators of mineral uptake. Intake of foods containing LDCs can improve the state of health and may prevent diseases as for example certain forms of cancer. Given the large number of different molecules belonging to LDCs, we focused our attention on fructans (inulin, fructo‐oligosaccharides), galacto‐oligosaccharides and resistant starches and their therapeutic and protective applications. Evidence is accumulating that LDCs can inhibit bacterial and viral infections by modulating host defense responses and by changing the interactions between pathogenic and beneficial bacteria. Animal studies and studies on small groups of human subjects suggest that LDCs might help to counteract colorectal cancer, diabetes and metabolic syndrome. The action mechanisms of LDCs in the human body might be broader than originally thought, perhaps also including reactive oxygen species scavenging and signaling events. Copyright

Sugar ratios, glutathione redox status and phenols in the resurrection species Haberlea rhodopensis and the closely related non-resurrection species Chirita eberhardtii.

Because of their unique tolerance to desiccation, the so-called resurrection plants can be considered as excellent models for extensive research on plant reactions to environmental stresses. The vegetative tissues of these species are able to withstand long dry periods and to recover very rapidly upon re-watering. This study follows the dynamics of key components involved in leaf tissue antioxidant systems under desiccation in the resurrection plant Haberlea rhodopensis and the related non-resurrection species Chirita eberhardtii. In H. rhodopensis these parameters were also followed during recovery after full drying. A well-defined test system was developed to characterise the different responses of the two species under drought stress. Results show that levels of H₂O₂ decreased significantly both in H. rhodopensis and C. eberhardtii, but that accumulation of malondialdehyde was much more pronounced in the desiccation-tolerant H. rhodopensis than in the non-resurrection C. eberhardtii. A putative protective role could be attributed to accumulation of total phenols in H. rhodopensis during the late stages of drying. The total glutathione concentration and GSSG/GSH ratio increased upon complete dehydration of H. rhodopensis. Our data on soluble sugars suggest that sugar ratios might be important for plant desiccation tolerance. An array of different adaptations could thus be responsible for the resurrection phenotype of H. rhodopensis.

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.

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.

Chilling tolerance and early vigour-related characteristics evaluated in two Miscanthus genotypes

A long growing season, mediated by the ability to grow at low temperatures early in the season, can result in higher yields in biomass of crop Miscanthus. In this paper, the chilling tolerance of two highly productive Miscanthus genotypes, the widely planted Miscanthus × giganteus and the Miscanthus sinensis genotype ‘Goliath’, was studied. Measurements in the field as well as under controlled conditions were combined with the main purpose to create basic comparison tools in order to investigate chilling tolerance in Miscanthus in relation to its field performance. Under field conditions, M. × giganteus was higher yielding and had a faster growth rate early in the growing season. Correspondingly, M. × giganteus displayed a less drastic reduction of the leaf elongation rate and of net photosynthesis under continuous chilling stress conditions in the growth chamber. This was accompanied by higher photochemical quenching and lower nonphotochemical quenching in M. × giganteus than that in M. sinensis ‘Goliath’ when exposed to chilling temperatures. No evidence of impaired stomatal conductance or increased use of alternative electron sinks was observed under chilling stress. Soluble sugar content markedly increased in both genotypes when grown at 12°C compared to 20°C. The concentration of raffinose showed the largest relative increase at 12°C, possibly serving as a protection against chilling stress. Overall, both genotypes showed high chilling tolerance for C4 plants, but M. × giganteus performed better than M. sinensis ‘Goliath’. This was not due to its capacity to resume growth earlier in the season but rather due to a higher growth rate and higher photosynthetic efficiency at low temperatures.

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.

The X-ray diffraction structure of a glycosyl hydrolase family 32 protein: 1-fructan exohydrolase IIa ofCichorium intybus

Glycosyl Hydrolase Family 32 protein: 1-Fructan Exohydrolase IIa of Cichorium Intybus. Maureen Verhaest, Wim Van den Ende, Camiel De Ranter, André Van Laere and Anja Rabijns, Laboratorium voor Analytische Chemie en Medicinale Fysicochemie, K.U. Leuven, E. Van Evenstraat 4, 3000 Leuven, Belgium, and Laboratorium voor Moleculaire Plantenfysiologie, K.U.Leuven, Kasteelpark Arenberg 31, 3001 Heverlee, Belgium. E-mail: Anja.Rabijns@pharm.kuleuven.ac.be