Fructan Breakdown Research Articles

The RNA landscape of the human commensal Segatella copri reveals a small RNA essential for gut colonization

The bacterium Segatella copri is a prevalent member of the human gut microbiota associated with health and disease states. However, the intrinsic factors that determine its ability to colonize the gut effectively remain largely unknown. By extensive transcriptome mapping of S.copri and examining human-derived samples, we discover a small RNA, which we name Segatella RNA colonization factor (SrcF), and show that SrcF is essential for S.copri gut colonization in gnotobiotic mice. SrcF regulates genes involved in nutrient acquisition, and complex carbohydrates, particularly fructans, control its expression. Furthermore, SrcF expression is strongly influenced by human microbiome composition and by the breakdown of fructans by cohabitating commensals, suggesting that the breakdown of complex carbohydrates mediates interspecies signaling among commensals beyond its established function in generating energy. Together, this study highlights the contribution of a small RNA as a critical regulator in gut colonization.

Cloning and characterization of a novel fructan 6-exohydrolase strongly inhibited by sucrose in Lolium perenne.

The first 6-fructan exohydrolase (6-FEH) cDNA from Lolium perenne was cloned and characterized. Following defoliation, Lp6 - FEHa transcript level unexpectedly decreased together with an increase in total FEH activity. Lolium perenne is a major forage grass species that accumulates fructans, mainly composed of β(2,6)-linked fructose units. Fructans are mobilized through strongly increased activities of fructan exohydrolases (FEHs), sustaining regrowth following defoliation. To understand the complex regulation of fructan breakdown in defoliated grassland species, the objective was to clone and characterize new FEH genes in L. perenne. To find FEH genes related to refoliation, a defoliated tiller base cDNA library was screened. Characterization of the recombinant protein was performed in Pichia pastoris. In this report, the cloning and enzymatic characterization of the first 6-FEH from L. perenne is described. Following defoliation, during fructan breakdown, Lp6-FEHa transcript level unexpectedly decreased in elongating leaf bases (ELB) and in mature leaf sheaths (tiller base) in parallel to increased total FEH activities. In comparison, transcript levels of genes coding for fructosyltransferases (FTs) involved in fructan biosynthesis also decreased after defoliation but much faster than FEH transcript levels. Since Lp6-FEHa was strongly inhibited by sucrose, mechanisms modulating FEH activities are discussed. It is proposed that differences in the regulation of FEH activity among forage grasses influence their tolerance to defoliation.

Carbohydrate transporting membrane proteins of the rumen bacterium, Butyrivibrio proteoclasticus

The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.

Crystallization and preliminary X-ray diffraction study of fructan 1-exohydrolase IIa from Cichorium intybus.

Fructan 1-exohydrolase IIa (1-FEH IIa), a plant enzyme involved in fructan breakdown, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. The crystals are tetragonal, belonging to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 139.83, b = 139.83, c = 181.94 A. Calculation of the Matthews coefficient indicates there to be two or three molecules in the asymmetric unit. Synchrotron radiation was used to collect a complete native data set to a resolution of 2.35 A.

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.

Low-temperature Storage of Rooted Chrysanthemum Cuttings: Relationship to Carbohydrate Status of Cultivars

Low-temperature storage potential of rooted cuttings of garden chrysanthemum [Dendranthema ×grandiflorum (Ramat.) Kitamura] cultivars and its relationship with carbohydrate reserves were evaluated. Storage of chrysanthemum cuttings at -1 and -3 °C resulted in freezing damage. Visual quality of rooted cuttings stored at 0 or 3 °C varied among cultivars. Quality of `Emily' and `Naomi' cuttings was reduced within a week by dark storage at 0 or 3 °C due to leaf necrosis, while `Anna' and `Debonair' cuttings could be held for 4 to 6 weeks without significant quality loss. In `Anna' and `Debonair', low-temperature storage reduced the number of days from planting to anthesis regardless of storage duration. However, flowers of plants grown from stored cuttings were smaller than those of nonstored cuttings. At the beginning of storage, `Emily' and `Naomi' had lower sucrose, glucose, and fructose (soluble sugars) content compared to `Anna' and `Debonair'. Regardless of temperature, leaf soluble sugar was significantly reduced by dark storage for 4 weeks. In stems, sucrose and glucose were reduced while fructose generally increased during low-temperature storage probably due to the breakdown of fructans. Depletion of soluble sugars and a fructan-containing substance during low-temperature dark storage was greater in `Emily' and `Naomi' than in `Anna' and `Debonair'. Low irradiance [about 10 μmol·m-2·s-1 photosynthetically active radiation (PAR) from cool-white fluorescent lamps] in storage greatly improved overall quality and delayed the development of leaf necrosis in `Naomi'. Cuttings stored under light were darker green and had a higher chlorophyll content. Leaf and stem dry weights increased in plants stored under medium and high (25 to 35 μmol·m-2·s-1 PAR) irradiance while no change in dry weight was observed under dark or low light. Results suggest that the low-temperature storage potential of chrysanthemum cultivars varies considerably, and provision of light is beneficial in delaying the development of leaf necrosis and maintaining quality of cultivars with short storage life at low temperatures.

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei, a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.

Changes in gene‐expression in response to sucrose accumulation in leaf tissue of Lolium temulentum L.

summarySucrose accumulation and subsequent fructan synthesis was initiated in detached, mature leaves of Lolium temulentum by incubating them either in the light or with 200 mM sucrose in the dark. Fructan synthesis was also induced in intact seedlings by lowering root and shoot meristem temperature to 5 °C while maintaining aerial parts of the plant at 20 °C in the light. In this system, subsequent breakdown of fructans was induced by raising root and meristem temperature to 20 °C in the dark. Products of in vitro translations showed that transcripts encoding polypeptides of 17 and 35 kDa increased in sucrose accumulating‐tissue. Transcripts encoding 28 and 32 kDa polypeptide products appeared to be associated with leaf tissue actively exporting carbohydrate. Labelling studies in vivo in mature leaf tissue showed transient increases in the synthesis of polypeptides with MR 59, 60 and 70 kDa during the first 6 h of sucrose accumulation. Polypeptides with an MR of 74 and 27 kDa showed increased amounts later in induction.Cordycepin and cycloheximide were fed to mature leaf tissue at different stages during induction of sucrose accumulation in the light. Both control and cordycepin‐treated tissue showed higher levels of monosaccharide accumulation compared with cycloheximide‐treated tissue. Cordycepin applied during the first 2 h after excision blocked subsequent synthesis of all fructans. When applied at two‐hour‐stages between 2 and 8 h, subsequent accumulation of 1–kestose occurred without accumulation of higher fructans. Application after 8 h had no effect upon the pattern of fructan accumulation when compared to untreated controls. Application of cycloheximide during the first 8 h following excision blocked all fructan accumulation. Subsequent administration had no effect on the pattern of fructan accumulation. The results are discussed in relation to the changes in gene expression associated with induction of fructan synthesis.

Fructose‐containing carbohydrates in the tuberous root of Gomphrena macrocephala St.‐Hil. (Amaranthaceae) at different phenological phases

ABSTRACT Gomphrena macrocephala St.‐Hil. (Amaranthaceae) is a perennial herb that grows spontaneously in the cerrado and is characterized by well‐defined phenological phases throughout the year. Soluble carbohydrates are the main reserve compounds of the tuberous root and constitute approximately 50% of the dry weight. These sugars were partially characterized as fructans forming a single homologous series, different from inulin, the most common fructan of dicotyledons. The mean molecular weight of polysaccharides was high and reached 37 kDa in the dormant phase. Fructan spherocrystals were detected in the tuberous root after treatment with ethanol, being associated with the parenchyma of secondary xylem. The content, composition and mean molecular weight of fructans were related to phenology. In late dormancy, there was a marked increase in monosaccharides, particularly fructose, and a concomitant decline of polysaccharides, probably as a result of fructan breakdown. During sprouting and in the vegetative phase, the contents of oligosaccharides and low molecular weight polysaccharides increased. A gradual rise in the molecular weight of polysaccharides occurred during the reproductive phase and at early dormancy, concurrently with decreasing levels of oligosaccharides. The capacity of G. macrocephala to accumulate readily accessible sugars, such as fructans, instead of starch, in response to environmental changes, may be of considerable advantage, since the cerrado is often subjected to seasonal drought and burnings.

Fructan Mobilization and the Involvement of Two .BETA.-Fructofuranosidases in Lycoris radiata Sprouting.

Seasonal changes of oligofructans, sucrose, glucose, and fructose contents in the various tissues of Lycoris radiata were followed by HPLC analyses. Oligofructans were found in all tissues except the scape, where large and nearly equal amounts of glucose and fructose were present. At the sprouting stage, oligofructans in the bulb greatly decreased with increases of both fructose content and β-fructofuranosidase activity. The bulb enzyme responsible for the fructan breakdown was a fructan exohydrolase that was inactive against sucrose, and the scape tissue contained a β-fructofuranosidase (EC 3.2.2.26) highly active toward sucrose. These results show a role of the accumulated fructans as an energy source for sprouting of the scape during the non-photosynthetic stage, the energy probably being supplied as sucrose.

アスパラガス貯蔵根における発芽前後の糖の変化

Changes in carbohydrates of the storage roots of asparagus on sprouting were studied.1. In the storage roots, fructose, sucrose and fructans were present consistently, but glucose was rarely found. Fructans as storage carbohydrates were identified from 3 to 13 degrees of polimerization.2. There was a carbohydrate concentration gradient along the storage root 2 days after sprouting. Concentrations of fructose and sucrose increased toward the root apex, but those of fructans decreased.3. In rhizomes, concentrations of fructose and glucose were almost equal, and the concentration of sucrose was 5 times that of hexose.4. Before sprouting, concentrations of fructose and sucrose in the storage roots decreased, but those of fructans did not change remarkably by 1 day after sprouting.5. After sprouting, the concentration of fructose in the storage roots began to increase from 5th day at 20°C and from 8th day at 10°C. The increasing rate was higher at 20°C. Concentrations of sucrose and fructans did not change much.6. In the detached root segments, no changes were detected in carbohydrate concentrations regardless of temperature.7. Exogenously applied plant hormones such as GA3, GA4 and IAA did not influence carbohydrate concentrations in the detached root segments, regardless of temperature. It was suggested that breakdown of fructans in intact storage roots was not influencedby plant hormones.