Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.     

Characterization of fructan from mature leaf blades and elongation zones of developing leaf blades of wheat, tall fescue, and timothy

Water-soluble carbohydrate composition of mature (ceased expanding) leaf blades and the elongation zone of developing leaf blades was characterized in wheat (Triticum aestivum L.), tall fescue (Festuca arundinacea Schreb.), and timothy (Phleum pratense L.). These species were chosen because they differ in mean degree of polymerization (DP) of fructan in the mature leaf blade. Our objective was to compare the nature and DP of the fructan. Vegetative plants were grown with a 14-hour photoperiod and constant 21°C at the leaf base. Gel permeation chromatography of leaf blade extracts showed that the apparent mean fructan DP increased in the order wheat < tall fescue < timothy. Apparent mean DP of elongation zone fructan was higher than that of leaf blade fructan in wheat and timothy, but the reverse occurred for tall fescue. Low DP (≤10) and high DP (>10) pools were found in both tissues of tall fescue and wheat, but concentration of low DP fructan was very low in either tissue of timothy. All three species have high DP fructan. Comigration with standards on thin-layer chromotography showed that wheat contained 1-kestose and a noninulin fructan oligomer series. Tall fescue contained neokestose, 1-kestose and higher oligosaccharides that comigrated with neokestose-based compounds and inulins. Thin-layer chromatography showed that small amounts of fructose-containing oligosaccharides were present in timothy.     

Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots

Highly water soluble fructans have been isolated from Echinacea purpurea (L.) Moench. roots by hot water extraction and precipitation at three different ethanol concentrations (80% v/v, 60% v/v and 40% v/v). The structure of the fructans has been characterised by three analytical methods: GC of silylated oxime derivatives and partially methylated alditol acetates, respectively, as well as 13C NMR analysis. The mean degree of polymerisation (mean DP) of each fructan has been determined by the glucose/fructose ratio. E. purpurea fructans represent linear inulin-type fructans with almost exclusively beta-(2-->1)-linked fructosyl units, terminal glucose and terminal fructose. Small proportions of beta-(2-->1,2-->6)-linked branch point residues were detected. The mean DP of the fructan fractions depends on the ethanol concentration used for precipitation: the lower the ethanol concentration the higher the mean DP. Corresponding results were found with all of the three analytical methods: 80% ethanol-insoluble fructan from E. purpurea shows an average mean DP of 35, 60% ethanol-insoluble fructan of 44 and 40% ethanol-insoluble fructan of 55. The applied methods provide sufficient sensitivity to determine not only the composition and structure but also the mean degree of polymerisation of fructans.     

Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase in Winter Oat: Responses to Second-Phase Cold Hardening

Changes in apoplastic carbohydrate concentrations and activities of carbohydrate-degrading enzymes were determined in crown tissues of oat (Avena sativa L., cv Wintok) during cold hardening. During second-phase hardening (−3°C for 3 d) levels of fructan, sucrose, glucose, and fructose in the apoplast increased significantly above that in nonhardened and first-phase-hardened plants. The extent of the increase in apoplastic fructan during second-phase hardening varied with the degree of fructan polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP > 7). Activities of invertase and fructan exohydrolase in the crown apoplast increased approximately 4-fold over nonhardened and first-phase-hardened plants. Apoplastic fluid extracted from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by malate dehydrogenase activity. The significance of these results in relation to increases in freezing tolerance from second-phase hardening is discussed.     

Effects on the Stem of Winter Barley of Manipulating the Source and Sink during Grain-filling: II. CHANGES IN THE COMPOSITION OF WATER-SOLUBLE CARBOHYDRATES OF INTERNODES

Field-grown barley plants were manipulated by removing earsand by shading to promote and to reduce, respectively, the storageof carbohydrates in their stems. Water-soluble carbohydrate(WSC) was extracted and separated by HPLC into glucose, fructose,sucrose, and fructan of degree of polymerization (DP) 3, 4,5, > 5, and measured in both the penultimate and fourth internodesfrom the ear to determine the effects of the manipulations. During the accumulation of WSC, the mass of fructan with a DPgreater than 5 continued to increase whilst the mass of fructansof DP 3 to 5 reached a maximum and then remained constant. Fructanaccumulated in internodes while they were extending althoughmost of the fructan in an internode accumulated after it wasfully extended. When WSC was mobilized from the stem, the massof glucose, sucrose and fructan decreased but the mass of fructosefirst increased then decreased, indicating that fructan washydrolysed at a faster rate than its product, fructose, couldbe utilized. Plants shaded to 50% of incident light from 14 d after anthesisaccumulated the same mass of WSC in the stem as controlplantsin one crop, whereas in another crop, plants shaded to 9% ofincident light from 11 d after anthesis accumulated less WSCthancontrol plants. WSC in the stem was lost from the more intenselyshaded plants earlier than from control plants. Plants de-eared at anthesis and at 21 d after anthesis accumulateda similar mass of WSC to control plants, although plants de-earedat 9 d after anthesis, in another crop, accumulated a greatermass of WSC than control plants. Although control plants mobilizedalmost all of their stored WSC, de-eared plants retained 43–55%of the WSC stored in their penultimate internode and did notlose any stored WSC from their fourth internode. In the penultimateinternodes of de-eared plants the mass of glucose, sucrose andfructan decreased and this was balanced in part by an increasein the mass of fructose. In the fourth internode, the mass ofWSC remained constant but the mass of accumulated fructose wasequal to the decrease in the mass of fructan. Results are discussed in relation to current knowledge of fructanmetabolism and to contributions of stored WSC to grainyield. Key words: Winter barley, water-soluble carbohydrate, fructan, de-earing, shading     

Analysis of nonstructural carbohydrates in storage organs of 30 ornamental geophytes by high-performance anion-exchange chromatography with pulsed amperometric detection

A comprehensive analysis of nonstructural carbohydrates in storage organs (bulbs and corms) of 30 ornamental geophytes was conducted by employing a variety of extraction techniques followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Among species, starch, fructan, glucomannan and soluble sugars accounted for 50-80% of storage organ dry weight (DW). Starch ranged from 24 to 760 mg g(-1) DW, fructan (commonly occurring with starch) from 25 to 500 mg g(-1) DW, and glucomannan from 15 to 145 mg g(-1) DW. An acid hydrolysis protocol for concurrent determination of fructan and glucomannan was developed. The average degree of polymerization (DP) of ethanol and water-soluble fructan and the man : glu ratio of glucomannan also varied between species. The 80% ethanol fraction contained soluble sugars and short-chain fructans (< 25 DP), whereas water extracts contained soluble sugars, fructans (both short- and long-chain, 相似文献   

Fructans in crested wheatgrass leaves

Crested wheatgrass is an important cool-season grass that has become naturalized in many semiarid regions of the western U.S. It provides ground cover and reduces soil erosion caused by water and wind. Additionally, crested wheatgrass produces important forage for livestock and wildlife on 6 to 8 million hectars of western rangeland. It is well adapted to semiarid cold desert regions because of its cool temperature growth and drought tolerance. Understanding the biosynthesis of fructans in crested wheatgrass is important because of their likely role in both cool temperature growth and drought tolerance. Recent research described a major gene (6-SFT) in crested wheatgrass that is involved in fructan biosynthesis. 1-kestotriose, the major DP3 fructan in crested wheatgrass, serves as the substrate for the two major DP4 fructans, 1&6-kestotetraose and 1,1-kestotetraose. The three major DP5 fructans are 1&6,1-kestopentaose, 1,1&6-kestopentaose and 1,1,1-kestopentaose. The major DP6 fructan is 1&6, 1&6-kestohexaose. We postulate that 1&6,1&6-kestohexaose is synthesized from the addition of a fructose to 1&6, 1-kestopentaose. This paper provides structures of the various DP 3, 4, 5 and 6 fructan types produced by crested wheatgrass and provides suggested biosynthetic pathways for all major fructan linkage types present.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.     

Barley yellow dwarf virus: effects on carbohydrate metabolism in oat (Avena sativa) during cold hardening

Barley yellow dwarf virus (BYDV) causes significant losses in yield and in overwintering ability of winter cereals. Mechanisms by which the physiology of plants is affected by the virus are not clear. To see how carbohydrates in the crown of winter cereals were affected by BYDV, fructan isomers of degree of polymerization (DP) 3–5, fructan DP>6 and the simple sugars, glucose, fructose and sucrose, were measured before and during cold hardening in three oat ( Avena sativa L.) cultivars, 'Wintok', 'Coast Black' and 'Fulghum'. On a fresh weight basis fructan DP>6 decreased by 50% in infected 'Wintok' and 'Coast Black' and by 25% in 'Fulghum'. Two DP3, one DP4 and one DP5 isomer were significantly higher than non-infected controls. The percentages of simple sugars in infected crowns were significantly higher than controls in all three cultivars in every week except the first week of hardening. Crude enzyme extracts from BYDV infected plants incubated with sucrose suggested higher invertase and lower sucrose-sucrosyl transferase activity. When incubated with 1-kestose and neokestin, no significant difference was found in fructose fructosyl transferase or in hydrolase activity. The activity of unidentified enzymes catalysing the synthesis of larger (DP>5) fructan was altered by BYDV. The decrease of carbohydrates in the crown induced indirectly by BYDV may alter the plant's capacity to regenerate tillers in the spring. The ability of plants to prevent or tolerate carbohydrate fluctuations induced by BYDV infection may be an important genetically regulated characteristic for developing virus-resistant cultivars.     

Levans in Excised Leaves of<Emphasis Type="Italic">Dactylis glomerata</Emphasis>: Effects of Light,Sugars, Temperature and Senescence

Dactylis glomerata (orchardgrass) accumulates a single series of levans and the high DP polymers might be correlated with an increased stress resistance. A single levan series could be induced in excised orchardgrass leaves, without any 1 -kestose accumulation, strongly suggesting that fructan synthesis occurs independently of 1-SST activity. This elegant excised leaf system was used to study fructan metabolism regulation as affected by environmental conditions and exogenous sugar treatments. In contrast to the well-studied barley excised leaf system, fructan biosynthesis could not be rapidly induced in the light without exogenous sugar and only a limited fructan synthesis was observed in the dark with sugar. It can be concluded that both light and sugar are needed to achieve an optimal fructan synthesis. To induce fructan biosynthesis, sucrose could be replaced by a combination of glucose and fructose. Fructans were found to be a surplus pool of sucrose when a threshold sucrose concentration is surpassed. A metabolic switch to fructan degradation was observed when induced orchardgrass leaves were incubated in the dark at 30°C. Interestingly, fructans persisted during senescence of sugar-induced orchardgrass leaves. On the longer term, these fundamental regulatory insights might help to create superior grasses for future feed and/or biomass production.     

Molecular cloning and characterization of a high DP fructan: fructan 1-fructosyl transferase from Viguiera discolor (Asteraceae) and its heterologous expression in Pichia pastoris

Inulin-type fructans are stored in the tuberous roots of the Brazilian cerrado plant Viguiera discolor Baker (Asteraceae). In Cynara scolymus (artichoke) and Echinops ritro (globe thistle), the fructans have a considerably higher degree of polymerization (DP) than in Cichorium intybus (chicory) and Helianthus tuberosus (Jerusalem artichoke). It was shown before that the higher DP in some species can be attributed to the properties of their fructan: fructan 1-fructosyl transferases (1-FFTs; EC 2.4.1.100), enzymes responsible for chain elongation. Here, we describe the cloning of a high DP (hDP) 1-FFT cDNA from V. discolor and its heterologous expression in Pichia pastoris . Starting from 1-kestose and Neosugar P (a mixture of oligo-inulins from microbial origin) as substrates, the recombinant enzyme produces a typical hDP inulin profile in vitro, closely resembling the one observed in vivo. The enzyme shows no invertase activity and sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity in vitro. Pattern evolution during incubation suggests that inulins with DP ≥ 6 are much better substrates than sucrose or lower DP oligo-fructans. Because hDP inulin-type fructans show superior properties for specific food and non-food applications, the hDP 1-FFT gene from V. discolor has potential for the production of hDP inulin in vitro or in transgenic crops.     

高等植物果聚糖研究进展

果聚糖是高等植物重要的贮藏碳水化合物,因植物种类和发育阶段而异,主要存在5种类型的结构：线型菊糖型果聚糖、菊糖型果聚糖新生系列、线型梯牧草糖型果聚糖、混合型梯牧草糖型果聚糖和梯牧草糖型果聚糖新生系列。果聚糖的代谢模型随着代谢酶—蔗糖：蔗糖果糖基转移酶、蔗糖：果聚糖_6_果糖基转移酶、果聚糖：果聚糖果糖基转移酶、果聚糖：果聚糖_6_果糖基转移酶、果聚糖外水解酶等的发现、纯化和克隆日趋清晰。此外,果聚糖分子生物学研究也取得了一定的进展。     

高等植物果聚糖研究进展

果聚糖是高等植物重要的贮藏碳水化合物 ,因植物种类和发育阶段而异 ,主要存在 5种类型的结构 :线型菊糖型果聚糖、菊糖型果聚糖新生系列、线型梯牧草糖型果聚糖、混合型梯牧草糖型果聚糖和梯牧草糖型果聚糖新生系列。果聚糖的代谢模型随着代谢酶—蔗糖 :蔗糖果糖基转移酶、蔗糖 :果聚糖_6_果糖基转移酶、果聚糖 :果聚糖果糖基转移酶、果聚糖 :果聚糖_6_果糖基转移酶、果聚糖外水解酶等的发现、纯化和克隆日趋清晰。此外 ,果聚糖分子生物学研究也取得了一定的进展     

具有生物活性的川牛膝果聚糖的结构

从传统中药川牛膝(Cyathula offcinalis kuan)中分离提取到了一种具有生物活性的多糖RCP.核磁共振、甲基化分析、还原裂解和GC-MS分析揭示了RCP是一高度分支的果聚糖,它以(2→1)连接为骨架,其上有大量的(2→6)连接的分支,且属于新蔗果三糖系列.在93.17%果糖残基中,24.15%是末端果糖,26.24%是1-连接果糖,20.46%是6-连接果糖.在6.83%的葡萄糖残基中,2.14%是末端葡萄糖,4.69%是6-连接葡萄糖.RCP的平均聚合度是15.     

Comparative studies of the degradation of grass fructan and inulin by strains of Lactobacillus paracasei subsp. paracasei and Lactobacillus plantarum

Four strains of Lactobacillus paracasei subsp. paracasei and Lact. plantarum are investigated within 16 d in order to determine the formation of metabolites during the degradation of grass fructan and inulin as well as the subsequent fermentation to lactic acid. The decrease of the total content of fructans throughout the entire time of investigation shows differences specific for strains as for either fructan substrate. The strain Lact. plantarum V 54/6 completely degrades the grass fructan and inulin within no longer than 13 d. The utilization of fructan by the other strains is temporally delayed, and in a smaller degree of degradation, especially remarkable for inulin cleavage. The structural modifications of decomposed fructans are characterized by a noticeable shift of the mean DP from approximately 80 to the oligomeric range analysed by anion exchange chromatography. Additionally, a newly formed series of peaks of oligomeric saccharides was detected during the degradation of grass fructan and inulin. Part of the fructose that is derived from cleavage of fructans is fermented immediately by the LAB strains into differently high amounts of lactic acid. The abundance of formed fructose is enriched in the medium to a varying extent, depending on the strain as well as the substrate used. From these results a number of fructan degradative enzymes in lactobacilli have been concluded to possibly vary their modes of regulation: strain specific exo- and endohydrolases with different activities against β-2,1 and β-2,6 linked fructan.     

Growth, fructan yield, and quality of chicory (Cichorium intybus L.) as related to photosynthetic capacity, harvest time, and water regime

Fructans are polymers that are widely used in several industrial applications. In the last few years they have received increasing interest because of their positive effects on health. At present, fructans are mostly supplied by chicory, which is only grown and processed in The Netherlands, France, and Belgium. It would therefore be an attractive concept to expand its cultivation to the southern European countries, although water shortage and high temperatures may hinder its growth and yield. So far, few experiments have been carried out on the effects of water, so the present research was focused on the course of growth and fructan quality in rainfed (W(0)) and well-watered (W(1)) conditions. The positive effects of water restoration mostly concerned the above-ground dry weight (ADW), whereas the root dry weight (RDW) was less influenced. No significant differences on RDW were found in 1999, whereas it was 14% higher (P <0.01) in W(1) in 2000. The effect of water was very clear on assimilate allocation: the overall priority at the whole plant scale seemed to be root structures, then storage reserves, and finally ADW. Therefore, the fructan content was higher in W(0), and insignificant differences between W(0) and W(1) were found on fructan yield at the final harvests. The only significant effect of the water regime on fructans was to speed up their storage. The leaf photosynthetic capacity (A) was poorly affected by water availability, whereas it appeared consistently modulated by leaf temperature and leaf nitrogen content. Stomatal conductance appeared to be mostly affected by the soil water content and it was mostly related to A up to about 300 mmol m(-2) s(-1). The fructan chain length (DP) was not affected by water regime. Besides, DP classes showed a normal statistical distribution; skewness and kurtosis significantly changed only when the harvest was very late. Equally, a very late harvest time significantly lowered DP.     

N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft

Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.