Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.)

Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve inulin yield and quality in chicory.     

The temporal development in a hybridizing population of wild and cultivated chicory (Cichorium intybus L.)

Hybridization and its possible impacts is a subject of increased attention in connection with the risk of unintended gene flow from cultivated (including genetically modified) plants to wild relatives. Whether such gene flow by hybridization is likely to take place depends among other things on the persistence of the hybrids in a natural environment over time. To evaluate this, we studied an experimental hybridizing population of wild and cultivated chicories (Cichorium intybus) relative to a previous study on the same population 2 years earlier. We compared the genetic composition, morphology and fitness traits of plants from 2004 to the plants in the same plot in 2002. The majority of the plants in 2004 was more morphologically and genetically intermediate than in 2002. This indicates that no selection towards being wild-like or cultivar-like was present over the period of 2 years. Furthermore, no distinct fitness differences existed between the plants of 2004, probably due to most of the plants being intermediate. No hybridization barriers appeared to be present between wild and cultivated chicories beyond the F1 generation, since F2 hybrids and backcrosses were in abundance; in fact, hybrids of probably fourth or fifth generation were present. In conclusion, all results indicate that no barriers exist to the temporal persistence of chicory hybrids in a natural environment.     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Drought induces fructan synthesis and 1-SST (sucrose: sucrose fructosyltransferase) in roots and leaves of chicory seedlings (Cichorium intybus L.)

 Seeds of Cichorium intybus L. var. foliosum cv. Flash were sown in acid-washed vermiculite and grown in a controlled-environment growth chamber. After 1 month of growth, plantlets did not contain sucrose:sucrose 1-fructosyltransferase (1-SST), the key enzyme in fructan biosynthesis. No fructan could be observed. Some of the plants were submitted to drought for 2 weeks. Glucose, fructose and sucrose concentrations increased in roots and leaves of stressed plants and the fructan concentration in roots and leaves was ten times higher than in control plants. The onset of fructan synthesis coincided with the increase in 1-SST activity in roots. Expression of the 1-SST gene could be observed in roots and leaves of stressed plants. Received: 12 July 1999 / Accepted: 16 October 1999     

Development of SSR markers and construction of a consensus genetic map for chicory (Cichorium intybus L.)

A consensus genetic map for chicory (2n = 2x = 18) was obtained after the integration of molecular marker data of two industrial chicory progenies (K28K59, Rubis118) and one witloof chicory progeny (BR). As a limited number of co-dominant markers was available at the beginning of this work, three different microsatellite-enriched libraries were produced from genomic DNA, resulting in 420, 719 and 1,251 sequences, respectively. The level of informative Simple Sequence Repeat (SSR) sequences from the three libraries ranged from 28 to 40%, thus defining a set of 730 SSR markers available for polymorphism screening. A subset of 81 Sequence-Tagged Sites (STS) developed from EST, cDNA, genes, and non-coding sequences was screened through Single Strand Conformational Polymorphism (SSCP) analysis, leading to 46 polymorphic loci integrated in the genetic maps. Markers were grouped and ordered on 9 homologous Linkage Groups (LG) for each of the three maps: 274 markers in K28K59, 282 markers in Rubis118, 178 markers in BR. Co-linear regions between maps were identified through 193 ‘bridge’ markers that allowed the integration of the 9 homologous LG in a consensus map containing 472 markers and covering 878 cM. Comparison across maps revealed the presence of 4 conserved regions with significant distorted markers, also defined as Segregation Distortion Regions (SDR), affected by gametic or zygotic selection factors. Marker distribution was not always uniform; 6 LG possessed homologous clustered regions in all maps. The consensus map could be the starting point for the identification and the cloning of major genes and QTL in fundamental and applied genetic areas in chicory.     

Inclusion of chicory (Cichorium intybus L.) in pigs' diets affects the intestinal microenvironment and the gut microbiota

The content and composition of prebiotic plant fiber in the diet is important in promoting gut-related health. This study investigated the effects of the dietary inclusion of chicory forage and roots on the intestinal microenvironment of pigs. Thirty-seven-week-old pigs were fed 1 of 5 diets for 18 days, including a cereal-based control diet and 4 diets with the inclusion of 80 and 160 g kg(-1) of body weight chicory forage (CF80 and CF160), 80 g kg(-1) chicory root (CR80), and a mix of 80 g kg(-1) forage and 80 g kg(-1) chicory root (CFR). The animals maintained good performance and health irrespective of diet. Bacterial community structure and diversity in ileal and colonic samples was assessed using terminal restriction fragment length polymorphism (T-RFLP), combined with cloning and sequencing. Samples clustered perfectly according to gut segment with a higher bacterial diversity in colon than ileum. Distal ileum was dominated by lactic acid bacteria (LAB), and the relative amount of this group was increased by the CF160 and CFR diets. The colonic bacterial community was dominated by butyrate-producing bacteria and Prevotella. The increased relative abundance of butyrate-producing bacteria in the colon was positively correlated with the molar proportion of acetic acid and furthermore linked to the chicory forage diets (CF80 and CF160). Diets including chicory roots (CR80 and CFR) were correlated with a higher colonic abundance of Megasphaera elsdenii. The fermentation products and pH in digesta responded to diet type and were correlated with shifts in the microbiota, showing that chicory influences the intestinal microenvironment of pigs.     

海水灌溉欧洲菊苣盐肥耦合效应

2008年在江苏北部沿海滩涂进行田间试验研究了不同浓度海水灌溉下欧洲菊苣(Cicherium intybus L.)的盐肥耦合效应。结果表明:1)20%海水灌溉下欧洲菊苣肉质根和地上部分生物产量与淡水处理相比没有显著差异,而40%海水灌溉下产量均显著下降;N3(氮用量90kg.hm-2)水平与N1(氮用量0kg.hm-2)水平相比,欧洲菊苣肉质根产量与地上部分生物产量可以显著提高;同样,P3(P2O5用量45kg.hm-2)水平与P1(P2O5用量0kg.hm-2)水平相比,欧洲菊苣产量亦显著提高。2)各浓度海水灌溉下,随着施氮、磷量的增加菊苣主茎普遍增长和增粗。3)经过海水与氮肥及磷肥的交互作用对总产量影响的分析,可以看出W1N3(淡水,氮用量90kg.hm-2)和W1P3(淡水,P2O5用量45kg.hm-2)是优化组合。4)经过海水与氮肥及磷肥的交互作用对籽粒产量影响的分析,可以看出W2N3(20%海水,氮用量90kgN.hm-2)和W1P2(淡水,P2O5用量22.5kg.hm-2)是优化的组合。处理因子分析表明:海水、氮肥、磷肥对欧洲菊苣产量具有显著效应,以海水影响最大;总产量的优化组合为W1N3P...     

Calcium (45Ca) accumulation and transport in chicory (Cichorium intybus L.) root during bud development (forcing)

The lower part (4 cm) of the witloof chicory tap-root (15 cm) was immersed in a complete nutrient solution for 21 days, in the darkness at 18°C and at high RH. This process of forcing which leads to the emergence of an etiolated bud (chicon) was associated with a decrease in root dry weight. Although the amount of calcium in the root and the root cationic exchange capacity remained constant during forcing, the net uptake of calcium, negligible at the onset of forcing, progressively increased to a rate after ten days of 45 mol day^–1. Absorption of ⁴⁵Ca remained at a constant high rate, while the initially low upward migration of ⁴⁵Ca within the root and the chicon accelerated markedly. This upward migration was associated with a progressive decline in the release of newly absorbed ⁴⁵Ca. The data support the hypothesis that calcium acquisition by witloof chicory root is predominantly determined by calcium efflux. As the forcing progressed, the influx remained almost constant while a large decrease in the efflux led to a net uptake of calcium. Upward translocation was probably linked to the formation of new negative exchange sites within the growing chicon. The hypothesis that calcium movement occurred along a preferential pathway (xylem vessels) or involved a mass movement through the root is discussed.     

In vivo and in vitro flowering response of chicory (Cichorium intybus L.): influence of plant age and vernalization

Chicory plants (Cichorium intybus L. var foliosum cv Flash) were tested with and without a 4-week-long cold treatment for in vivo and in vitro flowering potential every 2 weeks during the growing season. One hundred percent of the plants harvested 112 days or later after sowing and then vernalized flowered in vivo. In vitro, no vernalization was needed to initiate flowering-stems on chicory explants taken from roots of 100 days old and older. 5-Azacytidine, a DNA demethylation agent, increased the flowering percentage on explants from young, vernalized roots but could not induce more than 15% flowering on young, nonvernalized roots. The greater flowering potential of chicory root explants in vitro when compared to plants of the same age tested in vivo was clearly established. This result suggests that some negative control on flowering was removed when root explants were excised and the main plant body discarded. Received: 31 August 1998 / Revision received: 27 October 1998 / Accepted: 10 November 1998     

Some enzyme changes in chicory (Cichorium intybus) during forcing

Differences in beet weight and sugar concentration of four commercial varieties grown on two types of soil and at different planting density were related to changes in the numbers, sizes and sugar storage capacities of the storage root cells. Much of the variation in beet weight between varieties and soil type was attributable to differences in cell size, less to differences in their number. Differences in weight of beet grown at different densities were caused equally by changes in number of cells and their size. Variation in sugar concentration of the beet was, in all cases, determined more by differences in cell size than in their capacity tostore sugar, but the two factors were not wholly independent of each other.     

Effect of salt stress on growth parameters, enzymatic antioxidant system, and lipid peroxidation in wild chicory (Cichorium intybus L.)

Efficient utilization of saline land for food cultivation can increase agricultural productivity and rural income. To obtain information on the salt tolerance/susceptibility of wild chicory (Cichorium intybus L.), the influence of salinity (0–260 mM NaCl) on chicory seed germination and that of two salinity levels of irrigation water (100 and 200 mM NaCl) on plant growth, antioxidative enzyme activity, and accumulation of proline and malondialdehyde (MDA) were investigated. The trials were performed outdoors, in pots placed under a protective glass covering, for two consecutive years. Seeds showed a high capacity to germinate in saline conditions. The use of 100 mM NaCl solution resulted in 81 % germination, whereas seed germinability decreased below 40 % using salt concentrations above 200 mM NaCl. Wild chicory showed tolerance to medium salinity (100 mM NaCl), whereas a drastic reduction in biomass was observed when 200 mM NaCl solution was used for irrigation. MDA, present in higher amounts in leaves than in roots, decreased in both tissues under increasing salinity. Proline content increased remarkably with the level of salt stress, more so in roots than in leaves. In salt stress conditions, the activity of antioxidant enzymes (APX, CAT, POD, SOD) was enhanced. The electrophoretic patterns of the studied enzymes showed that the salinity of irrigation water affected only the intensity of bands, but did not activate new isoforms. Our results suggest that wild chicory is able to grow in soil with moderate salinity by activating antioxidative responses both in roots and leaves.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

In situ gynogenetic haploid plants of chicory (Cichorium intybus L.) after intergeneric hybridization with Cicerbita alpina Walbr.

The possibility of obtaining haploid plants of chicory (Cichorium intybus L.) was investigated through intergeneric hybridization. Chicory plants (industrial chicory and Chioggia) were pollinated with pollen of Lactuca tatarica L. and Cicerbita alpina Walbr. Many achenes contained embryos which were rescued in vitro. Only a few embryos developed into plants which were then acclimatized in soil. Among them, three expressed a chicory phenorype and were haploidAbbreviations DAPI 4,6-diamidinophenylindole - DH doubled haploid - DNA deoxyribonucleic acid - GUS ß glucuronidase - RFLP Restriction Fragment Length Polymorphism - X-Gluc 5-bromo-4-chloro-3-indolyl-glucuronide - mtDNA mitochondrial DNA