The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5Ј-and 3Ј-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

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.

Fructans are fructose-based oligo-and polymers that serve as reserve carbohydrates in many plant species. The biochemistry of fructan biosynthesis in dicots has been resolved, and the respective cDNAs have been cloned. Recent progress has now succeeded in elucidating the biochemistry and molecular biology of fructan biodegradation in chicory, an economically important species used for commercial inulin extraction. Unlike fructan biosynthetic genes that originated from vacuolar-type invertase, fructan exohydrolases (FEHs) seem to have evolved from a cell-wall invertase ancestor gene that later obtained a low iso-electric point and a vacuolar targeting signal. Expression analysis reveals that fructan enzymes are controlled mainly at the transcriptional level. Using chicory as a model system, northern analysis was consistent with enzymatic activity measurements and observed carbohydrate changes throughout its development.KEY WORDS: 1-FEH, 1-FFT, 1-SST, Chicory (Cichorium intybus L.), fructan, fructosyltransferase, inulin, invertase DOMAINS: agronomy, biotechnology, plant sciences, molecular biology, gene expression INTRODUCTIONFructans occur as a reserve carbohydrate in about 15% of flowering plant species [1]. Fructans are defined as oligo-or polymeric carbohydrates in which fructosyl linkages predominate [2]. They Van den Ende et al.: Fructan Enzymes in Chicory TheScientificWorldJOURNAL (2002) 2, 1281-1295 1282 are built up by further adding fructofuranosyl units (β(2→1) or β(2→6) linkages) to three basic trisaccharides (1-kestose, 6-kestose, and neokestose), which themselves are synthesized by linking a fructose moiety to one of the three primary hydroxyl groups of sucrose [3]. Dicotyledonous species store inulin-type fructan consisting of linear β(2→1) linked fructofuranosyl units. The most extensively studied species include Jerusalem artichoke (Helianthus tuberosus) and chicory (Cichorium intybus), which belong to the Asteraceae. However, inulin-type fructans also occur in some other dicot families (Boraginaceae, Campanulaceae). Levan-type fructan consists of linear β(2→6) linked fructofuranosyl units. More complex and branched fructan types, including graminan or mixed-type fructan as well as neokestose-based fructans, are common in monocotyledonous species [4]. Fructans are used for food and non-food applications [5]. So far, chicory is the only crop that is used for commercial fructan extraction. During the last decade, annual inulin production from chicory roots increased from 1,000 to more than 100,000 tons. Therefore, this review mainly focuses on inulin-type fructans and on inulin metabolism in chicory roots.Besides their function as a reserve carbohydrate, fructans might fulfill other, perhaps more specific, roles in plants. Some data suggest a correlation with drought resistance, frost tolerance, and osmoregulation [1,6,7]. The molecular mechanism explaining the protective function of fructans is still obscure. However, it has recently been demonstrated that fructans stabilize liposomes during free...

A 1‐FEH II (1‐fructan exohydrolase, EC 3.2.1.80) was purified from forced chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70‐fold purification and a specific activity of 52 nkat mg−1 protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS‐PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent Km of 58 mM for 1‐kestose (Kes) and 64 mM for 1,1‐nystose (Nys). The purified 1‐FEH II hydrolyzed the β(2?1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β(2?6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1‐FEH II activity but sucrose (Suc) was a strong inhibitor of this 1‐FEH II (Ki=5.9 mM). The enzyme was partially inhibited by Na‐EDTA and CaCl2 (1 mM).

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.