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).

Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels.

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Abstract. 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 pmol.(mg protein)-l.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 [3-fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5-50~ Therefore enzyme assays were performed at 0~ 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.Key words: Cichorium -Fructan: fructan fructosyl transferase -Fructan -Inulin -1-Kestose Sucrose 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; Sue = sucrose

A genuine 1‐SST (sucrose:sucrose 1‐fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63‐fold purification and a specific activity of 4.75 U (mg protein)−1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS‐PAGE apparent molecular masses of 49 kDa (α‐subunit) and 24 kDa (β‐subunit) were found. Further specification was obtained by MALDI‐TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS‐boiled chicory root extract and chicken‐raised polyclonal antibodies against the purified 1‐SST, indicating that the enzyme is a heterodimer in vivo. The N‐terminus of chicory root 1‐SST α‐subunit was shown to be highly homologous with the cDNA‐derived amino acid sequences from barley 6‐SFT and a number of β‐fructosyl hydrolases (in‐vertases and fructan hydrolases). However, chicory root 1‐SST properties could be clearly differentiated from those of chicory root 1‐FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1‐kes‐tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1‐SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1‐FFT and barley 6‐SFT, the enzyme also showed some β‐fructofuranosi‐dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β‐fructofuranosidase activity were also detected using 1‐kestose as the sole substrate.