Transgenic potato tubers accumulate high levels of 1‐kestose and nystose: functional identification of a sucrose sucrose 1‐fructosyltransferase of artichoke (Cynara scolymus) blossom discs

Abstract

SummaryBy screening a cDNA library of artichoke (Cynara scolymus} blosssom discs for fructosyltransferases, we isolated a clone designated Cy21. The deduced amino acid sequence shows homology to acid J~-fructosyl hydrolases and to the sucrose-fructan 6-fructosyltransferase (6-SFT) of barley. Transiently expressed in Nicotiana tabacum

Transgenic potato ( Solanum tuberosum ) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke ( Cynara scolymus ) roots

“…performance liquid anion exchange chromatography (HPAEC) using a CarboPac PA-100 column on the Dionex DX-500 gradient chromatography system (17). Leaf and tuber discs were homogenized in liquid nitrogen and then extracted twice with ethanol at 80°C for 30 min.…”

“…Désirée) and analyzed the inulin pattern of leaves and tubers. In a first transformation step, a chimeric construct containing the 1-SST gene (17) under the control of the constitutive cauliflower mosaic virus 35S promoter was introduced into wild-type potato plants. In 19 of 41 antibiotic-resistant plants 1-SST mRNA accumulation and oligofructan synthesis could be observed.…”

Transport of 1-kestose across the tonoplast of Jerusalem artichoke tubers

“…This is despite the fact that genetically modified tissues with 1-SST have been able to synthesize 1-kestose (Hellwege et al, 1997;Vijn et al, 1998). Synthesis of 1-kestose in the cytosol and transport into the vacuole offered an appealing possibility in light of the capacity of sucrose carriers to transport other related molecules (Keller, 1992;Greutert and Keller, 1993).…”

“…This step is catalyzed by sucrose:sucrose fructosyl transferase (1-SST; EC 2.4.1.99), a vacuolar enzyme isolated and purified from a wide variety of tissues (Pollock and Suzuki, 1986;Praznik et al, 1990;Angenent et al, 1993;Koops and Jonker, 1996;. Although demonstrated both in vivo (Cairns et al, 1989;Hellwege et al, 1997) and in vitro (Bancal and Gaudillere, 1993;Cairns, 1993;Penson and Cairns, 1994;Lu¨scher et al, 1996;Itaya et al, 1997;Vijn et al, 1998), synthesis of 1-kestose by 1-SST has remained ambiguous in light of the enzyme's unusually high K m for sucrose which varies from 75 mM up to over 500 mM depending on the tissue investigated and assay conditions (Chatterton et al, 1988;Cairns and Ashton, 1994). For example, in developing Jerusalem artichoke tubers, the K m for sucrose has been estimated to be approximately 500 mM (Koops and Jonker, 1996).…”

“…The in vivo synthesis of 1-kestose by transformed potato tubers (Hellwege et al, 1997) was facilitated by the higher vacuolar sucrose concentration and by long term incubation. Based on these observations, his results, and those of others, Cairns (1999) suggested that 1-kestose may be synthesized elsewhere and transported into the vacuole where elongation of fructan chains takes place.…”