Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1‐FFT cDNAs
Abstract:A newly isolated cDNA clone, Cy3, encoding the fructan fructan 1-fructosyltransferase (1-FFT) from artichoke was expressed using tobacco protoplasts as expression system. Analysis of the inulin molecules synthesized upon incubation of protoplast extracts with a mixture of oligofructans (DP3^5) shows the production of inulins with a degree of polymerization (DP) of up to 23, whereas parallel experiments performed using a 1-FFT cDNA from Jerusalem artichoke led to the production of fructans with a DP of up to on… Show more
“…Alternatively, distinct enzymatic properties should be taken into account. We have reported that 1-FFT of globe artichoke shows a preference for higher DP substrates as acceptor of transferred fructosyl residues than does the enzyme from Jerusalem artichoke (18). Under conditions where high invertase activity prevents accumulation of lower molecular weight inulin molecules, which are the main products formed by the H. tuberosus 1-FFT, the C. scolymus enzyme would be more efficient in synthesizing higher-order inulin molecules, which are less accessible to invertases (31).…”
Section: Accumulation Of High Molecular Weight Inulin In Plants Exprementioning
confidence: 93%
“…The 1-SST (17) and 1-FFT cDNAs (18) were subcloned separately into the binary vector pBinAR, a derivative of the pBin19 vector (19). The cDNAs contain endogenous signal sequences for a predicted vacuolar localization of the encoded enzymes.…”
Section: Methodsmentioning
confidence: 99%
“…In 19 of 41 antibiotic-resistant plants 1-SST mRNA accumulation and oligofructan synthesis could be observed. Of those we chose three transgenic lines and transformed them with the 1-FFT gene (18), again under the control of the 35S promoter. We obtained 10 transgenic lines accumulating high molecular weight inulin.…”
Section: Inulin Synthesis In Transgenic Potatoesmentioning
The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.
“…Alternatively, distinct enzymatic properties should be taken into account. We have reported that 1-FFT of globe artichoke shows a preference for higher DP substrates as acceptor of transferred fructosyl residues than does the enzyme from Jerusalem artichoke (18). Under conditions where high invertase activity prevents accumulation of lower molecular weight inulin molecules, which are the main products formed by the H. tuberosus 1-FFT, the C. scolymus enzyme would be more efficient in synthesizing higher-order inulin molecules, which are less accessible to invertases (31).…”
Section: Accumulation Of High Molecular Weight Inulin In Plants Exprementioning
confidence: 93%
“…The 1-SST (17) and 1-FFT cDNAs (18) were subcloned separately into the binary vector pBinAR, a derivative of the pBin19 vector (19). The cDNAs contain endogenous signal sequences for a predicted vacuolar localization of the encoded enzymes.…”
Section: Methodsmentioning
confidence: 99%
“…In 19 of 41 antibiotic-resistant plants 1-SST mRNA accumulation and oligofructan synthesis could be observed. Of those we chose three transgenic lines and transformed them with the 1-FFT gene (18), again under the control of the 35S promoter. We obtained 10 transgenic lines accumulating high molecular weight inulin.…”
Section: Inulin Synthesis In Transgenic Potatoesmentioning
The ability to synthesize high molecular weight inulin was transferred to potato plants via constitutive expression of the 1-SST (sucrose:sucrose 1-fructosyltransferase) and the 1-FFT (fructan: fructan 1-fructosyltransferase) genes of globe artichoke (Cynara scolymus). The fructan pattern of tubers from transgenic potato plants represents the full spectrum of inulin molecules present in artichoke roots as shown by high-performance anion exchange chromatography, as well as size exclusion chromatography. These results demonstrate in planta that the enzymes sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1-fructosyltransferase are sufficient to synthesize inulin molecules of all chain lengths naturally occurring in a given plant species. Inulin made up 5% of the dry weight of transgenic tubers, and a low level of fructan production also was observed in fully expanded leaves. Although inulin accumulation did not influence the sucrose concentration in leaves or tubers, a reduction in starch content occurred in transgenic tubers, indicating that inulin synthesis did not increase the storage capacity of the tubers.
“…11,16) Sprenger et al 16)ˆr st reported the cloning of a plant gene of the fructan biosynthesis enzyme 6-SFT from Hordeum vulgare. Since then, several genes encoding enzymes in fructan biosynthesis have been cloned from various plants: 1-SST from C. intybus; 19) 1-SST and 1-FFT from Cynara scolymus 20,21) and from H. tuberosus; 22) 1-SST and 6G-FFT from Allium cepa; 18,23) and 1-SST from Festuca arundinacea; 24) and 6-SFT from Agropyron cristatum. 12) We have identiˆed cDNAs for 1-SST and 6-SFT from wheat, and this is theˆrst report of cloning 1-SST and 6-SFT from the same plant.…”
performance anion exchange; PAD, pulsed amperometric detector; DP, degree of polymerization; LT 50 , temperature at which 50z of plants are killed by freezing; Wft1, wheat fructosyltransferase 1; Wft2, wheat fructosyltransferase 2
“…Fungal as well as plant genes have successfully been expressed in yeast systems (20,31), and several plant fructosyltransferases have been expressed in transformed plants or protoplasts (7,16,38,39,47). Because plant fructosyltransferases are vacuolar enzymes (9), it can be assumed that posttranslational modification is necessary for enzymatic function and thus prevents functional expression in bacterial systems.…”
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