DNA polymerase activity in embryos of Allium cepa seed

A low level of DNA polymerase (deoxyribonucleosidetriphosphate: deoxynucleotidyltransferase; E.C. 2.7.7.7) activity can be detected by autoradiography after incubating frozen sections of unimbibed onion seed embryos in an aqueous solution of tritium labelled deoxynucleoside triphosphate. The enzyme is sensitive to sulfhydryl reagents, and incorporation was reduced by high levels of deoxyribonuclease, but inclusion of cycloheximide, ribonuclease, or nucleoside triphosphate did not change activity. On germination, activity remained relatively constant and at a low level until 30 hr after imbibition when it began to increase significantly.     

Oligosaccharide intermediates of fructan synthesis in Lolium temulentum

Evidence is presented that fructan accumulation in leaves of Lolium temulentum plants grown at 5° proceeded via the synthesis of trisaccharide intermediates. Studies on the oligosaccharide components of this tissue indicated that the major intermediate was probably 1^F-fructosyl sucrose (isokestose) but that two distinct series of oligofructosides could be isolated. One of these had chromatographic properties identical to the 1,2-linked inulin series from Helianthus tuberosus. The relationship of this synthetic pattern to the structure of grass fructans and their accumulation in other species is discussed.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Turnover des flavonols dans les tuniques d'Allium cepa en survie

During ageing of isolated onion scales there is an accumulation of flavonols which increase continuously. This is not light-dependent but is associated with a high level of respiratory gaseous exchanges. Phenylalanine-[U-¹⁴C] is actively incorporated in these flavonols but experiments of pulse labelling show that they have either a very slow or no turnover. This suggests that enzymes able to destroy flavonols are lacking in this material.     

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.     

Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose:fructan 6-fructosyltransferase

We have recently cloned a cDNA encoding sucrose:fructan 6-fructosyltransferase (6-SFT), a key enzyme of fructan synthesis forming the β-2,6 linkages typical of the grass fructans, graminans and phleins [Sprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11652–11656]. Here we report functional expression of 6-SFT from barley in transgenic tobacco and chicory. Transformants of tobacco, a plant naturally unable to form fructans, synthesized the trisaccharide kestose and a series of unbranched fructans of the phlein type (β-2,6 linkages). Transformants of chicory, a plant naturally producing only unbranched fructans of the inulin type (β-2,1 linkages), synthesized in addition branched fructans of the graminan type, particularly the tetrasaccharide bifurcose which is also a main fructan in barley leaves.     

C-galactosylflavones from Polygonatum multiflorum

8-C-Galactosylapigenin and 6-C-galactosyl-8-C-arabinosylapigenin were isolated from the leaves of Polygonatum multiflorum (L.) All. Structural assignments for the latter compound were made on the basis of mass, CD and ¹³C-NMR spectra.     

Molecular characterization and expression of a cDNA encoding fructan:fructan 6G-fructosyltransferase from asparagus (Asparagus officinalis)

* Fructan:fructan 6G-fructosyltransferase (6G-FFT) catalyses a transfructosylation from fructooligosaccharides to C6 of the glucose residue of sucrose or fructooligosacchrides. In asparagus (Asparagus officinalis), 6G-FFT is important for the synthesis of inulin neoseries fructan. Here, we report the isolation and functional analysis of the gene encoding asparagus 6G-FFT. * A cDNA clone was isolated from asparagus cDNA library. Recombinant protein was produced by expression system of Pichia pastoris. To measure enzymatic activity, recombinant protein was incubated with sucrose, 1-kestose, 1-kestose and sucrose, or neokestose. The reaction products were detected by high performance anion-exchange chromatography. * The deduced amino acid sequence of isolated cDNA was similar to that of fructosyltransferases and vacuolar type invertases from plants. Recombinant protein mainly produced inulin neoseries fructan, such as 1F, 6G-di-beta-D-fructofuranosylsucrose and neokestose. * Recombinant protein demonstrates 6G-FFT activity, and slight fructan:fructan 1-fructosyltransferase (1-FFT) activity. The ratio of 6G-FFT activity to 1-FFT activity was calculated to be 13. The characteristics of the recombinant protein closely resemble those of the 6G-FFT from asparagus roots, except for a difference in accompanying 1-FFT activity.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.     

Sesquiterpene lactones,flavonoids and anthraquinones from Asphodeline globifera and Asphodeline Damascena

The known compounds chrysoeriol, apigenin, luteolin, acacetin, scutellarein, 6-methoxyluteolin, apigenin 7-glucoside, luteolin 7-glucoside, esculetin, chrysophanol, asphodeline, mircocarpin, sitosterol, 1-β-acetoxyeudesman-4(15),7(11)dien-2α,12-olide and 1-β-acetoxy-8β-hydroxyeudesman-4(15),7(11)-dien-8α,12-olide were isolated from Asphodeline globifera and A. damascena. A new sesquiterpene lactone 1-β-acetoxy-8β-ethoxyeudesman-4(15),7(11)dien-8α, 12-olide was also characterized. These are the first reports of sesquiterpene lactones in Asphodeline and in the Liliaceae.