Andries J. Koops

Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus colombia. II. Purification of sucrose:sucrose 1-fructosyltransferase and reconstitution of fructan synthesis in vitro with purified sucrose:sucrose 1-fructosyltransferase and fructan-fructan 1-fructosyltransferase

Sucrose:sucrose 1-fructosyltransferase (1-SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianthus tuberosus that were harvested in the accumulation phase. Gel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27- and a 55-kD fragment. 1-SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1-kestose (GF2). Other reactions catalyzed by 1-SST at a lower rate were self-transfructosylations with GF2 and 1,1-nystose (GF3) as substrates yielding GF3 and 1,1,1-fructosylnystose, respectively, as products. 1-SST also catalyzed the removal of the terminal fructosyl unit from both GF2 and GF3, which resulted in the release of sucrose and GF2, respectively, and free Fru. The purified enzyme did not display [beta]-fructosidase activity. An enzyme mixture of purified 1-SST and fructan:fructan 1-fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.

Uptake of [14C]sucrose in isolated minor-vein networks of Commelina benghalensis L

Maceration with pectinase (4.5h) of Commelina benghalensis L. leaves stripped at either side yielded isolated vein networks consisting of four to five secondary veins and tertiary cross veins (=minor veins). Examination with Evans Blue and injection of Fluorescein F showed that 80% of the veins were viable. Proof of normal functioning of isolated minor veins was that [14C]sucrose fed to an apical vein network attached to the remaining intact part of the leaf was absorbed and finally arrived in the petiole. Sucrose uptake by veins obeyed Michaelis-Menten kinetics (Km 5·10-4 mol l-1; Vmax (light) 3.2 μmol h-1 g-1 fresh weight, Vmax (dark) 1.5 μmol h-1 g-1 fresh weight). A linear component, not inhibited by carbonylcyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid, was present. Maximal uptake took place at 5 mmol l-1 K+; concentrations of K+ higher than 10 mmol l-1 decreased the rate of uptake. The uptake rates by isolated veins and veins in situ (in disks) were in the same order of magnitude. Altogether, isolated veins promise to be a useful system for the study of loading.

The composition of phytosterols, latex triterpenols and wax triterpenoids in the seedling of Euphorbia lathyris L.

Abstract In the etiolated seedling of Euphorbia lathyris L., three groups of triterpenoids were found, located at different sites. The extracted triterpenoids were purified by TLC and HPLC and identified by GC/MS. Latex consisted mainly of triterpene alcohols. From these, guimarenol, 24-CH3-lanosterol and hopenol-B were not earlier identified in E. lathyris. The epicuticular wax contained mainly triterpene ketones, which were identified as taraxeron, hopenone-I, hopenone-II, hopenone-B, 3-oxo-22-hydroxyhopane, simiarenone and fernenone. Other components of the epicuticular wax were triterpenols, n-alkanes and aliphatic alcohols. The phytosterols were identified as cholesterol, campesterol, stigmasterol, sitosterol and isofucosterol.

Triterpenoid biosynthesis in the seedling of Euphorbia lathyris L. from sucrose and amino acids

Abstract The triterpenoid biosynthesis from sucrose and several amino acids was investigated with 9-day-old Euphorbia lathyris seedlings. Tracer experiments with [U-14C]sucrose demonstrated that sucrose was mainly incorporated into latex triterpenols. Several [U-14C]amino acids were found to be efficient precursors in the synthesis of sterols and the wax triterpene ketones. The incorporation data of sucrose and amino acids into triterpenoids together with the daily uptake of these substrates from the endosperm were used to estimate the daily triterpenoid production from each substrate species. Sucrose was quantitatively the most important precursor in triterpenoid biosynthesis. About 10% of the sterols and wax triterpenoids were produced from the amino acids.

Triterpenoid biosynthesis in the etiolated seedling of Euphorbia lathyris L.. Developmental changes and the regulation of local triterpenoid production

Summary Intact seedlings of Euphorbia lathyris were used to measure the daily production of sterols, latex triter-penols and wax triterpene ketones, and to estimate the participation of sucrose and threonine in the synthesis of these lipids. In the early stage of seedling development, 3–4 days after seed imbibition (DAI), the biosynthesis of these triterpenoids is limited by enzymatic production capacity. From 5 DAI onwards, the triterpenoid production seemed to be restricted by the availability of sugars. Threonine played only a marginal role in the synthesis of triterpenoids. From 5-8 DAI, most of the triterpenoids are synthesized directly from sucrose taken up from the endosperm by the cotyledons. From 8–12 DAI, sugars from the hypocotyl and root became another source of carbon for triterpenoid synthesis. In 9-day-old seedlings, the production of latex triterpenols and triterpene ketones was most prominent in the rapidly elongating parts of the hypocotyl. The activity of sterol synthesis was most pronounced in the root tip.

The Quantitative Contribution of Sucrose and Threonine to Triterpenoid Production in the Etiolated Seedling of Euphorbia lathyris L.

Summary Seedlings, 9–10-days old, were used to estimate their potential to synthesize sterols, latex triterpenols and wax triterpene ketones from sucrose and threonine. The endosperms of these seedlings were removed and replaced by solutions with various concentrations of 14 C-sucrose or 14 C-threonine. The triterpenoid synthesis from these substrates was measured after 24 h of incorporation. The maximum production of triterpenols and triterpene ketones from exogenously supplied sucrose in such a seedling matched the daily synthesis of these triterpenoids in the seedling with endosperm. About 50% of the daily triterpenoid synthesis is derived from sucrose, in the production period taken up by the cotyledons from the endosperm or a substituting solution. The other part was mainly produced from sugars deposited in the seedling in previous days of uptake. The potential of the seedling to synthesize triterpenoids from externally supplied threonine is less than 10 % of that from sucrose. In the intact seedling, only a small fraction of the potential production capacity from threonine is used for triterpenoid synthesis.