Metabolisme van galactosyl-oligosachariden in de Caryophyllaceae.

The raffinose family oligosaccharides (RFOs) are the most widespread galactosyl-sucrose oligosaccharides in the plant kingdom. Raffinose (Gal-α(1→6)-Glc-α(1→2)ß-Fru), the smallest member of the family, can be further elongated by the additional transfer of galactosyl residues to the galactose of raffinose by α(1→6) linkages. In the Caryophyllaceae, however, these typical α(1→6) oligomers of raffinose appear to be replaced by other types of galactosyl-oligosaccharides (GOS). During this project, the structure and metabolism of these GOS were further elucidated using Stellaria media, a typical representative of the Caryophyllaceae. Next to raffinose the tetrasaccharide lychnose, an α-(1→1) galactosyl extension on the fructose of raffinose, is the predominant GOS in S. media. Its structure was unambiguously determined by biochemical approaches and NMR. Lychnose synthesis occurs by lychnose synthase, a raffinose:raffinose 1Fru galactosyltransferase, using only raffinose both as donor and acceptor substrate. After partial purification of the native enzyme, lychnose synthase was characterized, cloned and heterologously expressed. Although concentrations up to 375 mM were used, no saturation could be observed. Sucrose inhibits the biosynthesis reaction which is optimal at pH 5.4 - 6.0. The two other GOS accumulating in S. media are mediose, an α(1→4) galactosyl extension on the glucose of raffinose and stellariose, an α(1→4) galactosyl extension on the glucose of lychnose. Mediose and stellariose are both synthesized by a raffinose:lychnose 4Glc galactosyltransferase or stellariose synthase, which catalyzes the direct transfer of a galactose residue of raffinose to raffinose or lychnose, respectively. This enzyme however, was neither characterized nor cloned. Most likely, these GOS are subject to partial trimming by endogenous acid and alkaline α‑galactosidases. Lychnose can be broken down into the trisaccharide Glc-α(1→2)ß-Fru-α(1→1)-Gal, which structure was determined by NMR. An alkaline α-galactosidase of Silene dioica was cloned and heterologously expressed in E. coli. Finally, S. media plants were grown and acclimated (4°C) under laboratory conditions to see whether lychnose metabolism affects its frost tolerance. During low temperature acclimation a consecutive, temporal increase in the raffinose and lychnose concentration could be observed, resulting in a distinct positive correlation with increased freezing tolerance.status: publishe