Disaccharide-mediated regulation of sucrose : fructan-6-fructosyltransferase, a key enzyme of fructan synthesis in barley leaves

Previous work has indicated that sugar sensing may be important in the regu lation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fruc tosyltransferase (1-SST; EC 2.4.1.99) and Suc:fructan-6-fructosyltransferas e (6-SFT; EC 2.4.1.10) were analyzed. Upon supply of Sue, fructan pool size s increased markedly. Within 24 h, 1-SST activity was stimulated by a facto r of three and 6-SFT-activity by a factor of more than 20, compared with co ntrol leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increase d in the presence of the invertase inhibitor 2,5-dideoxy-2,5-imino-D-mannit ol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose st imulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc -alpha-1 and 1-alpha-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxyl amine A, a potent trehalase inhibitor. Tre by itself, however, in the prese nce or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized , such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fruct an synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-M an isomerase (EC 5.3.1.8) activity was detected. These results indicate tha t the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Sue, and also that t he structurally related disaccharide Tre can replace Suc as a regulatory co mpound.