Fructan Synthesis in Excised Barley Leaves (Identification of Two Sucrose-Sucrose Fructosyltransferases Induced by Light and Their Separation from Constitutive Invertases)

Abstract

Excised leaves of barley (Hordeum vulgare L.) exposed to continuous light accumulate large amounts of soluble carbohydrates. Carbohydrates were analyzed in deionized extracts by high-pressure liquid chromatography on an anion exchange column coupled with pulsed amperometric detection. During the first few hours of illumination, the main sugar to accumulate was sucrose. The levels of glucose and fructans (oligofructosylsucroses) increased later. The trisaccharide 1-kestose (1-kestotriose) predominated initially among the fructans. Later, 6-kestose (6-kestotriose) and tetra- and pentasaccharides accumulated also. Total extracts from barley leaves were chromatographed on a MonoQ column, and each fraction was assayed for enzymes of interest by incubation with 200 mM sucrose for 3 h, followed by carbohydrate analysis. Freshly excised leaves yielded two peaks of invertase, characterized by formation of fructose and glucose, but had almost no trisaccharide-forming activities. In leaves exposed to continuous light, two new enzyme activities appeared that generated fructan-related trisaccharides and glucose from sucrose. One of them was a sucrose-sucrose fructosyl-1-transferase (1-SST), producing 1-kestose exclusively: the peak fractions of this activity contained almost no invertase. The other was a sucrose-sucrose fructosyl-6-transferase (6-SST), producing 6-kestose. It comigrated with one of the constitutive invertases on MonoQ but was separated from it by subsequent chromatography on alkyl Superose. Nevertheless, the preparation retained invertase activity, suggesting that this enzyme may act both as fructosidase and fructosyltransferase. When incubated with 1-kestose in addition to sucrose, this enzyme formed less 6-kestose but instead produced large amounts of the tetrasaccharide bifurcose (1&6-kestotetraose), the main fructan tetrasaccharide accumulating in vivo. These results suggest that two inducible enzymes, 1-SST and 6-SST, act in concert to initiate fructan accumulation in barley leaves.