Regulation of Sucrose-Sucrose-Fructosyltransferase in Barley Leaves

Abstract

The activity of sucrose-sucrose-fructosyltransferase (SST), a vacuolar enzyme strongly induced by light in excised leaves of barley (Hordeum vulgare L.), rapidly declined even in continuous light upon feeding of cycloheximide (CHI). The rate of decline was similar to that observed in light-treated leaves that were placed into darkness, in the presence or absence of CHI. The protease inhibitor leupeptin totally stopped the decline in SST activity in the dark and caused a substantial increase in the rate of induction of SST activity by light. Feeding of sucrose prevented or even reversed the SST activity decay induced by darkness in the absence of CHI but did not stabilize SST activity in the presence of CHI. The results suggest that SST is continuously subjected to rapid, constant proteolytic degradation in the vacuole, and that the enhancement of SST activity in the light or upon feeding sucrose in the dark is due exclusively to de novo protein synthesis.Fructans are polymers of fructose with a terminal glucose that function as major vacuolar storage carbohydrates in the vegetative organs of many important crop plants, most notably the forage grasses and cereals (9, 1 1). SST2, the first enzyme in the pathway of fructan synthesis, catalyzes the formation of the trisaccharide isokestose from two molecules of sucrose (9,11,14). The isokestose is then utilized by fructan-fructanfructosyltransferases to form fructans of varying degrees of polymerization.In previous work, barley leaves were used to study regulation of fructan biosynthesis. SST emerged as a key regulatory enzyme because SST activity and the rate of fructan synthesis were closely correlated under various conditions (1 1-13), and because SST activity increased rapidly upon feeding of sugars (14) or treatments that cause accumulation of photosynthate, such as illumination of excised leaves ( 13) or cold treatments (8,12), and decreased equally rapidly upon treatments lowering the photosynthate level (13). It has been suggested that a threshold level ofsucrose in the tissue is the key for induction of SST and fructan biosynthesis (5,6,13,15).Like the fructans, SST is localized in the vacuole (4, 12). The rapid changes in its activity are a paradigm of the dynam-' Supported by the Swiss National Science Foundation.2Abbreviations: SST, sucrose-sucrose-fructosyltransferase; CHI, cycloheximide.ics of the vacuole (2), but little is known about the regulatory mechanisms involved. Inhibitor studies have provided evidence that induction ofSST occurs at the level oftranscription and requires protein synthesis (3,13). However, the decay of SST activity is not affected by CHI, an inhibitor of protein synthesis, indicating that protein synthesis is not necessary for down-regulation of the enzyme (13).Here, we further exploit excised barley leaves as a model system and present evidence that SST is continuously degraded by leupeptin-sensitive proteinases. Regulation appears to occur exclusively by changes in the rate of its synthesis on the background of rapi...

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

“…Based on this fact and on detailed work with barley leaves (Wagner et al, 1983;Wagner and Wiemken, 1987;Obenland et al, 1991), our group previously suggested SST to be the key enzyme for fructan accumulation in the grasses also, and we reported the partia1 purification of two SST activities from barley protoplasts, one producing 1 -kestose and the other 6-kestose (Wagner and Wiemken, 1987). However, these studies were subject to recent criticism (Cairns et al, 1989;Cairns and Ashton, 1991;Pollock and Cairns, 1991;Winters et al, 1992), which focused on two main points.…”

Fructan exohydrolase from grasses

“…During periods of fructan accumulation, SST activity increases Cairns & Pollock, 19886;Smouter & Simpson, 19916;Obenland et al, 1991) and the evidence indicates that FEH activity in some grasses may be reduced significantly. The concentration of sucrose in leaves and shoots of Lolium spp.…”

“…Although FEH activities in tissues from both seedlings and reproductive plants of L. rigidum are similar, rates of invertase activity vary considerably, being highest in young tissues and lowest in mature plant parts (Bonnett & Simpson, 1993). If the shoot tissues of grasses have been induced to accumulate fructans, the activity of sucrose-sucrose fructosyl transferase (SST) is induced to relatively high levels (Wagner, W'iemken & Matile, 1986;Cairns & Pollock, 19886;Obenland et al, 1991) and SST and FEH may be present simultaneously Smouter & Simpson, 1991 b). This situation does not occur in specialized storage organs of some species because SST and FEH are not active at the same time.…”

Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan‐polymerizing enzyme preparation

“…Its accumulation triggers the onset of fructan synthesis, it provides the substrate for polymerization (Cairns & Pollock, 1991) and there is evidence that it stabilizes the enzyme(s) of fructan synthesis against proteolytic degradation, a proposed feature of the metabolic control of fructan metabolism (Obenland et al, 1991, Cairns & Ashton, 1994. The presence of sucrose in vitro stabilizes small fructans against hydrolysis and is crucial to their accumulation.…”