Vacuoles from Sugarcane Suspension Cultures

Abstract

Vacuoles, isolated from sugarcane (Saccharum sp.) cells, took up 3-0 methylglucose and sucrose and the evidence suggests specific transport systems for these sugars. There was no evidence of sugar efflux from preloaded vacuoles. Vacuoles in situ accumulated 3-0 methylglucose, sucrose, glucose, and fructose, as shown by incubation of protoplasts with labeled sugar and subsequent analysis of vacuolar and cytoplasmic radioactivity. During the initial minutes of incubation, the amount and concentration of labeled sugar was higher in the cytoplasm than in the vacuole, but subsequently there was active uptake and accumulation into the vacuole. The rate of hexose transfer into the vacuole in situ approached that of hexose uptake by isolated vacuoles; however, the rate of sucrose uptake by isolated vacuoles was below the in situ rate. The site of sucrose synthesis was in the cytoplasm.The importance of uptake and accumulation of sugars into vacuoles of plant cells has long been recognized but only recently have methods become available to study this organelle. In sugarcane, where vacuoles accumulate the economically important product, sucrose, the process of loading and storage of sugars is of particular interest.The uptake of sucrose into vacuoles from red beets (2, 1 1) and of 3-OMG:3 into vacuoles from pea leaf mesophyll cells (4) has recently been reported. Evidence for a specific transport mechanism in isolated vacuoles was shown in both instances. In addition, arginine transport across the tonoplast has been characterized in isolated yeast vacuoles (1), and transport of basic amino acids has been studied in vacuoles from Hevea latex (5).For the present investigation, two approaches were used to study sugar accumulation into vacuoles isolated from cell suspensions. One method involved the direct measurement of sugar uptake into isolated vacuoles; the other method involved the measurement of sugar accumulation by vacuoles in situ; results from the two methods were compared. MATERIALS AND METHODSPlant materials used and procedures for isolation of protoplasts and vacuoles were described previously (10). In Vitro Uptake of Sugars. Cells from 8-d-old cultures were depleted of sugars for 18 h before enzymic release of protoplasts. During this period, sucrose, glucose, and fructose concentrations were reduced by about 50%. The sugar uptake system changed from a steady state to a net uptake state with concomitant changes in uptake kinetic parameters (6). Vacuoles were isolated and resuspended in White's basal salt medium (7) containing 0.4 M mannitol and the solution was adjusted to pH 5.5. The vacuole suspension (5 ml) was transferred to a 25-ml Erlenmeyer flask and the flasks were placed on a rotary shaker (100 rpm) at 25°C. Uptake was initiated by the addition of 14C sugars. One-ml aliquots were withdrawn at intervals (usually 1, 3, 5, and 7 min after addition of substrate) and layered over a silicone oil mixture AR 20:AR200 (SWS Silicones Corp., Adrian, MI). Vacuoles were separated from the incubation medium b...

Influence of Mannose on the Apoplasmic Retrieval Systems of Source Leaves

“…At the end of a 30-min uptake period only 5% of the label resided in the 'cytosolic compartment' of sugar beet source leaves. This result is similar to the findings of Thom et al (29) for their study of efflux of sugars from cultured sugar cane suspension cells. They reported that while most of the label was in the cytoplasm following the initial minutes ofuptake, the majority oflabel was subsequently located in the vacuole.…”

A Novel Arabidopsis Vacuolar Glucose Exporter Is Involved in Cellular Sugar Homeostasis and Affects the Composition of Seed Storage Compounds

“…Hence, vacuoles provide additional space for transient and long-term storage of sugars (Martinoia et al, 2007). In support of this aspect, several studies have provided biochemical evidence for the uptake of both Suc and hexoses into vacuoles (Thom et al, 1982;Rausch, 1991;Keller, 1992), and both passive diffusion and active transport have been determined as uptake mechanisms for these sugars (Thom and Komor, 1984;Martinoia et al, 1987Martinoia et al, , 2000. Furthermore, determination of subcellular sugar concentrations in a variety of plants via nonaqueous fractionation (Gerhardt and Heldt, 1984) revealed that in leaves, Glc is predominantly found in the vacuole, while Suc is mainly found in the cytosol or is evenly distributed (Wagner, 1979;Heineke et al, 1994;Pollock et al, 2000;Voitsekhovskaja et al, 2006).…”

“…While considerable effort has been expended in elucidating the flux of carbon in and out of the starch pool in chloroplasts (Zeeman et al, 2007), our current knowledge of carbon allocation in the vacuole and the corresponding transport steps is rather limited. However, understanding the details of vacuolar sugar partitioning is of major relevance, since several agriculturally important plants, like sugar beet (Beta vulgaris; Doll et al, 1979;Briskin et al, 1985;Getz, 1991;Getz and Klein, 1995) and sugarcane (Saccharum officinarum; Thom et al, 1982), accumulate considerable amounts of sugars in the vacuoles of storage organs.…”

A Group Translocator for Sucrose Assimilation in Tonoplast Vesicles of Sugarcane Cells

“…The transport of sugars can proceed not only across the plasmalemma but also across the tonoplast (5). Sugarcane cells grown in suspension cultures utilize a proton-antiport mechanism to accumulate glucose across the tonoplast (9), but the rate of sucrose uptake by this mechanism is marginal and cannot account for the rate at which sucrose appears inside the vacuole (8). While exogenously added MgATP stimulates sucrose uptake into red beet vacuoles (2), a similar stimulation in sugarcane vacuoles has not been observed.…”