The mechanism of sugar uptake by sugarcane suspension cells

The accumulation of tetraphenylphosphonium (TPP@), 5,5'-dimethyloxazolidine-2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPPF accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells.Kinetic analyses of sugar uptake into protoplasts isolated from a rapidly growing soybean cotyledon revealed that at low external sugar concentrations, sucrose is preferentially transported over glucose (14,22). This difference in sugar uptake is mainly due to the presence of a saturable uptake component for sucrose but not for glucose (14,22). The responses of sucrose uptake into protoplasts (14,22) and excised intact soybean cotyledons (9,26) to the external pH, temperature, and several metabolic inhibitors suggested a proton cotransport mechanism in sucrose uptake, but the evidence was circumstantial at best. Electrogenic sucrose/proton transport predicts that the addition of sucrose could cause (a) a transient depolarization of the membrane potential because of the initial influx of the charged proton, (b) a transient acidification of the internal cytoplasm resulting from proton entry, and (c) alkalinization of the external medium due to proton disappearance from the medium. These changes should both be transient (because of the re-establishing of the proton gradient by an active proton pump) and specific for sucrose.In this study, we used TPP+2 (2,6,7,12,(18)(19)(20)(21)27), DMO (3,6,24,25), and external pH monitor (6-8, 17, 18, 31) plasts isolated from developing soybean cotyledons. The stoichiometry of sucrose and proton in the system was also estimated. The results strongly support a sucrose/proton cotransport in soybean cotyledons. MATERIALS AND METHODSProtoplasts were isolated from developing soybean (Glycine max L. Merr cv Wye) cotyledons as described previously (14) and suspended for all experiments in a basic medium of 0.5 M sorbitol, 10 mM CaCl2, and 25 mM Mes-KOH (pH 6.0). Sucrose uptake into the protoplasts was also according to Lin et al. (14) except as noted.The electric potential difference between the protoplasts and the surrounding medium (membrane potential of the protoplast) was calculated using the Nernst equation (15) from the distribution of TPP+ between protoplasts and medium. After incubation with labeled [3H]TPP+ (diluted with 8 gM unlabeled TPP+, final specific radioactivity 125 gCi/mmol) for various time...

Section: Methodssupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Energetics of Sucrose Transport into Protoplasts from Developing Soybean Cotyledons

Lin¹

1985

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…2B), and had a similar pH optimum (about 5.5). However, since protoplasts from sugarcane suspension cultures do not take up sucrose (6), a comparison of vacuoles with protoplasts was not possible in this case.…”

Section: Methodsmentioning

confidence: 99%

Vacuoles from Sugarcane Suspension Cultures

Thom

Komor²,

Maretzki³

1982

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...

“…A strong line of evidence for the involvement of a H+-sugar co-transport is the sugar-induced rapid and transient alkalinization of the incubation solution (10,14). In contrast to sucrose (3) Facilitated diffusion ofglucose, commonly observed in animal systems (24) has not been clearly shown to occur in higher plants (13).…”

mentioning

confidence: 99%

Facilitated Transport of Glucose in Isolated Phloem Segments of Celery

Daie¹,

Wilusz²

1987

In isolated phloem segments of celery (Apium graveolns L.), a tissue highly specific for sucrose and mannitol uptake, glucose uptake occurs at very low rates and exhibits biphasic kinetics. Nonpenetrating inhibitors such as parachloromercuribenzene sulfonic acid did not inhibit glucose uptake. However, uptake was greatly inhibited by penetrating inhibitors such as N-ethylmaleimide and carbonylcyanide-m-chlorophenyl hydrazone. Carbonylcyanide-m-chlorophenyl hydrazone inhibition of uptake was reversed by washing and addition of thiol reagents to uptake solutions. Phlorizin, a competitive inhibitor of glucose caused moderate inhibition of uptake only after 3 hours of tissue exposure. Low pH, fusicoccin, and low turgor which enhance H+-sugar cotransport did not alter uptake rates. Furthermore, glucose did not induce alkalinization of the uptake media. Efflux analysis indicated that the presence of 50 millimolar unlabeled glucose in the wash media enhanced exchange of the labeled glucose across the tonoplast. Results indicate that the glucose carrier is not located at the plasmalemma but appears to be present at the membrane of an intracellular compartment, most likely the tonoplast. Carrier-mediated glucose transport in this tissue is proposed to be a facilitated diffusion.