Margaret Thom

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the Km value was higher (70 μM) than for net influx (24 μM); the exchange efflux is proposed to be mediated by the same transport system with a Km value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H+ per sugar; during the onset of hexose transport there was a K+ exit of 0.94 K+ per sugar for charge compensation. (It was assumed that the “real” stoichiometries are 1 H+ and 1 K+ per sugar.) The Km values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.

Nutrient uptake and accumulation by sugarcane cell cultures in relation to the growth cycle

Growth characteristics and nutrient changes in medium and cells of batch-grown sugarcane cultures were investigated over a period of 14 days. Amino acids, PO43− and K+ were substantially removed from the medium during the first seven days of culture; a strong preference for uptake of organic nitrogen over inorganic nitrogen was observed. Sodium uptake increased during the time when K+ was becoming deficient in the medium. The main anions taken up were SO42− and PO43−. Strong acidification and a virtually total extracellular hydrolysis of sucrose in the medium during the first seven days of culture were also observed.Tapering off of the rapid growth phase was accompanied by an increase of intra-cellular sucrose and a decrease of intracellular protein. As cells went from rapid growth into stationary phase, cytoplasmic space of the cells decreased slightly in favor of vacuolar space. Overall cell volume stayed constant throughout the growth cycle, except during a short period before onset of rapid growth.Transport of the glucose analog 3-O-methyl glucose remained constant in terms of Km value but the Vmax was slightly higher in rapidly growing cells.

H+-sugar antiport as the mechanism of sugar uptake by sugarcane vacuoles

The uptake of 3‐O‐methylglucose (3‐OMG) by vacuoles isolated from sugarcane cells caused a depolarization of the internally positive membrane potential and a decrease of the pH gradient. The uptake of sugar was accompanied by a corresponding efflux of protons out of vacuoles with a stoichiometry of 1:1. The data support a model whereby sugar is transported via an H+‐antiport system.

Tissue Distribution and Characterization of Sucrose Synthase Isozymes in Sugarcane

Summary The two isozymic forms of sucrose synthase in sugarcane have been investigated to determine their respective tissue distribution in the plant and differences in their biochemical characteristics. Separation of the isozymic forms was aided by the application of immuno-separation for one form (SS2), whereas the other was purified from older internode tissue in which only one form is present. All other tissue examined contained both forms of this enzyme. The two isoforms of sucrose synthase from sugarcane resemble those found in maize sufficiently to enable the use of maize monoclonal antibodies for their detection. The two sucrose synthase isoforms also resemble each other very closely, as shown by their kinetic and pH characteristics.

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[14C]glucose in inhibited by high concentrations of osmoticum.

Sucrose metabolism in sugarcane cell suspension cultures

Abstract Enzymes of sucrose metabolism in sugarcane cell suspension cultures were followed throughout a cell culture cycle (14 days) to monitor relative changes in the activities of enzymes directly involved in sucrose synthesis and hydrolysis. Doubling of initial sucrose-phosphate synthase (SPS) activity corresponded to the time when the sucrose accumulation rate had reached its peak. Sucrose synthase (SS), measured in the direction of synthesis, reached a high rate of activity during the period of maximal sucrose increase. Activity of SS in the direction of sucrose hydrolysis was much lower and decreased throughout the cell culture cycle. Invertase, measured at pH 7.0, followed a similar pattern to that for sucrose hydrolysis by SS. Acid invertase, assayed at pH 5.5, exhibited a high rate of activity at the beginning of the culture cycle and was followed by a sharp decline during sucrose accumulation. The rate of maximal sucrose accumulation was determined to be approximately 167 nmol h−1 (g fresh wt.)−1. The average activity of SPS during the same period was 8970 nmol h−1 (g fresh wt.)−1. Therefore, all sucrose synthesis in the cell cultures can be ascribed to SPS activity, whereas SS most likely functions in a degradative capacity.

Products of arginine catabolism in growing cells of sugarcane

Abstract On incubating sugarcane cells of variety H 50–7209 in a synthetic liquid medium in the presence of 14C-arginine, a substantial proportion of the label was found in N-carbamylputrescine, while a much smaller proportion appeared in guanidine. Large amounts of 14C-arginine were found in the ethanol-soluble fraction of the cells, and labelled arginine, proline and glutamic acid were incorporated in the proteins. The N-carbamylputrescine which began to accumulate within 10 min after the addition of 14C-arginine to the cell cultures was not excreted, nor did it appear to be further metabolized by these cells. Evidence was found that N-carbamylputrescine is formed more readily from citrulline than from arginine in sugarcane cells.

Evidence for Direct Uptake of Sucrose by Sugarcane Stalk Tissue

Summary Sucrose uptake was studied in slices of sugarcane stalk tissue to determine whether sucrose can be taken up intact from the apoplast. Fluorosucrose, an analog of sucrose but a poor substrate for invertase, was taken up at a rate as high as or higher than sucrose. The distribution of radioactivity in the hexose moieties of sucrose after [ 14 C]fructosyl sucrose was supplied externally to the tissue showed that some sucrose can be hydrolyzed before or during uptake, thus resulting in randomization of label in the tissue. The uptake of sucrose showed biphasic kinetics, was sensitive to a protonophore, sulfhydryl inhibitor, and an arginyl residue modifier. There was little competition by fructose, glucose, or raffinose for sucrose uptake. The data suggest that although a site for sucrose uptake exists in stalk tissue of sugarcane, the cell wall bound invertase can alter the nature of the sugar taken up.

Isolation of membranes from sugarcane cell suspensions: Evidence for a plasma membrane enriched fraction

Abstract Membranes in a post-mitochondrial supernanant from sugarcane cells grown in suspension culture were separated by sucrose density centrifugation and examined for presence of plasma membrane. Membrane vesicles from the fraction differed in size, binding properties toward naphthylphthalamic acid (NPA) or helminthosporoside and in some marler enzyme activities. Phosphotungstic acid (PTA) staining failed to distinguish between plasma membrane and other types of membranes in these preparations, but binding and enzymatic activities indicated enrichment of plasma membrane in the 38% sucrose fraction.

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction, oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the β-configurated pyridine nucleotide and the α-configurated form. Since NADH-linked dehydrogenases usually do not work with α-NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.