T.E. Humphreys

Involvement of sucrose synthase in sucrose catabolism

Abstract Maize scutellum slices incubated in water utilized sucrose at a maximum rate of 0.12,μmol/min per g fr. wt of slices. When slices were incubated in DNP, there was a three-fold increase in the rate of sucrose utilization. Sucrose breakdown in higher plants can be achieved by pathways starting with either invertase or sucrose synthase (SS). Invertase activity in scutellum homogenates was found only in the cell wall fraction, indicating that SS was responsible for sucrose breakdown in vivo . SS in crude scutellum extracts broke down sucrose to fructose and UDPG at 0.39,μmol/min per g fresh wt of slices. The UDPG formed was not converted to UDP + glucose, UMP + glucose-1-P, UDP + glucose-1-P or broken down by any other means by the crude extract in the absence of PPi. In the presence of PPi, UDPG was broken down by UDPG pyrophosphorylase which had a maximum activity of 26 μmol/min per g fr. wt of slices. Levels of PPi in the scutellum could not be measured using the UDPG pyrophosphorylase: phosphoglucomutase: glucose-6-P dehydrogenase assay because they were too low relative to glucose-6-P which interferes in the assay. An active inorganic pyrophosphatase was present in the scutellum extract which could prevent the accumulation of PPi in the cytoplasm. ATP pyrophosphohydrolase, which hydrolyses ATP to AMP and PPi, was found in the soluble portion of the scutellum extract. The enzyme activity was increased by fructose-2,6-bisP and Ca 2+ . In the presence of both activators, enzyme activity was 1.1 μmol/min per g fr. wt of slices, a rate sufficient to supply PPi for the breakdown of UDPG. These results indicate that sucrose breakdown in maize scutellum cells occurs via the SS: UDPG pyrophosphorylase pathway.

Sucrose transport at the tonoplast

Abstract Sucrose that leaked from maize scutellum slices upon transfer of slices from a hexose or hexitol solution to water or upon placing the slices in a buffered EDTA solution was considered to be cytoplasmic in origin; residual (after leakage) tissue sucrose was considered to be stored in the vacuoles. This paper presents a study of the movement of sucrose across the tonoplast between the vacuoles and the cytoplasmic compartment. It is concluded that; (a) sucrose transport into the vacuoles is directly linked to sucrose synthesis in such a way that free sucrose is not an intermediate in the coupled process, (b) cytoplasmic sucrose is not (cannot be?) stored, (c) sucrose transport out of the vacuoles is linked to the metabolic demand for sugar, and (d) the transport process removing sucrose from the vacuoles does not release free sucrose into the cytoplasm. The sucrose fluxes at the plasmalemma and at the tonoplast are calculated, and the transport processes at the two membranes are compared.

Aerobic fermentation and phosphofructokinase in tissue slices of the corn scutellum

Abstract The study of gas exchange in tissue slices of corn scutellum incubated either in water or 0·1 M fructose showed that the presence of the hexose increased the rate of glycolysis in this tissue 4- to 7-fold depending on the experimental conditions, and that this increase was quantitatively linked with an aerobic alcoholic fermentation. The properties of crude phosphofructokinase (PFK) were studied and compared with the properties of PFKs from other higher plants, one of which does not carry out aerobic fermentation in the presence of exogenous hexose. Corn scutellum PFK appears to be similar in its properties to the PFKs of other higher plants. The tissue levels of certain metabolic intermediates, some of which influence PFK activity (citrate, ATP, inorganic orthophosphate, and fructose-1,6-diphosphate), were determined and were found not to vary in amount under regimes of low and high glycolytic activity. No evidence was found to support a regulatory role for PFK on the rate of glycolysis in corn scutellum slices. Alternative mechanisms for the control of glycolysis in scutellum slices are proposed. These controls may involve the reactions leading to the metabolic utilization of stored sucrose and the intracellular distribution of adenine nucleotides and inorganic phosphate.

The effect of divalent cations on the leakage of sucrose from corn scutellum slices

Abstract During the incubation of corn scutellum slices in fructose, the presence of Ca 2+ , Mn 2+ or Mg 2+ in the bathing medium reduced the leakage ofsucrose (leakage A). Ca 2+ was particularly effective in this respect. Following a period of incubation in fructose, the subsequent leakage of sucrose into water (leakage B) was greatly reduced by the addition of either Ca 2+ or Mn 2+ to the aqueous bathing medium, whereas the addition of Mg 2+ to the bathing medium only slightly decreased this leakage. In order for Ca 2+ to be effective in inhibiting the leakage of sucrose, it had to be present in the medium into which leakage was occurring. During Ca 2+ -inhibition of leakage B, the sucrose of the synthesis compartment was stored in an area of the cell from which it would not leak into water or EDTA. Possible mechanisms for the effect of Ca 2+ on sucrose leakage are discussed.

Sugar uptake in the maize scutellum

Abstract Characteristics of the uptake of sucrose, glucose and fructose by maize scutellum slices are presented. Sugars were taken up at almost a constant rate until the bathing solution was nearly depleted, even when initial sugar concentrations were well below those which saturated the uptake mechanisms. DNP, phloridzin, uranyl ion, and anoxia were more inhibitory to the uptake of sucrose than to the uptake of hexoses. Maltose was taken up without hydrolysis. Turanose was not taken up, but it slightly inhibited the uptake of sucrose. The uptake of sucrose, glucose and fructose was accompanied by aerobic fermentation. The following conclusions are drawn; (a) sucrose is taken up actively without inversion; (b) hexoses are taken up by two processes, diffusion and active transport operating simultaneously; (c) the active uptake mechanisms for both sucrose and the hexoses are coupled to glycolysis.

Leakage and storage of sucrose during sucrose synthesis in the corn scutellum

Abstract During incubation of corn scutellum slices in fructose solutions (0·1–0·9 M) sucrose leaked (leakage A) from the slices, and additional leakage (leakage B) occurred upon dilution of the fructose solution to 0·1 M or less. Sucrose was also stored within the slices, and net sucrose synthesis (stored plus leaked) was 20–25 μmoles/hrg fresh wt. Fructose concentration of the bathing solution had little effect on net sucrose synthesis or on the amount of sucrose leakage A. However, with increased fructose concentration, the amount of sucrose stored decreased while the amount of sucrose leakage B increased. The rate of sucrose leakage B was strongly inhibited by fructose, galactose, polyhydric alcohols and 2,4-dinitrophenol. After incubation of the slices in 14C-fructose, leakage A and leakage B sucrose were found to be quite different with respect to specific activity and distribution of 14C between the fructose and glucose moieties. From these results and the results of inhibition studies, it is concluded that the two types of leakage have different origins within the cell.

A model for sucrose transport in the maize scutellum

Abstract A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N -ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.

The effect of d-mannose on sucrose storage in the corn scutellum: Evidence for two sucrose transpsort mechanisms☆

Abstract Evidence is presented that tissue slices of the corn scutellum contain two distinct mechanisms for the storage of sucrose. The first mechanism is responsible for the storage of exogenously supplied sucrose and is strongly inhibited by the presence of d -mannose in the sucrose-containing bathing medium. The second mechanism is responsible for the transport of sucrose (newly synthesized from hexose) from the synthesis compartment of the cell into storage. This mechanism is not inhibited by mannose. Although the intercompartmental storage of newly synthesized sucrose is unaffected by the presence of mannose, the synthesis of the disaccharide from exogenously supplied fructose is strongly inhibited when mannose is present in the bathing medium. In addition, treatment of tissue slices with mannose resulted in a drastic reduction in the ATP level of the tissue. This reduction in ATP was not accompanied by an equivalent increase in ADP or AMP. It is suggested that the effect of mannose in inhibiting the storage of exogenous sucrose and in inhibiting the synthesis of sucrose from exogenous fructose arises from ATP deficiency. This deficiency is presumably the result of mannose and mannose-6-phosphate inhibitions of glycolysis at the level of hexokinase and phosphoglucose isomerase, respectively, and/or the trapping of inorganic phosphate in a pool of non-metabolizable mannose-6-phosphate. Possible mechanisms for sucrose storage are discussed.

Glucose uptake by the corn scutellum.

Abstract The rate of uptake of glucose into corn scutellum slices was dependent both on glucose concentration and the time the slices were incubated in water prior to the addition of glucose. As the length of the incubation period was increased, so was the rate of glucose uptake. This effect was nullified by high glucose concentrations (e.g. 4·9 × 10−2 M) during the uptake period. Incubation in the absence of glucose caused the total reducing sugars and glucose to decrease in the tissue during the first hour and increase slightly thereafter. The sucrose and glucose-6-phosphate contents decreased throughout the 4-hr period of measurement. During incubation in the presence of different concentrations of glucose, the contents of reducing sugars, glucose, sucrose and glucose-6-phosphate decreased more slowly than during incubation in water or were maintained at levels equal to or greater than initial levels. Mannose inhibited glucose uptake. During incubation with mannose the slices accumulated mannose and mannose-6-phosphate, and evidence is presented that both mannose and mannose-6-phosphate are responsible for the inhibition of glucose uptake. It is suggested that glucose-6-phosphate competitively inhibits an enzymatic step (hexokinase-catalyzed reaction ?) associated with net glucose uptake.

The storage of exogenous sucrose by corn scutellum slices

Abstract Corn scutellum slices stored sucrose when incubated in either sucrose or fructose, and similar maximum rates of net storage were obtained with 0·4 M sucrose or 0·2 M fructose. It is concluded that exogenous sucrose is stored without prior inversion. This conclusion is based on the following: (1) The amount of extracellular inversion (as measured by the appearance of glucose and fructose in a 0·3 M sucrose bathing solution) was too low to support a net sucrose synthesis and storage, and (2) sucrose storage in slices bathed in optimal concentrations of fructose or glucose was increased by the addition of sucrose to the bathing medium. Further evidence for the existence of a transport system for exogenous sucrose was obtained in the demonstration of an exchange of sucrose between the bathing solution and the storage compartment. The rate of exchange was increased as the concentration of exogenous sucrose was increased, was doubled in the presence of citrate-phosphate buffer, was little affected by pH in the range 5·0–7·3 and showed no dependence on the net amount of sucrose stored.