L.A. Garrard

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.

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.

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.

Effect of gibberellic acid on starch degrading enzymes in leaves of Digitaria decumbens

Abstract Tropical ‘Pangola’ digitgrass was treated with gibberellic acid (GA) and subjected to 30° or 10° nights. The starch degrading enzymes of the leaves were separated by polyacrylamide gel electrophoresis. Densitometer tracings of gels negatively stained by starch-iodine showed the presence of seven bands regardless of treatment of plants. GA treatment increased starch degrading enzyme activity in plants subjected to 10° to the level of activity found in 30° untreated (control) plants and, additionally, enhanced enzyme activity in plants at 30°. GA treatment of 10° plants decreased sucrose and starch levels when compared to levels found in untreated 10° plants. The action of GA in reversing the effects of 10° nights on ‘Pangola’ leaves was found to be the result of a quantitative increase in activity of existing enzyme forms rather than production of isozymes.

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.

Sucrose leakage from the maize scutellum: Evidence for the participation of the phloem

Abstract Sucrose leaked from maize scutellum slices when they were incubated in fructose. The rate of leakage declined with time, but the initial rate could be maintained in the presence of HCl, ATP or certain buffers. The leakage process was a labile one and the ability of cells to leak (but not produce) sucrose declined during incubation of the slices in water at 30°. The leakage process was protected, however, during incubation in ice water. Sucrose leakage from whole scutella had properties similar to leakage from slices but had only about one-third the rate. The whole scutellum leaked appreciable sucrose only after the root—shoot axis was removed. The compounds which increased leakage also have the ability to displace or to form complexes with cations, and it appears that their effect on leakage is due to the removal of Mg 2+ (and possibly Ca 2+ ) from the slices. Evidence is presented that there is a pool of leakable sucrose which, when the leakage process is functioning, can be emptied into the bathing solution. The pool level is maintained, however, when fructose is present as a source of sucrose. The results presented are consistent with the idea that sucrose leakage originates in the sieve tubes of the phloem and is the end result of a series of events which includes intercellular sucrose transport, vein loading and phloem transport.

Effect of hydrogen ion concentration on sucrose leakage from corn scutellum slices: Evidence for two kinds of sucrose pool within the slice☆

Abstract Incubation of corn scutellum slices in HCl concentrations above 0·01 M caused a marked disruption of cell function as evidenced by the copious leakage of sucrose from the tissue, the loss of the tissues capacity for the synthesis and storage of sucrose from exogenously supplied hexose and the failure of the tissue to store exogenously supplied sucrose. In contrast, treatment of the tissue in 0·01 M HCl both in the presence and the absence of fructose increased the leakage of sucrose without seriously impairing sucrose synthesis. In addition, pretreatment of the slices in 0·01 M HCl did not reduce sucrose synthesis and storage in the presence of exogenous fructose or inhibit the storage of exogenous sucrose. The leakage of sucrose into 0·01 M HCl or 0·01 M HCl plus fructose is not ascribed to a disruption of the plasmalemma but rather to the existence of two pools of stored sucrose within the tissue, one mobile in acid and the other immobile in acid. The location of these pools within the tissue and the movements of sucrose between pools and bathing solution are discussed in relation to previously conceived ideas of cellular compartmentation and observed anatomical features of the scutellum tissue.

The effect of tris(hydroxymethyl)aminomethane on sucrose storage in and leakage from corn scutellum slices

Abstract Sucrose leaked from slices of the corn scutellum incubated in tris(hydroxymethyl)aminomethane (tris) buffer (0·06 M, pH 7·5). When the slices were incubated in tris buffer plus fructose the amount of sucrose leakage was increased. The sucrose content of slices incubated in tris buffer plus sucrose declined nearly as much as those incubated only in tris indicating that tris almost completely inhibited the storage of exogenous sucrose. Evidence is presented that these results were due to the action of tris on the membrane separating the storage compartment of the scutellum cell from the cell exterior rather than on the membrane between the sucrose synthesis compartment and the cell exterior. Slices pretreated in tris buffer for 1 hr and then quickly washed with water were no longer leaky, but the inhibition of exogenous sucrose storage persisted. This inhibition could be reversed by H + and either Al 3+ or certain divalent cations.