Sugar uptake by maize endosperm suspension cultures

Maize (Zea mays L.) endosperm suspension cultures are a useful model system for studying biochemical and physiological events in developing maize endosperm. In this report, sugar uptake by the cultures is characterized. Uptake of ¹⁴C-labeled fructose and l-glucose was linear with time, while the rate of uptake of radioactivity from sucrose increased over a 120 min period. Both saturable and linear components of uptake were observed for fructose, glucose, sucrose, 1′-deoxy-1′-fluorosucrose, and maltose. Uptake of mannitol, sorbitol, and l-glucose took place at lower rates and was linear with concentration. Rates of incorporation of radioactivity from fructose and glucose exceeded that of sucrose at all concentrations tested. Kinetics of 1′-deoxy-1′-fluorosucrose uptake indicated that ¹⁴C from sucrose can be taken up by a saturable carrier of intact sucrose as well as by invertase hydrolysis and subsequent uptake of hexoses. Cell wall invertase was demonstrated histochemically. Further study of fructose uptake at a concentration at which the saturable component predominated revealed sensitivity to metabolic inhibitors, respiratory uncouplers, the nonpermeant sulfhydryl reagent p-chloromercuribenzenesulfonic acid, and nigericin. Uptake was not affected by valinomycin plus K⁺ and was stimulated by fusicoccin. Fructose and glucose uptake was not pH-sensitive below pH 7.0, whereas uptake of radioactivity from sucrose and 1′-deoxy-1′-fluorosucrose declined as the pH was increased above 5.0. Fructose uptake was not completely inhibited by glucose and vice versa, suggesting the presence of specific carriers. These results indicate that maize endosperm suspension cultures (a) absorb fructose via a typical, energy-requiring, carrier-mediated proton cotransport system; (b) possess saturable carriers for glucose and sucrose; and (c) also absorb sucrose via hexose uptake after sucrose hydrolysis by extracellular invertase.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar

Several weakly transported sugars were tested for transport by the Na⁺-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na⁺ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K_i for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na⁺-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na⁺-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.     

Structural Features of Sugars That Trigger or Support Conidial Germination in the Filamentous Fungus Aspergillus niger

The asexual spores (conidia) of Aspergillus niger germinate to produce hyphae under appropriate conditions. Germination is initiated by conidial swelling and mobilization of internal carbon and energy stores, followed by polarization and emergence of a hyphal germ tube. The effects of different pyranose sugars, all analogues of d-glucose, on the germination of A. niger conidia were explored, and we define germination as the transition from a dormant conidium into a germling. Within germination, we distinguish two distinct stages, the initial swelling of the conidium and subsequent polarized growth. The stage of conidial swelling requires a germination trigger, which we define as a compound that is sensed by the conidium and which leads to catabolism of d-trehalose and isotropic growth. Sugars that triggered germination and outgrowth included d-glucose, d-mannose, and d-xylose. Sugars that triggered germination but did not support subsequent outgrowth included d-tagatose, d-lyxose, and 2-deoxy-d-glucose. Nontriggering sugars included d-galactose, l-glucose, and d-arabinose. Certain nontriggering sugars, including d-galactose, supported outgrowth if added in the presence of a complementary triggering sugar. This division of functions indicates that sugars are involved in two separate events in germination, triggering and subsequent outgrowth, and the structural features of sugars that support each, both, or none of these events are discussed. We also present data on the uptake of sugars during the germination process and discuss possible mechanisms of triggering in the absence of apparent sugar uptake during the initial swelling of conidia.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Sugar transport in immature internodal tissue of sugarcane: I. Mechanism and kinetics of accumulation

Transmembrane sugar transport into immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L.) is a metabolically regulated process as evidenced by its sensitivity to pH, temperature, anaerobiosis, and metabolic inhibitors. All sugars studied—glucose, fructose, galactose, sorbose, glucose 6-phosphate, 3-O-methylglucose, and 2-deoxy-d-glucose—were apparently transported via the same carrier sites since they competed with each other for uptake. External concentrations of these sugars at one-half V_max were in the range of 3.9 to 8.4 nm. Preliminary data indicated that phosphorylation may be closely associated with glucose transport. The dominant intracellular sugar after 4-hours incubation was sucrose when glucose, glucose-6-P, or fructose was the exogenously supplied sugar; but when galactose was supplied, only 28% of intracellular radioactivity was in sucrose. Sorbose, 3-O-methylglucose, and 2-deoxy-d-glucose were not metabolized. Thus, by using these analogs, transport could be studied independently of subsequent metabolism, effectively eliminating a complicating factor in previous studies.     

Isolation of amyloplasts from developing maize endosperm

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4°C or 5 hours at 30°C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl₂. After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.     

The Effect of Sugars on the Binding of [Hg]-p-Chloromercuribenzenesulfonic Acid to Leaf Tissues

Replacement of mannitol with sucrose decreases the binding of [²⁰³Hg]-p-chloromercuribenzenesulphonic acid (PCMBS) to Vicia faba leaf discs without epidermis. This decrease is optimal for 20 minutes on incubation, is concentration-dependent, and is also found with maltose and raffinose. In parallel experiments, the addition of sucrose, maltose, and raffinose during PCMBS pretreatment was shown to increase subsequent uptake of [U-¹⁴C]sucrose. In contrast, d- or l-glucose, 3-O-methylglucose, galactose, fructose, palatinose, turanose, or melibiose had no effect either on PCMBS binding or on [¹⁴C]sucrose uptake. The sucrose-induced decrease of PCMBS binding is retained after a cold and ionic shock. Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant. Altogether, the data provide new support to the hypothesis that the active site of the sucrose carrier contains a group sensitive to PCMBS.     

Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake

Analysis of [³H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [¹⁴C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [¹⁴C]-(glucosyl)-1′-fluorosucrose was identical to the rate of [¹⁴C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast.     

Active Sugar Transport by the Small Intestine : The effects of sugars,amino acids,hexosamines, sulfhydryl-reacting compounds,and cations on the preferential binding of D-glucose to Tris-disrupted brush borders

Tris-disrupted and intact brush border membrane preparations from mucosa of hamster jejunum were capable of preferentially binding actively transported D-glucose in a similar manner. Density gradient centrifugation of the Tris-disrupted brush borders indicated that D-glucose was bound to a fraction containing the cores or inner material of the microvilli. The properties of this binding were examined with the Tris-disrupted brush border preparation. Actively transported sugars competitively inhibited preferential D-glucose binding, whereas no effect was observed with nonactively transported sugars. Neither actively nor nonactively transported amino acids affected D-glucose binding. D-Glucosamine, which is not actively transported, was inhibitory to preferential D-glucose binding as well as to the active transport of D-glucose by everted sacs of hamster jejunum. No inhibitory effect was observed with the same concentration of D-galactosamine. Preferential D-glucose binding was also inhibited by sulfhydryl-reacting compounds, Ca²⁺, and Li⁺ ions. On the other hand, Mg²⁺ was shown to be stimulatory and Na⁺, NH₄ ⁺, and K⁺ had no effect on this phenomenon. The results of these experiments suggest that preferential D-glucose binding to brush borders is related to the initial step in active sugar transport by the small intestine.     

Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H⁺-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-α-d-glucopyranoside, phenyl-α-d-thioglucopyranoside, and phenyl-α-d-4-deoxythioglucopyranoside (PDTGP; K_i = 67, 180, and 327 micromolar, respectively). The K_m for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-α-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-α-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-α-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-α-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K_i values ranging from 600 to 800 micromolar, and phenyl-α-d-thiomannopyranoside, phenyl-β-d-glucopyranoside, and phenylethyl-β-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-α-d-thioglucopyranoside and PDTGP, were transported.     

Ketose reductase activity in developing maize endosperm

Ketose reductase (NAD-dependent polyol dehydrogenase EC 1.1.1.14) activity, which catalyzes the NADH-dependent reduction of fructose to sorbitol (d-glucitol), was detected in developing maize (Zea mays L.) endosperm, purified 104-fold from this tissue, and partially characterized. Product analysis by high performance liquid chromatography confirmed that the enzyme-catalyzed reaction was freely reversible. In maize endosperm, 15 days after pollination, ketose reductase activity was of the same order of magnitude as sucrose synthase activity, which produces fructose during sucrose degradation. Other enzymes of hexose metabolism detected in maize endosperm were present in activities of only 1 to 3% of the sucrose synthase activity. CaCl₂, MgCl₂, and MnCl₂ stimulated ketose reductase activity 7-, 6-, and 2-fold, respectively, but had little effect on NAD-dependent polyol dehydrogenation (the reverse reaction). The pH optimums for ketose reductase and polyol dehydrogenase reactions were 6.0 and 9.0, respectively. K_m values were 136 millimolar fructose and 8.4 millimolar sorbitol. The molecular mass of ketose reductase was estimated to be 78 kilodaltons by gel filtration. It is postulated that ketose reductase may function to metabolize some of the fructose produced during sucrose degradation in maize endosperm, but the metabolic fate of sorbitol produced by this reaction is not known.     

Sugar transport across the peritubular face of renal cells of the flounder

The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose.     

Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway

Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv `Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co²⁺ ions. These three tested sugars also stimulated the conversion of l-[3,4-¹⁴C]methionine to [¹⁴C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [¹⁴C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO₂.     

Transport and metabolism of 1'-fluorosucrose, a sucrose analog not subject to invertase hydrolysis

The novel sucrose derivative 1′-fluorosucrose (α-d-glucopyranosyl-β- d-1-deoxy-1-fluorofructofuranoside) was synthesized in order to help define mechanisms of sucrose entry into plant cells. Replacement of the 1′-hydroxyl by fluorine very greatly reduces invertase hydrolysis of the derivative (hydrolysis at 10 millimolar 1′-fluorosucrose is less than 2% that of sucrose) but does not reduce recognition, binding, or transport of 1′-fluorosucrose by a sucrose carrier. Transport characteristics of 1′-fluorosucrose were studied in three different tissues. The derivative is transported by the sucrose carrier in the plasmalemma of developing soybean cotyledon protoplasts with a higher affinity than sucrose (K_m 1′-fluorosucrose 0.9 millimolar, K_m sucrose 2.0 millimolar). 1′-Fluorosucrose is a competitive inhibitor of sucrose uptake with an apparent K_i also of 0.9 millimolar, while the K_i of sucrose competition of 1′-fluorosucrose uptake was 2.0 millimolar. Thus, both sugars are recognized at the same binding site in the plasmalemma. Both sucrose and 1′-fluorosucrose show very similar patterns of phloem translocation from an abraded leaf surface through the petiole indicating that recognition of 1′-fluorosucrose by sucrose carriers involved in phloem loading is likely as well.     

Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway

We have used ¹³C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of ¹³C between carbons atoms 1 and 6 of [1-¹³C] or [6-¹³C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the ¹³C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the ¹³C sugars were supplied in vivo to the intact plant. The ¹³C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of ¹³C in all glucosyl carbons was quantified by ¹³C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background ¹³C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.     

C]Sucrose Uptake and Labeling of Starch in Developing Grains of Normal and segl Barley

Previous work showed that the segl mutant of barley (Hordeum vulgare cv Betzes) did not differ from normal Betzes in plant growth, photosynthesis, or fertility, but it produced only shrunken seeds regardless of pollen source. To determine whether defects in sucrose uptake or starch synthesis resulted in the shrunken condition, developing grains of Betzes and segl were cultured in [¹⁴C]sucrose solutions after slicing transversely to expose the endosperm cavity and free space. In both young grains (before genotypes differed in dry weight) and older grains (17 days after anthesis, when segl grains were smaller than Betzes), sucrose uptake and starch synthesis were similar in both genotypes on a dry weight basis. To determine if sucrose was hydrolyzed during uptake, spikes of Betzes and segl were allowed to take up [fructose-U-¹⁴C]sucrose 14 days after anthesis and the radioactivity of endosperm sugars was examined during 3 hours of incubation. Whereas less total radioactivity entered the endosperm and the endosperm cavity (free space) of segl, in both genotypes over 96% of the label of endosperm sugars was in sucrose, and there was no apparent initial or progressive randomization of label among hexose moieties of sucrose as compared to the free space sampled after 1 hour of incubation. We conclude that segl endosperms are capable of normal sucrose uptake and starch synthesis and that hydrolysis of sucrose is not required for uptake in either genotype. Evidence suggests abnormal development of grain tissue of maternal origin during growth of segl grains.     

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells

Addition of l-[U-¹⁴C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-¹⁴C]glutamate, and (c) efflux of [¹⁴C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H⁺/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H⁺/l-Glu symport may involve both H⁺ consumption during 4-aminobutyrate production and ATP-driven H⁺ efflux.