Sucrose Transport in Isolated Immature Barley Embryos

Techniques have been devised to select immature barley embryosat various stages in their development, and to study their accumulationof sucrose in vitro. Isolated embryos accumulate sucrose overa period of several hours of which some 80 per cent is conservedas a pool of free sucrose and the remainder utilized in macromolecularsynthesis. The rate of sucrose uptake increases with embryodevelopment, however the specific activity of uptake remainsconstant, indicating that the transport processes are fullyoperative early in embryogenesis. From the kinetics of sucroseuptake it is deduced that facilitated transport predominatesat sucrose concentrations of 50 mM, while at higher concentrationspassive diffusion makes an increasing contribution to sucroseaccumulation. The substrate specificity and the sensitivityof sucrose transport to uncoupling agents, in addition to thestability of the pool of accumulated sucrose, are all indicativeof active transport playing a major role in the sucrose assimilationof developing barley embryos. Hordeum distichum (L.) Lam, barley, embryo, sucrose transport     

In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos

In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m-chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented.     

Apoplastic Sugar Concentration and pH in Barley Leaves Infected with Brown Rust

Soluble sugars were extracted by low speed centrifugation fromthe apoplast of leaves of barley (Hordeum distichum L.) infiltratedwith water. Infection of the leaf with the brown rust fungus(Puccinia hordeii) resulted in a reduction in the concentrationof sucrose, glucose and fructose in the apoplast. Sugars werepresent in an apoplastic space occupying 12 and 17 cm³ m^–2of leaf area in healthy and infected tissue, respectively. Uptakeof hexoses by intercellular hyphae is suggested as a cause ofthis reduction. The pH of apoplastic sap extracted from rust-infectedleaves was increased to pH 7·3 from pH 6·6 incontrols. The effect of a reduced apoplastic sugar pool andincreased pH on export from infected leaves is discussed. Key words: Apoplast, barley (Hordeum distichum L.), brown rust (Puccinia hordeii Otth.), pH, sucrose, hexose     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Early carbon mobilization and radicle protrusion in maize germination

Considerable amounts of information is available on the complex carbohydrates that are mobilized and utilized by the seed to support early seedling development. These events occur after radicle has protruded from the seed. However, scarce information is available on the role of the endogenous soluble carbohydrates from the embryo in the first hours of germination. The present work analysed how the soluble carbohydrate reserves in isolated maize embryos are mobilized during 6-24 h of water imbibition, an interval that exclusively embraces the first two phases of the germination process. It was found that sucrose constitutes a very significant reserve in the scutellum and that it is efficiently consumed during the time in which the adjacent embryo axis is engaged in an active metabolism. Sucrose transporter was immunolocalized in the scutellum and in vascular elements. In parallel, a cell-wall invertase activity, which hydrolyses sucrose, developed in the embryo axis, which favoured higher glucose uptake. Sucrose and hexose transporters were active in the embryo tissues, together with the plasma membrane H(+)-ATPase, which was localized in all embryo regions involved in both nutrient transport and active cell elongation to support radicle extension. It is proposed that, during the initial maize germination phases, a net flow of sucrose takes place from the scutellum towards the embryo axis and regions that undergo elongation. During radicle extension, sucrose and hexose transporters, as well as H(+)-ATPase, become the fundamental proteins that orchestrate the transport of nutrients required for successful germination.     

A Comparative Study of Early Seed Development in Genotypes of Barley and Rye

Early seed development was studied in 17 genotypes of barley,Hordeum vulgare L., and 11 genotypes of rye, Secale cerealeL. The numbers of cells and nuclei in the embryos and endospermsof developing seeds were scored daily for 5 days after selfpollination. For embryos, the mean cell doubling times variedfrom 9.2–12.9 h for barley and 15.7–22.7 h for rye.Endosperm mitotic cycle times of both species were shortestover the first 24 h after pollination but then became longer.A non-linear correlation was found between the number of embryocells and the number of endosperm nuclei in barely and rye andis similar to that for other members of the Triticeae. Hordeum vulgare L., Secale cereale L., barley, rye, embryo, endosperm, mean cell doubling time     

Role of calcium in serum-stimulation of hexose transport in muscle cells

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.     

Sugar transport in chick embryo cardiac cells in culture. Analysis by countertransport,relationship to phosphorylation and effect of glucose starvation

Embryonic chick heart cells in culture transport 2-deoxy-D-glucose and 3-O-methyl-D-glucose very rapidly. By direct measurements of uptake, it was not possible to estimate accurately transport rates, nor, with 2-deoxyglucose, to discriminate clearly between its transport and phosphorylation. In contrast, the technique of countertransport made it possible to determine precisely initial transport velocity and to make the following observations: (1) phosphorylation, and not transport, is rate-limiting in 2-deoxyglucose uptake; (2) hexose transport is stimulated 5-fold by removal of glucose from culture medium; and (3) this stimulation is followed by an increase in phosphorylation, but the effect is much less pronounced (2-fold stimulation only). In conclusion, the adaptative regulation of glucose transport described in many fibroblast cell lines exists also in cardiac cells.     

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).     

Control of Net Uptake of Nutrients by Regulation of Influx in Barley Plants Recovering from Nutrient Deficiency

Rates of influx and net uptake of nitrate, phosphate and sulphatewere measured in intact barley plants, and concurrent effluxwas obtained by difference. Net uptake of these anions variedwidely depending on the nutrient status of the plants, and thedifferences in net uptake could be accounted for almost entirelyby changes in influx. Efflux played only a minor role in regulatingnet uptake of nitrate, phosphate or sulphate during recoveryfrom N-, P-, or S-deficiency. Nitrate influx and short-term ammonium absorption by N-deficientbarley plants were closely correlated, and varied in parallelwith rates of net uptake of nitrate or ammonium by similar plants.Again, it would seem that net uptake of ammonium is controlledpredominantly by changes in the rate of influx.Copyright 1993,1999 Academic Press Hordeum vulgare, barley, nutrient absorption, influx, nitrate, phosphate, sulphate, ammonium     

Influence of Genotype and Environment on Growth of Barley Embryos in vitro

Isolated embryos of three contrasting barley genotypes werecultured in vitro on a range of media comprising 16 combinationsof sucrose (3–12 per cent) and 2, 4-D (0–8 mg 1^–1)concentration. Cultures were incubated at a range of temperaturesfrom 5 to 25°C and were examined after 21 days when shootlengths as well as fresh and dry weights were recorded. Therelative influence on growth of increasing concentrations ofsucrose and 2, 4-D was investigated, as was their interactionwith the incubation temperature. The genotypes were found todiffer markedly in their response to these two media components,with each parameter of growth differentially affected. Resultsare discussed in relation to the known dormancy characteristicsof these genotypes. Hordeum vulgare L., barley, embryo, dormancy, 2, 4-dichlorophenoxy acetic acid, sucrose     

Effect of the Osmotic Environment on Assimilate Transport in Isolated Developing Embryos of Maize (Zea mays)

Embryos obtained from developing kernels of maize (Zea maysL.) were incubated in a bathing medium, to measure the effectof the osmotic environment on the balance between uptake andrelease of assimilates by the embryo. Net efflux of sucroseand amino acids from the embryo decreased with increasing mannitolconcentration in the bathing medium and net uptake of [¹⁴C]valine increased with increasing mannitol concentration. Therole of a high osmolality of the seed apoplast in seed developmentis discussed Zea mays, maize, embryo, seed development, assimilate transport, turgor-sensitive transport     

The Inhibitory Effect of Light on Proton-Coupled Hexose Uptake in Chlorella

The addition of 3-O-methyl-D-glucopyranose to wild-type cellsof Chlorella vulgaris (211-11h) grown in glucose medium in thedark induced a rapid alkalization of the external medium (protonuptake), whereas this pH shift did not occur in autotrophicallygrown cells. Light-irradiation inhibited the sugar-induced protonuptake, making the blue end of the visible spectrum very effectiveand the red end only slightly effective. This spectral dependencecorresponds to that of photoinhibition on hexose uptake in thesealgae. A similar photoinhibitory effect was observed in cellsof a colorless mutant of Chlorella vulgaris (M125). The activity of nitrate-proton symport in Chlorella vulgaris(211-11h) was also enhanced by the addition of glucose. Illuminationhad no inhibitory effect on this increased transport. The effectof light on the hexose uptake system is discussed. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Sugar transport into protoplasts isolated from developing soybean cotyledons : I. Protoplast isolation and general characteristics of sugar transport

A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Q₁₀ value and high activation energies).     

Comparing carbohydrate status during norway spruce seed development and somatic embryo formation

Summary The carbohydrate status of developing seeds of Picea abies was examined in order to provide a frame of reference for the evaluation of changes in carbohydrate content in maturing somatic embryos of the same species. Samples were taken at weekly intervals from 12 May 1998 (estimated time of pollination) until 20 October 1998. The total non-structural carbohydrate content was high (≈150–180 μg mg⁻¹ dry weight) at the time of the first samples and the carbohydrate spectrum consisted of sucrose, glucose, fructose, and pinitol. A dramatic decrease in carbohydrate content took place from June 6 onwards, that was accompanied by changes in carbohydrate partitioning to favor sucrose over hexoses and the disappearance of pinitol. Raffinose and stachyose were first detected on July 28, and their content gradually increased thereafter. Isolated embryos and remaining megagametophytes were analyzed starting with September 1. Carbohydrate content was higher in isolated zygotic embryo than in the rest of the seed, with a slowly increasing fraction of raffinose and stachyose. Comparisons of presented data with the results of our previous study of somatic embryo carbohydrate status (Lipavská et al., 2000) revealed the following common features: (1) a decrease in total carbohydrate content and (2) an increase in sucrose:hexose ratios in developing seeds and embryonal suspensor mass. Marked differences were observed in carbohydrate spectra: (1) somatic embryo development was not accompanied by pinitol accumulation in any phase; (2) mature zygotic embryos, in contrast to mature somatic embryos, contained raffinose and stachyose. These observations will provide a solid basis for improvement of protocols for somatic embryogenesis in Picea.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency