Hexose Transport in Isolated Immature Barley Embryos

The pathways of monosaccharide assimilation in developing barleyembryos have been investigated in an in vitro assay system,and the results are discussed in relation to the primary carbohydratenutrients of the developing and germinating embryo. Glucoseand fructose are both rapidly taken up by immature embryos,and at a substrate concentration of 100 mM their assimilationrate exceeds by threefold that of sucrose. The kinetics of hexoseuptake are complex, reflecting a combination of passive diffusionand metabolically mediated uptake mechanisms. The hexose transportenergetics were further studied with the glucose analogue 3-O-methyl-D-glucose,shown to be a non-metabolite in the barley embryo. The kineticsof 3-O-methyl-D-glucose influx and efflux indicated that passivediffusion accounts for a major part of hexose assimilation inbarley embryos. However, an active transport component was revealedby the sensitivity of 3-O-methyl-D-glucose uptake to uncouplingagents and the demonstration of competitive inhibition of uptakebetween glucose analogues. These observations supported theview that part of the observed energy-dependence of hexose uptakemight be asociated with a direct coupling of energy to transport.A high-affinity active group translocation mechanism for glucoseassimilation could be important in embryo metabolism duringthe first hours of germination prior to hydrolysis of endospermstarch reserves. Analysis of the fate of assimilated hexosesshowed that the rapid consumption of the hexose pool in thesynthesis of sucrose must contribute to the influx of hexosesby the maintenance of a hexose concentration gradient betweenthe embryo and its environment. Hordeum distichum(L.) Lam, barley, embryo, hexose transport     

Characterization of the active sucrose transport system of immature soybean embryos

Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [¹⁴C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35°C, saturating illumination (pH 6), and 21% oxygen, the apparent K_m for sucrose is approximately 8 millimolar and V_max is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.     

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     

Peptide transport by germinating barley embryos: Evidence for a single common carrier for di- and oligopeptides

Competition for uptake of a range of amino acids and peptides by germinating barley (Hordeum vulgare L.) embryos was studied. Peptides and amino acids show no competition and are apparently absorbed by independent transport systems. However, peptides of widely different structures do compete and it seems that only a single peptide transport system is present in barley embryos, capable of handling both di- and oligopeptides. The ability of physiological peptides to totally inhibit the uptake of glycylsarcosine indicates they share a common uptake system which previously has been shown to have the properties of an active transport process. The characteristics of the barley peptide transport system are compared with those found in other organisms.     

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     

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     

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associatedwith a large accumulation of glucose and fructose in the vacuolesof the fruit cells. These hexoses are derived from sucrose,which is released from the phloem and may be taken up by parenchymacells prior to hydrolysis. We have expressed two putative ripening-relatedsucrose transporters from grape berries, VvSUC11 (synonymouswith VvSUT1) and VvSUC12, in an invertase deficient yeast strainto characterize their transport activities. Sucrose was takenup by yeast transformed with either transporter at an optimumpH of <4.5 and with a Michaelis constant (K_m) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibitedby protonophores and by vanadate. This is consistent with anactive uptake mechanism involving proton cotransport, typicalof sucrose/H⁺symporters. The transporters from grape berrieswere functionally similar to Scr1, a sucrose transporter fromRicinus cotyledons. It is likely that in grape berries VvSUC11and VvSUC12 facilitate the loading of sucrose from the apoplastinto the parenchyma cells. Copyright 2001 Annals of Botany Company Fruit, grape berries, plasma membrane, sugars, sucrose transporters, Vitis vinifera     

Comparative Morphological Study of Zygotic and Microspore-derived Embryos ofBrassica napusL. as Revealed by Scanning Electron Microscopy

A comparative morphological study of microspore-derived (MD)and zygotic embryos ofBrassica napusL. was conducted, illustratingsubstantial similarities in external morphology of these embryosthroughout their development. Haploid embryos were producedfrom isolated microspores cultured on high molecular weightpolyethylene glycol (PEG), replacing sucrose as an osmoticum.Morphological changes during the time-course of microspore embryodevelopment induced on PEG (25%) and sucrose (13%) are describedin detail as revealed by scanning electron microscopy (SEM)and compared to the corresponding stages of zygotic embryosdevelopedin ovulo. At the heart, torpedo and early cotyledonarystages, microspore-derived (MD) embryos on PEG closely resembletheir zygotic counterparts. In contrast, the external morphologyof embryos induced on high sucrose medium differs from thatof PEG and zygotic embryos indicating that a high concentrationof sucrose in culture has a morphogenetic effect on MD embryodevelopment inB. napus. Fragments of the original pollen wallare regularly observed at the root pole region and at the tipsof suspensors in MD embryos throughout their development. Thissuggests that polarity in MD embryos might originate from structurallypolarized late uninuclear microspores and early bicellular pollen.Copyright1998 Annals of Botany Company Brassica napusL., scanning electron microscopy, microspore-derived embryo, zygotic embryo, morphology, microspore, suspensor, exine, sucrose, polyethylene glycol.     

Influence of Proline and Glycinebetaine on Salt Tolerance of Cultured Barley Embryos

Lone, M. I., Kueh, J. S. H., Wyn Jones, R. G. and Bright, S.W. J. 1987. Influence of proline and glycinebetaine on salttolerance of cultured barley embryos.—J. exp. Bot. 38:479–490. The addition of exogenous proline and glycinebetaine to culturedbarley (Hordeum vulgare L. cv. Maris Mink) embryos increasedshoot elongation under saline conditions. Inhibition of shootelongation by NaCl was relieved by proline when plantlets weregrown in deep crystallizing dishes but not in Petri dishes whereshoots come into direct contact with the medium. The effectof proline could be related to a decrease in shoot Cl^–and Na⁺ accumulation which was only observed in plantlets grownin crystallizing dishes. Proline but not betaine uptake intocultured plantlets was stimulated by NaCl while each organicsolute inhibited the endogenous synthesis of the other soluteunder salt stress. Comparison of the effects of exogenously supplied proline withenhanced endogenous proline accumulation in the mutant lineR5201 suggested that the increased proline accumulation in themutant is an order of magnitude too low to have a significantphysiological effect. The implications of the effect of prolineon ion transport, discrimination and accumulation are discussed. Key words: Salt tolerance, proline, ion transport, barley embryo culture     

Structure and Function of Transfer Cells in the Sporophyte Haustorium of Funaria hygrometrica Hedw: II. KINETICS OF UPTAKE OF LABELLED SUGARS AND LOCALIZATION OF ABSORBED PRODUCTS BY FREEZE-SUBSTITUTION AND AUTORADIOGRAPHY

Excised sporophytes of the moss Funaria hygrometrica Hedw. absorbexternally applied sugar through their basal haustorium. Influxof [³H]sucrose is inhibited by metabolic uncouplers, darkness,and by the photosynthetic inhibitor DCMU. The kinetics of uptakeof glucose and sucrose suggest a biphasic mechanism of absorption.Uptake of 3-O-methyl [³H]glucose shows no saturation characteristicsand a passive mechanism is indicated. Externally applied glucoseis rapidly converted to sucrose. Good retention of productsof short-term absorption and metabolism of [³H]glucose was achievedby freeze-substitution. Autoradiography showed dense and uniformlabelling of the transfer cells of the haustorium. V_max valuesfor uptake of sucrose and glucose, expressed in terms of theweight and external surface area of haustorium, are considerablygreater than typical values from other plant systems. However,if the surface area amplification that is brought about by thedevelopment of wall ingrowths in the transfer cells is takeninto account, fluxes per unit area of plasma membrane are reducedinto the range of typical values. The hypothesis that the surfacearea amplification that characterizes transfer cells is relatedfunctionally to processes of solute transport is therefore supportedby the data.     

Sucrose uptake by the phloem parenchyma of carrot storage root

The characteristics of sucrose uptake into the symplast of phloemtissue discs harvested from fresh, actively-growing carrot storageroots are described. Sucrose uptake exhibited a curvilinearresponse with increasing sucrose concentration. The inhibitorsp-chloromercuribenzenesulphonic acid (PCMBS) and carbonyl cyanidem-chlorophenylhydrazone (CCCP) decreased uptake and resultedin solely linear relationships between uptake and sucrose concentration.These results suggest that active carrier-mediated transportoccurs at the plasmalemma in addition to a diffusive mechanism.The former saturates at a lower concentration (approximately20 mM) than the latter which does not saturate below 100 mM.Though similar in their effect on the ethanol-soluble fraction,CCCP and PCMBS had different effects on the conversion of sucroseto ethanol-insoluble material. Varying the osmotic environment with different mannitol concentrationsdid not affect uptake between 0 and 400 mM mannitol, but didcause an increase at 600 mM mannitol: an effect which may havebeen an artefact of plasmolysis. Metabolic conversion to ethanol-insolubleforms remained unchanged from 0 to 250 mM mannitol and declinedabove this. Thus metabolism, but not uptake may be responsiveto changes in turgor. Key words: carrot, sucrose, uptake, transport, turgor     

The Effect of Sucrose Supply on the Energization of Amino Acid Uptake by Cotyledons of Euphorbia lathyris L

The cotyledons of Euphorbia lathyris L. take up sucrose andamino acids from the endosperm. The interaction between theuptake of sucrose and that of amino acids by cotyledons of intactseedlings was investigated. Sucrose (100 mol m^–3) reducedvaline uptake to 75% of the control rate; the active uptakecomponent of valine uptake was reduced from 45 to 25 % of thetotal uptake rate. In a reverse experiment, 100 mol m^–3valine inhibited sucrose uptake by 25%. At 500 mol m^–3sucrose, valine uptake was completely restored to the controlrate, whereas high valine concentrations failed to restore sucroseuptake. The stimulation of valine uptake by sucrose is linkedto the role of sucrose as a primary respiratory substrate. Whenthe cotyledons were bathed in sucrose concentrations rangingfrom 0 to 100 mol m^–3 (these concentrations are non-saturatingwith respect to sucrose uptake), a constant 1.8% of the sucrosetaken up was respired. The K_m of the concentration-dependentsucrose oxidation (44±6 mol m^–3) agreed reasonablywell with that for sucrose uptake (29±6 mol m^–3).When the external sucrose concentration was increased from 100to 600 mol m^–3, the sucrose uptake increased by 30% again,while sucrose oxidation was increased by 300%. This increasewas not due to an increased engagement of the alternative (cyanide-resistant)pathway for respiration. Alternative pathway, Euphorbia lathyris L., fermentation, seedling, sucrose uptake, valine uptake     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Energy Coupled Transport of Sorbitol and Other Sugars into the Protoplast Isolated from Apple Fruit Flesh

The uptake kinetics of sorbitol, sucrose, glucose and fructoseacross the plasma membrane using protoplasts isolated from applefruit flesh (Malus pumila Mill. var. domestica Schneid.) wasinvestigated. When sorbitol was taken up into the cell, PCMBS-sensitivesaturable transport was distinguishable from the diffusive transport.At a low sorbitol concentration, the saturable transport systemaccounted for more than 50% of the total uptake, whereas ata high concentration the diffusive transport system was moredominant. The saturable transport was suggested be a carrier-mediatedtransport system coupled with ATP because the system was inhibitedCCCP or orthovanadate. The K_m value for sorbitol was computedto be 3.6mM. A carrier-mediated transport system coupled withATP was also observed for glucose and fructose with correspondingK_m values of 5.0 and 2.5 mM. However, no saturable transportfor sucrose was observed over a range of 0.1 to 10 mM sucroseconcentration. The relationship among these sugar transportsystems across the plasma membrane, apoplastic unloading, andsugar accumulation vacuoles are discussed. ¹Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received April 8, 1988; Accepted June 8, 1988)     

Storage Lipid Accumulation by Zygotic and Somatic Embryos in Culture

In vitro accumulation of storage lipids occurs in zygotic andsomatic embryos of Brassicu napus L. The concentration of sucrosein the medium modified the pattern of storage lipid accumulationin zygotic and somatic embryos. The sucrose concentration atwhich the maximum amount of lipid is accumulated by the twotypes of embryos is different Analysis of fatty acid compositionshowed that the same fatty acids are present in embryos in vivoand those cultured in vitro although there are quantitativedifferences. The possibility of using this type of system forin vitro production of valuable plant metabolites is discussed Embryo cloning, somatic embryogenesis, in cilro culture, storage lipids, Brussica napus, oilseed rape     

Tonoplast stability and survival of isolated vacuoles in different buffers

The mechanism of sucrose transport into vacuoles isolated from leaf tissue has been studied only in barley (Hordeum vulgare) mesophyll cells. In this tissue, sucrose transport was reported to be a facilitated diffusion. We have observed a facilitated diffusion of sucrose into vacuoles isolated from this tissue. However, no pH dependence was observed. Evidence is presented indicating that the pH dependence of sucrose uptake into vacuoles may be an artifact, reflecting tonoplast instability and survival of isolated vacuoles in different buffers. Apparently vacuoles do not withstand exposure to some commonly used buffers.     

Wild Oat/Barley Interactions: Varietal Differences in Competitiveness in Relation to K+ Supply

Eight cvs of barley (Hordeum vulgare L.) were separately plantedwith Wild Oats (Avena fatua L., genetically pure line CS40)in a sand culture with two external K⁺ concentrations. Substantialdifferences were observed among barley cvs in their abilityto compete with wild oat. The variety Fergus was highly competitiveat both high and low [K⁺]_e, whereas Steptoe was competitiveonly at high [K⁺]_e, and Compana was only weakly competitivewith wild oat. The differences between barley cvs were relatedto their previously reported efficiencies of K⁺ uptake and utilization. Hordeum vulgare L., Avena fatua L., barley, wild oat, competition, K⁺ nutrition, utilization efficiency     

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     

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.     

The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium in the Elongating Stem ofPhaseolus vulgarisL. A Physiological Assessment

Plasmolytic disruption of plasmodesmata interconnecting metaphloemsieve element-companion cell complexes with small and largephloem parenchyma cells in the elongating region of internode2 ofPhaseolus vulgarisL. seedlings did not affect accumulationof phloem-imported¹⁴C-photosynthates and⁸⁶rubidium. The membrane-impermeantdye, 5(6) carboxyfluorescein, loaded into leaf phloem as themembrane-permeant diacetate ester, was found not to move radiallyout of the importing sieve elements in the internode elongationregion. In contrast, the apoplasmic tracer, Calcuofluor White,rapidly moved laterally throughout all tissues of the elongationzone. Hexoses, sucrose and potassium were identified as themain osmotica in internode apoplasmic sap. Label asymmetry in[¹⁴C](fructosyl)sucrose was retained on accumulation by excisedstem segments. Uptake of [¹⁴C]sucrose and⁸⁶rubidium by stemsegments exhibited saturation kinetics. Sucrose uptake was inhibitedby the slowly penetrating sulphydryl reagent, para-chloromercuribenzenesulphonicacid.In vitrorates of sucrose uptake, at apoplasmic concentrations,corresponded to its predictedin vivorate of delivery to thestem ground tissues from mature sieve elements when respiratorylosses were assumed to be confined to the stem phloem. For potassium,the total delivery rate could be accounted for by itsin vitrorateof uptake. Overall, it was concluded that radial transport,in the elongation zone of internode 2 ofPhaseolus vulgarisL.seedlings, follows an apoplasmic route from mature sieve elementsto stem ground tissues.Copyright 1998 Annals of Botany Company PhaseoluLes vulgaris, apoplasm, elongating stem, French bean, photosynthates, potassium, radial transfer, symplasm.