Protoplast hexose carrier activity is a determinate of genotypic difference in hexose storage in tomato fruit

Post-phloem sugar transport in developing tomato (Lycopersicon esculentum Mill. cv. Flora-Dade) fruit follows an apoplastic route during the rapid phase of sugar accumulation. The pathway is characterized by sugar retrieval by the storage parenchyma cells from the fruit apoplast. Two tomato genotypes differing in fruit hexose content were compared in terms of the transport and transfer processes controlling fruit sugar levels. The genotypic difference in fruit sugar content was independent of photoassimilate export from source leaves. Discs of pericarp tissue were cultured in a medium based on analyses of the fruit apoplastic sap. The cultured discs maintained a composition, a relative growth rate and a respiration rate similar to those of the pericarp tissue of intact fruit. Estimates of hexose fluxes into metabolic and storage pools suggested that membrane transport regulated the genotypic difference in hexose accumulation. Short-term [¹⁴C]hexose uptake experiments demonstrated a genotypic difference in V_max for glucose, fructose and 3-O-methyl-glucose, and this difference was abolished in the presence of the inhibitor p-chloromercuribenzenesulphonic acid (PCMBS). The results support the hypothesis that the activity of energized hexose carriers on the plasma membranes of storage parenchyma cells is a significant determinate of the genotypic difference in hexose accumulation.     

The cellular pathway of sucrose transport in developing cotyledons ofVicia faba L. andPhaseolus vulgaris L.: a physiological assessment

The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [¹⁴C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [¹⁴C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS apparent free space - CF 5-(6)-carboxyfluorescein - CFDA 5-(6)-carboxyfluorescein diacetate - Mes 2-(N-morpholino)ethanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SRG sulphorhodamine G The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs.     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

Sucrose uptake and partitioning in discs derived from source versus sink potato tubers

The uptake of sucrose into isolated discs cut from sink (growing) and source (sprouting) potato (Solanum tuberosum L.) tuber tissue was studied. The uptake of sucrose into sink-tuber discs demonstrated biphasic kinetics. The large saturable component was inhibited by incubation of the discs with p-chloromercuribenzene sulfonic acid (PCMBS) whilst both the saturable and linear components were inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP). By contrast, in source-tuber discs, the linear component represented the majority of sucrose taken up, the saturable component playing only a minor role. In source discs, only the saturable component of uptake was inhibited by either PCMBS or CCCP. A large proportion (up to 25%) of sucrose taken up into sink-tuber discs was converted to starch but as the tubers aged the proportion of sucrose converted to starch decreased to the level found in source-tuber discs (approx. 3%). By contrast with sink-tuber discs (see Oparka and Wright, 1988b, Planta 175, 520–526) sucrose uptake into source discs was insensitive to turgor and demonstrated an uptake pattern similar to that of CCCP-treated sink tissue. It is proposed that exogenous sucrose is taken into the storage parenchyma of sink-tuber discs by both a carrier-mediated and a diffusional process. By contrast, uptake into the storage parenchyma of source-tuber discs appears to be essentially diffusional. The turgor sensitivity of sucrose uptake into sink-tissue discs may be mediated via the plasmalemma H⁺-ATPase. As the tuber ages the sucrose-uptake activity decreases and the capacity of the storage parenchyma to synthesise starch is lost. The data are discussed in relation to the in-vivo mechanisms of sucrose transport in storage tissues.     

Asymmetry of glucose transport in the yeast, Kluyveromyces lactis

Uptake and efflux of 6-deoxy-d-[³H]glucose and of 2-deoxy-d-[¹⁴C]glucose by the yeast Kluyveromyces lactis was studied. The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-d-glucose 6-phosphate. In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-d-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar. In starved, poisoned cells containing negligible amounts of 2-deoxy-d-glucose 6-phosphate, 2-deoxy-d-glucose does leave the cells upon transfer to glucose-free medium. An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested. An internal pool of 6-deoxy-d-glucose does not affect the rate of uptake of 6-deoxy-d-[³H]glucose, nor does internal 2-deoxy-d-[¹⁴C]glucose 6-phosphate influence that rate. Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.     

Properties of proton and sugar transport at the tonoplast of tomato (Lycopersicon esculentum) fruit

Tonoplast vesicles were isolated from tomato (Lycopersicon esculentum Mill.) fruit pericarp and purified on a discontinuous sucrose gradient. ATPase activity was inhibited by nitrate and bafilomycin A₁ but was insensitive to vanadate and azide. PPase hydrolytic activity was inhibited by NaF but was insensitive to nitrate, bafilomycin A₁ vanadate and azide. Kimetic studies of PPase activity gave an apparent K_m, for PP₃ of 18 μM. Identical distributions of bafilomycin- and NO₃-sensitive ATPase activities within continuous sucrose density gradients, confirmed that bafilomycin-sensitive ATPase activity is a suitable marker for the tonoplast. By comparing the distribution of bafilomycin-sensitive ATPase activity with that of PPase activity, it was possible to locate the PPase enzyme exclusively at the tonoplast. The apparent density of the tonoplast did not change during fruit development. Measurements of tonoplast PPase and ATPase activities during fruit development over a 35-day period revealed an 80% reduction in PPase specific activity and a small decrease in ATPase specific activity. ATP- and PP₁-dependent ΔpH generation was measured by the quenching of quinacrine fluorescence in tonoplast vesicles prepared on a discontinuous Dextran gradient. No H⁺ efflux was detected on the addition of sucrose to energized vesicles. Therefore a H⁺/sucrose antiport may not be the mechanism of sucrose uptake at the tomato fruit tonoplast. Similar results were obtained with glucose, fructose and sorbitol. The lack of ATP (or PP₁) stimulation of [¹⁴C]-sucrose uptake also suggested that an antiport was not involved. Initial uptake rates of radiolabelled glucose and fructose were almost double that for sucrose. The inhibition of hexose uptake by p-chloromercuribenzene sulphonate (PCMBS) implicated the involvement of a carrier. Therefore storage of hexose in the tomato fruit vacuole and maintenance of a downhill sucrose concentration gradient into sink cells is likely to be regulated by the activity of sucrose metabolizing enzymes, rather than by energy-requiring uptake mechanisms at the tonoplast.     

Parachloromercuribenzenesulfonic Acid : a potential tool for differential labeling of the sucrose transporter

Vicia faba leaf discs without epidermis were pretreated with parachloromercuribenzenesulfonic acid (PCMBS), rinsed and incubated on [¹⁴C]sucrose (1 or 40 millimolar). Those sucrose concentrations were chosen as representative of the apparent uptake system 1 (1 millimolar) and system 2 (40 millimolar) previously characterized. Pretreatment with 0.5 millimolar PCMBS for 20 minutes inhibited system 1 and system 2 by about 70%.

Addition of unlabeled sucrose during PCMBS-pretreatment protected the carrier(s) from the inhibition, whereas glucose, fructose, and sucrose analogs were unable to afford protection. At 1 millimolar [¹⁴C]sucrose, the protection resulted in a small but consistent reduction of normal inhibition (from 63 to 45%) for sucrose concentrations of 50 millimolar and more during pretreatment. Contrarily, at 40 millimolar [¹⁴C]sucrose, the protection increased linearly with the sucrose concentration in the pretreatment medium, and complete prevention of inhibition was reached for 250 millimolar sucrose.

The protection was not due to exchange diffusion and was located in the veins. Michaelian kinetics indicated that PCMBS and sucrose compete with each other at the active site of the carrier.

Among 14 compounds tested (sugars, amino-acids, hormones, ³²P), sucrose uptake was by far the most sensitive to PCMBS. Sucrose preferentially protected its carrier(s) from inhibition. Treatment with 20 millimolar cysteine or 20 millimolar dithioerythreitol reversed inhibition by PCMBS pretreatment.

     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

The effect of hypoxia on the control of carbohydrate metabolism in ripening bananas

The aim of this work was to determine the effects of hypoxia on the major fluxes of carbohydrate metabolism in climacteric fruit of banana (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in air at 21°C in the dark. When the climacteric began, fruit were transferred to 15 or 10% oxygen and were analysed once the climacteric peak had been reached 8–12 h later. The rates of starch breakdown, sucrose, glucose and fructose accumulation, and CO₂ production were determined, as were the contents of hexose monophosphates, adenylates and pyruvate. In addition, the detailed distribution of label was determined after supplying [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]- and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol to cores of tissue under hypoxia. The data were used to estimate the major fluxes of carbohydrate metabolism. There was a reduction in the rate of respiration. The ATP/ADP ratio was unaffected but there was a significant increase in the content of AMP. In 15% oxygen only minor changes in fluxes were observed. In 10% oxygen starch breakdown was reduced and starch synthesis was not detected. The rate of sucrose synthesis decreased, as did the rate of re-entry of hexose sugars into the hexose monophosphate pool. There was a large increase in both the glycolytic flux and in the flux from triose phosphates to hexose monophosphates. It is argued that the increase in these fluxes is due to activation of pyrophosphate: fructose-6-phosphate 1-phosphotransferase, and that this enzyme has an important role in hypoxia. The results are discussed in relation to our understanding of the control of carbohydrate metabolism in hypoxia.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - PP_i inorganic pyro-phosphate We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. S.A.H. thanks the managers of the Brood bank Fund for a fellowship.     

Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

The rate of decarboxylation of [1′-¹⁴C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl₂, inhibited the development of an enhanced capacity to decarboxylate [1′-¹⁴C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-¹⁴C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.     

Effect of abscisic acid (ABA) on sugar accumulation in the flesh tissue of peach fruit at the start of the maturation stage

Experiments were conducted on¹⁴C-sorbitol, fructose, and glucose uptakeinto flesh discs, and sorbitol efflux from thediscs, with and without ABA application toexamine the effect of abscisic acid (ABA) onsugar accumulation in peach fruit flesh at thestart of the maturation stage in relation tomembrane transport. Total uptake of¹⁴C-sorbitol, fructose, and glucose intoflesh discs was effectively promoted by ABA ata concentration of 10^–5 M. PCMBS(p-chloromercuribenzensulfonicacid)-sensitive uptake, which was considered ascarrier-mediated uptake, of sorbitol into thediscs was clearly stimulated by ABA at10^–5 M, compared with glucose andfructose uptake. Sorbitol efflux from the discsacross the tonoplast was restricted by ABA at10^–5 M. ABA application todeveloping fruit increased sugar accumulationin the fruit. Estimated ABA concentration inthis fruit was approximately 10^–5 M. These results indicate that sugar accumulationin peach fruit flesh is stimulated by ABA at aconcentration of 10^–5 M both invitro and in vivo. ABA stimulatesuptake of sugars, especially sorbitol, into theflesh by enhancing carrier-mediated transportpossibly across both tonoplast and plasmamembrane.     

Stimulation of sugar exit from leaf tissues ofVicia faba L.

After removal of the lower epidermis, leaf discs ofVicia faba L. were loaded with either [¹⁴C]sucrose or [³H]3-O-methylglucose (3-O-MeG). The exit of preloaded sucrose was strongly stimulated when sucrose was present in the bathing medium, and the exit of 3-O-MeG was also markedly increased in the presence of 3-O-MeG. This specific stimulation exhibited single saturation dependence on the external concentration of sugar (K _m=9 mM for sucrose, 5 mM for 3-O-MeG), and was sensitive to low temperature, uncouplers and thiol reagents. Sucrose exit was never affected by 3-O-MeG in the bathing medium. Sucrose did not affect the exit of 3-O-MeG in fresh discs, but promoted this exit in discs previously aged for 12 h, indicating partial external hydrolysis of sucrose in the latter tissues. Ageing also dramatically increased the exit of 3-O-MeG induced by 3-O-MeG but had no effect on the exit of sucrose induced by sucrose. The ability of 53 compounds (pentoses, hexoses, hexose-phosphates, polyols, di- and trisaccharides, phenyl- and nitrophenyl-derivatives, sweeteners) to interact with the sucrose carrier and with the hexose carrier was tested. Sucrose, maltose, -phenylglucoside andp-nitrophenyl--glucoside interacted with the sucrose carrier.d-glucose,d-xylose,d-fucose,d-galactose,d-mannose, 3-O-MeG and 2-deoxyglucose interacted with the hexose carrier.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - 3-O-MeG 3-O-methylglucose - PCMBS p-chloromercuribenzenesulphonic acid     

The effect of glucose on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO₂ production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-¹⁴C]-, [1-¹⁴C]-, [3,4¹⁴C]and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - AEC adenylate energy charge We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. SAH thanks the managers of the Broodbank Fund for a fellowship.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

Two experimental systems were developed to study the uptake of sucrose by the dermal transfer cells of developing cotyledons of Vicia faba L. First, the in-vivo state was approximated by short-term (10 min) incubation of whole cotyledons in [¹⁴C]sucrose solutions. Under these conditions, a minimum of 67% of the ¹⁴C label entered the dermal transfer cell complex. Of this, at least 40% crossed the plasma membranes of the epidermal transfer cells. Second, a protocol was developed to enzymatically isolate and purify dermal transfer cell protoplasts. The yields of the transfer cell protoplasts were relatively low and their preparation incurred a significant loss of plasma membrane. However, the protoplasts remained viable up to 24 h following purification and proved to be a suitable system to verify transport properties observed with whole cotyledons. Using these two experimental systems, it was established that [¹⁴C]sucrose uptake by the dermal transfer cells exhibited features consistent with mediated energy-dependent transport. This included saturation kinetics, competition for uptake between structurally similar molecules, and inhibition of uptake by p-chloromercuribenzenesulfonic acid and several other metabolic inhibitors. For comparative purposes, sugar uptake by the storage parenchyma of the Vicia cotyledons was also examined. In contrast to the dermal transfer cell complex, sucrose uptake by the storage parenchyma displayed characteristics consistent with simple diffusion.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid The investigation was supported by funds from the Research Management Committee, the University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

Metabolism in slices from growing potato tubers responds differently to addition of sucrose and glucose

The short-term changes in metabolism that occurred after adding glucose or sucrose to freshly cut discs from growing potato (Solanum tuberosum L.) tubers were investigated. (i) When glucose was supplied, there was a marked increase in glycolytic metabolites, and respiration was stimulated. When sucrose was supplied, amounts of glycolytic metabolites including hexose phosphates and 3-phosphoglycerate (3PGA) were similar to or lower than in control discs incubated without sugars, and respiration did not rise initially above that in control discs. This different response to sucrose and glucose was found across the concentration range 5–200 mM. A larger proportion of the metabolised ¹⁴C was converted to starch when [¹⁴C] sucrose was supplied than when [¹⁴C] glucose was supplied. The different effect on metabolite levels, respiration and starch synthesis was largest after 20–30 min, and decreased in longer incubations. (ii) When 5 or 25 mM sucrose was added in the presence of [¹⁴C] glucose, it led to a decrease in hexose phosphates and 3PGA, and a small increase in the rate of starch synthesis compared to discs incubated with glucose in the absence of sucrose. These differences were seen in a 30-min pulse and a 2-h pulse. Whereas ADP-glucose levels after adding sucrose resembled those in control discs, glucose led to a decrease in ADP-glucose. This decrease did not occur when 5 or 25 mM sucrose was added with the glucose. (iii) To check the relevance of these experiments for intact tubers, water or 100 mM mannitol, sucrose or glucose were supplied through the stolon to intact tubers for 24 h. A 0.2 mM solution of [¹⁴C] glucose was then introduced into the tubers, and its metabolism investigated during the next 30 min. Labelling of starch was increased after preincubation with sucrose, and significantly inhibited after preincubation with glucose. (iv) It is concluded that glucose and sucrose have different effects on tuber metabolism. Whereas glucose leads to a preferential stimulation of respiration, sucrose preferentially stimulates starch synthesis via a novel mechanism that allows stimulation of ADP-glucose pyrophosphorylase even though the levels of hexose phosphates and the allosteric activator 3PGA decrease. Received: 9 October 1997 / Accepted: 3 February 1998     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm

The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [¹⁴C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [¹⁴C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [¹⁴C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [¹⁴C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm.     

C]GA(12)-Aldehyde, [C]GA(12), and [H]- and [C]GA(53) Metabolism by Elongating Pea Pericarp

Gibberellins (GAs) are either required for, or at least promote, the growth of the pea (Pisum sativum L.) fruit. Whether the pericarp of the pea fruit produces GAs in situ and/or whether GAs are transported into the pericarp from the developing seeds or maternal plant is currently unknown. The objective of this research was to investigate whether the pericarp tissue contains enzymes capable of metabolizing GAs from [¹⁴C]GA₁₂-7-aldehyde ([¹⁴C]GA₁₂ald) to biologically active GAs. The metabolism of GAs early in the biosynthetic pathway, [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂ald, was investigated in pericarp tissue isolated from 4-day-old pea fruits. [¹⁴C]GA₁₂ald was metabolized primarily to [¹⁴C]GA₁₂ald-conjugate, [¹⁴C]GA₁₂, [¹⁴C]GA₅₃, and polar conjugate-like products by isolated pericarp. In contrast, [¹⁴C]GA₁₂ was converted primarily to [¹⁴C]GA₅₃ and polar conjugate-like products. Upon further investigations with intact 4-day-old fruits on the plant, [¹⁴C]GA₁₂ was found to be converted to a product which copurified with endogenous GA₂₀. Lastly, [²H]GA₂₀ and [²H]GA₁ were recovered 48 hours after application of [²H]- and [¹⁴C]GA₅₃ to pericarp tissue of intact 3-day-old pea fruits. These results demonstrate that pericarp tissue metabolizes GAs and suggests a function for pericarp GA metabolism during fruit growth.     

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.     

Comparison of ripening processes in intact tomato fruit and excised pericarp discs

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L^*a^*b^* color space, changing from green (a^* < −5) to red (a^* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO₂ production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a^* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a^* = −5), appeared as discs turned yellow-red (a^* = 5), and increased another eight-fold as discs became full red (a^* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a^* = 0) to red.