JXB Advance Access originally published online on May 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1691-1700,
July 1, 2003
© 2003 Oxford University Press
Sugar uptake and proton release by protoplasts from the infected zone of Vicia faba L. nodules: evidence against apoplastic sugar supply of infected cells
Received 21 January 2003; Accepted 10 April 2003
Institute of Plant Nutrition, Interdisciplinary Research Center (IFZ), Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
* Present address and to whom correspondence should be sent: Biology Department, Area 9, University of York, PO Box 373, York YO10 5YW, UK. Fax: +44 (0)1904 32 8666. E-mail: ep5{at}york.ac.uk
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Abstract |
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Symbiotic dinitrogen fixation of legume nodules is fuelled by phloem-imported carbohydrates. These have to pass several cell layers to reach cells infected with Rhizobium bacteroids. It is unclear whether apoplastic steps are involved in carbohyd rate translocation within the nodule. Protoplasts were isolated from the infected and uninfected cells of the central tissue of Vicia faba nodules using a recently developed protocol. These protoplasts were used to elucidate pathways for sugar transport in this tissue. Both types of protoplasts released protons into the medium. Acidification was inhibited by vanadate and erythrosin B. However, it was stimulated by fusicoccin only in uninfected cells. A symport of sugars with protons can therefore be energized in both cell types. Uptake of 14C-labelled sugars was determined using a phthalate centrifugation technique. Uninfected protoplasts accumulated glucose through high-affinity H+/glucose-symport that was not competitively inhibited by fructose or sucrose. Uninfected protoplasts also absorbed sucrose with biphasic kinetics. At 0.1, 1, and 10 mM sucrose, uptake was inhibited by CCCP. Fusicoccin did not stimulate the linear phase of sucrose uptake. Glucose inhibited sucrose uptake nearly completely. This was not related to sucrose cleavage in the medium because sucrose was absorbed at a much higher rate than glucose, and glucose concentration did not increase in sucrose-containing protoplast suspensions. By contrast with uninfected protoplasts, infected cells did not show transporter-mediated glucose or sucrose uptake. The findings underline a role of uninfected cells in sugar translocation. Infected cells are not apoplastically supplied with sugars and possibly depend on uninfected cells for carbon supply.
Key words: Faba bean, glucose uptake, membrane transport, metabolite transport, plasma membrane, proton release, root nodule, sucrose uptake.
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Introduction |
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Leguminous plants are able to form root nodules containing rhizobial bacteria, that are capable of reducing atmospheric N2 to NH3. To carry out symbiotic nitrogen fixation, legume nodules are phloem-supplied with photosynthesis-derived sucrose (Streeter, 1995). The phloem is part of the nodule vascular network, which is located within the inner cortex and enclosed by an endodermis with Casparian band (Brown and Walsh, 1994; Abd-Alla et al., 2000; Hartmann et al., 2002). This apoplastic barrier requires solutes to cross the endodermis in a symplastic way. Since there are no apoplastic barriers in the nodule centre, further movement to cells infected with rhizobial bacteria may be symplastic or apoplastic. Symplastic distribution of sugars within the nodule’s central tissue is supported by the presence of rays of uninfected cells interconnected by a high number of plasmodesmata (Brown et al., 1995). Infected cells adjacent to this putative solute route may be supplied symplastically, but non-adjacent cells are symplastically isolated (Abd-Alla et al., 2000). It is therefore likely that plasma membrane sugar transporters are active in infected cells, allowing these cells to take up sugars from the apoplast. Uninfected cells may also possess transport systems to retrieve leaked sugars, as it is the case for phloem cells (Kühn et al., 1999).
The processes of dinitrogen fixation and assimilation may be affected by transport steps that limit solute fluxes. However, the transport pathways of imported carbohydrates within the nodule centre are not known and sugar fluxes across plasma membranes of nodule cells have not yet been determined. The aim, therefore, was to compare the plasma membrane transport characteristics of both cell types. The preparation of pure plasma membrane vesicles from nodule tissue was not possible, because plasma and peribacteroid membranes cannot be separated (F Yan, unpublished data). In addition, membrane vesicles from root nodules would originate from infected as well as uninfected cells. For that reason, it was decided to use protoplasts to study sugar transport. The isolation of infected protoplasts from root nodules has already been performed in the 1970s (Davey et al., 1973). Infected nodule protoplasts described in this and subsequent studies were of irregular, non-spherical shape (for a review see Peiter et al., 2003). However, closer examination revealed that the plasma membrane of infected protoplasts isolated according to conventional protocols was not intact (Peiter et al., 2003). A new procedure for the gentle isolation and separation of infected and uninfected protoplasts from root nodules of Vicia faba L. has been developed (Peiter et al., 2003). In the present study this material was used to examine glucose and sucrose transport. It has been shown for many plant systems that sugar uptake across the plasma membrane is dependent on the electrochemical proton gradient, generated by the activity of a plasma membrane H+-ATPase (Bush, 1993). Therefore, the ability of infected and uninfected protoplasts to create a proton motive force was also examined. The sugar uptake studies indicate a possible role of uninfected cells in the carbon supply of infected cells.
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Plant material
Vicia faba L. cv. Troy plants were inoculated with Rhizobium leguminosarum bv. viceae strain 6044 (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany) and grown in a pH-controlled nutrient solution as described previously (Hartmann et al., 2002). Cultivation took place in a walk-in growth chamber with 16 h day length. Temperatures were 22/17 °C day/night. Plants were illuminated by sodium high pressure discharge lamps (Philips SON-T AGRO 400, 500–900 µE m–2 s–1 at plant height). Nodules of 7–8-week-old plants were used for experiments.
Isolation and separation of protoplasts
Protoplasts were isolated from the central tissue of V. faba nodules by a newly developed procedure based upon osmotic expansion of protoplasts within digested coherent tissue (Peiter et al., 2003). In brief, nodules were dissected into the cortex and the central infected tissue. Meristematic and senescent parts were discarded and the remaining block of central tissue was placed onto a nylon screen submerged in suspension medium (Peiter et al., 2003). The screen with collected tissue pieces was transferred into enzyme suspension (Peiter et al., 2003), containing Cellulase Onozuka RS, Pectinase, Driselase, and Pectolyase. After digestion, the screen with the still coherent tissue was removed from the enzyme suspension and placed into the uppermost, slightly hypotonic layer of a combined osmolarity-density step gradient. The screen with remaining tissue was removed after the release of protoplasts. Infected and uninfected protoplasts were separated by low-speed centrifugation (15 g) of the gradient and subsequently collected from the gradient interfaces. Protoplasts to be used for experiments were washed three times by underlaying the suspension with new medium (Peiter et al., 2003).
Measurement of net proton release by infected and uninfected protoplasts
All steps were carried out at 20 °C. Infected and uninfected protoplasts isolated from 100 nodules and suspended in suspension medium SM 2b (Peiter et al., 2003) were aliquoted (3x1 ml). Immediately before measurement of net proton release, protoplasts were pelleted by slow centrifugation (infected fraction: 1 min at 8 g; uninfected fraction: 1 min at 80 g), the medium was removed and protoplasts were washed three times in 4 ml test medium (2.5 mM K2SO4, 1.0 mM MgSO4, 0.5 mM CaCl2, 390 mM mannitol). After the last pelleting, all supernatant was removed and 500 µl of test medium were added. Test medium pH changes were measured by means of a pH micro-electrode (InLab 423, Mettler, Giessen, Germany) connected to a precision pH meter (CG 805, Schott, Mainz, Germany). After determination of medium acidification by untreated protoplasts, these were washed as described above and incubated in treatment solution (Fig. 1). A comparison of net proton release into control and treatment solution shows relative treatment effects. If the test medium contained effectors, the solution of the first incubation (control) contained the effector solvent. It was determined in pre-experiments that net proton release did not change in two subsequent incubations of untreated protoplasts.
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Uptake of 14C-labelled glucose and sucrose by infected and uninfected protoplasts
Uptake studies were carried out with freshly isolated protoplasts. Accumulation of D-(U-14C) glucose (14C-Glc; Amersham Pharmacia Biotech (APB), Freiburg, Germany), D-(U-14C) sucrose (14C-Suc; APB), and (14C-carboxyl)-dextran-carboxyl (Biotrend, Köln, Germany) was examined. Specific activities of 14C-Glc, 14C-Suc, and 14C-dextran were 74 kBq ml–1 in suspensions of uninfected protoplasts and 37 kBq ml–1 in suspensions of infected protoplasts. Protoplast volume was determined using 3H2O (370 kBq ml–1; APB) as described by Fieuw and Willenbrink (1991). Uptake experiments with membrane-impermeable 14C-dextran (MW 70 000) were carried out to determine membrane leakiness. The ratio of 14C/3H quotients in medium and protoplasts at different incubation times, indicating membrane integrity, did not change during the incubation of infected and uninfected protoplast fractions (data not shown).
Protoplast yields varied among experiments. Further, protoplast suspensions were not completely homogenized to avoid physical damage. Therefore, protoplast volumes used in the uptake experiments varied. Per experiment 50–100 nodules were dissected, yielding 20–70 nl uninfected and 100–300 nl infected protoplasts per nodule. Per sample (100 µl protoplast suspension), 50–150 µl uninfected or 300–900 µl infected protoplasts were used. Metabolite uptake was started by simultaneously adding labelled and non-labelled metabolites to the protoplast suspension. 3H2O was added at least 30 min and effectors exactly 3 min before the start of uptake. If effectors were tested, control solutions contained the effector solvent. During metabolite uptake, the protoplast suspension was incubated at 25±1 °C. The assay was terminated by a modified oil centrifugation method (Klingenberg and Pfaff, 1967) as follows: an aliquot (100 µl) of protoplast suspension was transferred into a micro tube (6x46 mm; 400 µl; Sarstedt, Germany), in which a mixture (200 µl) of di-isoheptyl phthalate and dibutyl phthalate had been layered onto a HClO4 cushion (60 ml l–1; 10 µl). Protoplasts were separated from medium by centrifugation (1 min, 12 000 rpm; 2-MK centrifuge, Sigma, Osterode, Germany) through the phthalate layer. Phthalate density was 1.037 kg l–1 for infected and 1.009 kg l–1 for uninfected protoplasts. After uptake experiments, tubes were frozen (–20 °C). For liquid scintillation counting of protoplast pellets, the tip of the frozen tube was cut off and the content was shaken into 200 µl sodium dodecyl sulphate (SDS; 10 g l–1). After centrifugation (5 min, 13 000 rpm), 170 µl of the upper aqueous phase were mixed with 3 ml liquid scintillation cocktail (Lumasafe, Canberra-Packard, Dreieich, Germany). No activity was found in the non-aqueous phthalate phase. For the determination of 3H and 14C activities in protoplast suspensions, 10 µl were added to 3 ml scintillation cocktail. Activities (decays min–1) were determined using a liquid scintillation counter (2700 TR; Canberra-Packard) with automatic correction for background and quench. Energy windows for 3H and 14C were set to 0–12 keV and 12–156 keV, respectively.
Calculation of uptake rates
The accumulation of metabolites by protoplasts was calculated from 14C/3H activity ratios in the medium and protoplasts, and the metabolite concentration in the medium:
Uptake rates were related to plasma membrane surface area (mm2), which was calculated using protoplast diameters determined in representative micrographs. Infected protoplasts had a diameter of 52.2±0.4 µm (±SE; n=200), uninfected ones measured 35.3±0.6 µm (±SE; n=200). Thus 1 ml of infected and uninfected protoplasts contained 115 mm2 and 170 mm2 plasma membrane, respectively. Kinetic data were determined using SigmaPlot 7.0 (Enzyme Kinetics Module 1.1) software. Data were fitted by non-linear regression to a Michaelis–Menten equation and by linear regression after Eadie–Hofstee transformation.
Sucrose cleavage in suspensions of infected and uninfected protoplasts
Infected and uninfected protoplasts were isolated from the central tissue of 80 nodules and suspended in SM 2b medium (uninfected fraction) or SM 2b medium with 10% Ficoll (infected fraction) (Peiter et al., 2003). Assays were started by the addition of sucrose to a final concentrations of 10, 100, or 1000 µM and terminated by centrifugation of the protoplasts into phthalate oil after 5, 10, and 15 min as described above. Phthalate tubes were frozen in liquid N2 after centrifugation. The concentrations of glucose and fructose in the incubation medium were determined enzymatically (R-biopharm, Darmstadt, Germany).
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Net proton release by infected and uninfected protoplasts
Infected and uninfected protoplasts were able to acidify an unbuffered incubation medium. This net proton release was prevented by osmotically destroying the protoplasts in a hypertonic medium (Fig. 1A, B). The effect of effectors on medium acidification was determined by initially incubating protoplasts without, and subsequently with effector. Fusicoccin stimulated net proton release from uninfected protoplasts, but had no effect on infected proto plasts (Fig. 1C, D). Erythrosin B and vanadate inhibited net proton release from both cell types (Fig. 1E–H).
Glucose uptake by infected and uninfected protoplasts
Kinetics of glucose uptake: Net uptake of 14C-glucose by infected and uninfected protoplasts from medium containing 10 µM to 1 mM glucose was examined. In initial experiments, 14C accumulation was determined in triplicate after 5 to 15–30 min incubation. Uptake was linear over this period of time (Fig. 2). Due to a limited availability of material and a low variation within separate experiments, uptake kinetics were determined in the following experiments from two exposure times (5, 10 min) and 16–20 concentrations. Uninfected protoplasts accumulated 14C with a considerably higher rate than infected protoplasts (Fig. 3A). Since uptake rates of uninfected protoplasts varied considerably between experiments, exact kinetic data could not be deduced. However, saturation kinetics are clearly apparent (Fig. 3B). Non-linear fitting of all uptake rates of uninfected protoplasts to a Michaelis–Menten equation (Fig. 3A) revealed an affinity (Km) of 17±7 µM Glc and a maximum velocity (Vmax) of 0.18±0.014 pmol Glc mm–2 plasma membrane min–1 (±SE). No clear kinetic data could be obtained for infected protoplasts (Fig. 3B insert).
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Mechanism and specificity of glucose uptake: Uninfected protoplasts were able to accumulate 14C activity from glucose more than 10-fold with respect to the external concentration (Fig. 3C). In contrast, infected protoplasts did not accumulate 14C, even after 30 min incubation. In uninfected protoplasts the protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced glucose uptake by about 84%, whereas it was stimulated over 2-fold by the P-type H+-ATPase stimulator, fusicoccin (Table 1). The low uptake rates of infected protoplasts were neither inhibited by CCCP, nor stimulated by fusicoccin (Table 1). The specificity of 14C-glucose uptake by uninfected protoplasts was examined by including a 10-fold higher concentration of competing sugar in the medium (Table 2). Glucose uptake was not inhibited by fructose or sucrose.
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Sucrose uptake by infected and uninfected protoplasts
Kinetics of sucrose uptake: Net uptake of 14C sucrose by infected and uninfected protoplasts was examined at concentrations ranging from 10 µM to 40 mM. Linearity of uptake was checked by sampling after 5–15 min uptake (Fig. 4). At high sucrose concentrations, there was sometimes a slight decrease in uptake after 15 min (Fig. 4C). This may be due to 14C efflux and can lead to an underestimation of uptake rates. At all concentrations uptake rates were considerably higher in uninfected protoplasts than in infected ones (Fig. 5A–C). As determined from Michaelis–Menten plots (Fig. 5A–C, dashed lines) and an Eadie–Hofstee plot (Fig. 5D, dashed line), linear regression (y=0.31x; r2=0.98) fitted well to the data. However, neither linear regression (Fig. 5C, dashed line) nor non-linear fitting to a Michaelis–Menten equation (Fig. 5, solid line) of the whole data set fitted well to uptake rates at low sucrose concentrations. This is also evident from an Eadie–Hofstee plot (Fig. 5E). Instead, a high-affinity uptake with Michaelis–Menten type kinetics (r2=0.95; Km 299±70 µM Suc; Vmax 0.23±0.03 pmol Suc mm–2 plasma membrane min–1; ±SE) is apparent at low sucrose concentration (Fig. 5E, dash-dotted line). By contrast with uninfected protoplasts, sucrose uptake by infected protoplasts was extremely low even at high concentrations (up to 40 mM) and was only weakly concentration-dependent (Fig. 5A–C). This is reflected in the weak correlation of the linear regression (y=0.035x; r2=0.36) and the scatter after transformation to an Eadie–Hofstee plot (Fig. 5D insert).
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Mechanism and specificity of sucrose uptake: As described above for glucose, uninfected protoplasts also accumulated 14C from sucrose against an activity gradient (Fig. 5F). This was not the case for infected protoplasts. Effects of CCCP and fusicoccin were examined at sucrose concentrations of 0.1, 1, and 10 mM to account for the possible involvement of multiple uptake systems (Table 3). At all concentrations, sucrose uptake was strongly inhibited by CCCP. Fusicoccin stimulated sucrose uptake slightly at low sucrose concentration (Table 3). A comparison of single experiments at 0.1 mM sucrose indicated that the degree of stimulation was negatively correlated to the uptake rate of the control (data not shown). The uptake of 14C-labelled sucrose (1 mM) was strongly inhibited by glucose (10 mM; Table 2).
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Sucrose cleavage in suspensions of infected and uninfected protoplasts: To address the question of whether sucrose is cleaved before uptake by protoplasts, infected and uninfected protoplasts were incubated in sucrose-containing suspension media, as used for uptake experiments. Sampling after 5, 15, and 30 min showed that monosaccharide concentrations did not change over that incubation period (data not shown). About 10 µM glucose and fructose were detected in media without protoplasts (Fig. 6A). This concentration was close to the detection limit of the enzymatic assay. Addition of 100 or 1000 µM sucrose to media containing uninfected protoplasts led to slightly increased glucose, but not fructose concentrations (Fig. 6B). Glucose concentrations were always higher in suspensions of infected protoplasts than in uninfected fractions (Fig. 6C). In media without added sucrose, suspensions of infected protoplasts contained c. 20 µM glucose. The addition of sucrose slightly increased the glucose and fructose concentrations.
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Discussion |
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The aim of the present work was to elucidate potential sugar transport pathways within the infected tissue of Vicia faba nodules by means of uptake studies with protoplasts isolated using a new technique (Peiter et al., 2003). It has been shown previously that infected and uninfected nodule protoplasts isolated according to this protocol are osmotically active and exclude the dye propidium iodide, indicating the intactness of their plasma membrane (Peiter et al., 2003). However, it is essential that subsequent experimental procedures would not lead to cell damage. Incubation with 3H2O and membrane-impermeable 14C-dextran (Getz et al., 1987; Fieuw and Willenbrink, 1991) showed that the cell integrity of infected and uninfected protoplasts remained at a constantly high level (data not shown).
Infected and uninfected cells isolated from the central nodule tissue release protons
Sugar uptake across the plant plasma membrane is usually driven by the proton-motive force (Bush, 1993), which is generated by MgATP-fuelled H+-pumps in the plasma membrane (Mengel and Schubert, 1985). Both infected and uninfected protoplasts released protons into their surrounding medium (Fig. 1). This was inhibited by osmotically bursting the protoplasts and by the addition of erythrosin B or vanadate, strongly indicating an H+-ATPase-mediated proton release by both protoplast types. Proton release by uninfected protoplasts was stimulated by the fungal toxin fusicoccin, a commonly observed feature of plant tissues (Marré, 1979). Unexpectedly, fusicoccin had no effect on net H+ release by infected protoplasts. The specific stimulation of P-type H+-ATPases by fusicoccin is due to the stabilization of a phosphorylation-dependent complex with 14-3-3 protein (Maudoux et al., 2000). Specific dephosphorylation of the H+-ATPase or fusicoccin-independent stable binding of 14-3-3 are therefore possible causes for the lack of fusicoccin stimulation on infected protoplasts. Alternatively, differently fusicoccin-sensitive H+-ATPase isoforms may be expressed in both cell types. The presence of different isoforms is supported by the findings of Campos et al. (1996), who detected a plasma membrane H+-ATPase (BHA1) in uninfected, but not in infected cells of the central tissue of Phaseolus vulgaris nodules.
Uninfected cells isolated from the central tissue of the nodule take up glucose
Uninfected protoplasts from the central tissue of V. faba nodules accumulated glucose with high affinity (Km 17±7 µM) and saturable kinetics (Fig. 3). Strong inhibition by the protonophore CCCP and stimulation by fusicoccin (Table 1) indicated a dependence of uptake on the electrochemical proton gradient accross the plasma membrane. These results suggest that glucose uptake into uninfected cells is mediated by high-affinity glucose/H+-symport, as has been shown before in intact plant tissue (Stanzel et al., 1988a) and protoplasts (Lin et al., 1984a, b; Getz et al., 1987; Fieuw and Willenbrink, 1991; Ritte et al., 1999) of various species. In competition experiments (Table 2), glucose uptake by uninfected protoplasts was not affected by fructose. This is in accordance with the observation that plant monosaccharide transporters generally prefer glucose over fructose (Fieuw and Willenbrink, 1991; Ritte et al., 1999). In some systems (Getz et al., 1987), fructose did not compete at all with glucose for uptake, as was the case in the present work.
Glucose uptake by uninfected protoplasts showed a considerable degree of variation between experiments (Fig. 3). This may have been due to a variable representation of different developmental stages of the nodule, which are present side-by-side in indeterminate nodules formed by V. faba (Abd-Alla et al., 2000). A developmentally regulated transporter may be differentially expressed in the nodule. Similarly, tissue- and development-specific expression has been demonstrated for the V. faba monosaccharide transporter VfSTP1 in developing seeds (Weber et al., 1997). Kinetics of this transporter are very similar to the glucose uptake system found in the present study.
Uninfected cells isolated from the central nodule tissue take up sucrose
By contrast with glucose uptake, protoplasts accumulated sucrose with an apparently biphasic kinetic (Fig. 5). Biphasic and multiphasic kinetics of sucrose uptake are common to various plant systems (Maynard and Lucas, 1982a, b; Lin et al., 1984b; Stanzel et al., 1988a, b; Ritte et al., 1999), including leaf tissue (Delrot, 1981) and cotyledons (McDonald et al., 1996a) of V. faba. The linear uptake phase of uninfected protoplasts was inhibited by CCCP (Table 3), indicating a dependence on the electrochemical proton gradient. However, this system was not stimulated by fusicoccin (Table 3). Similar characteristics (a CCCP effect but no fusicoccin effect) have also been observed for low-affinity sucrose uptake by V. faba cotyledons (McDonald et al., 1996b). The underlying system may be a sucrose/H+-symporter, for which the electrochemical gradient generated by unstimulated H+-ATPase is not limiting. At low sucrose concentrations, a non-linear high-affinity system was present in addition to the linear system (Fig. 5D). This transporter was also inhibited by CCCP (Table 3).
The uptake of sucrose was almost completely inhibited by the addition of glucose to the medium (Table 2). This was presumably not due to direct competition for uptake, since sucrose transporters do not transport glucose (Bush, 1993). It was examined whether enzymes released from damaged protoplasts cleaved sucrose (Fig. 6), resulting in the uptake of monosaccharides. Monosaccharide concentrations in suspensions of uninfected protoplasts were only marginally above the trace levels detected in media without protoplasts and did not increase with sucrose concentration (Fig. 6B). Further, monosaccharide concentrations did not change over a 30 min incubation. This suggests that there was no measurable extracellular invertase activity under these conditions. In addition, uptake rates at high substrate concentration were much higher for sucrose (Fig. 5A) than for glucose (Fig. 3A), and sucrose did not inhibit the uptake of 14C-glucose (Table 2). These findings negate an involvement of extracellular sucrose cleavage in the low-affinity phase of 14C-sucrose uptake by uninfected protoplasts. Similarly, uptake of 14C-sucrose into Pisum sativum guard cell protoplasts was strongly inhibited by glucose without extracellular sucrose cleavage (Ritte et al., 1999). For the high-affinity component, the present data cannot fully exclude that sucrose uptake may be (partly) due to an uptake of trace amounts of 14C-glucose present after sucrose cleavage by enzymes released from broken cells. However, the Km values of the high-affinity glucose and sucrose uptake systems differ by nearly a factor of 10 (Figs 3, 5).
Infected cells do not actively accumulate apoplastic sugars
Unlike uninfected protoplasts, infected ones did not accumulate glucose against a concentration gradient (Fig. 3C). Their very low and inconsistent glucose uptake was neither inhibited by NEM (data not shown) or CCCP (Table 1), nor stimulated by fusicoccin (Table 1) and most likely represented passive, unspecific, influx. Further, infected protoplasts did not show sucrose uptake in the concentration range tested (<40 mM; Fig. 5). A missing proton motive force was not the reason for the absent activity of sugar transporters in this cell type (Fig. 1). Generally, the activity of sugar transporters can be diversely regulated, for example, by transcription, mRNA stability, translation, as well as by post-translational processes (Delrot, 2000). In response to interactions with microbes, induction and blocking of sugar transporters have been demonstrated (Truernit et al., 1996; Bourque et al., 2002). For example, expression of the Arabidopsis thaliana monosaccharide transporter AtSTP4 is induced by elicitors (Truernit et al., 1996). By contrast, cryptogein, a proteinaceous elicitor, completely blocked glucose uptake by Nicotiana tabacum cells in a phosphorylation-dependent manner (Bourque et al., 2002). Activity of a Beta vulgaris sucrose transporter was also blocked by a phosphatase inhibitor (Roblin et al., 1998). The present experiments do not allow a conclusion as to whether sugar transporters are not expressed in infected cells or whether they are blocked by post-translational modification.
Differential activity of sugar transporters in infected and uninfected cells affects carbon flows in the infected zone
The absence of active sugar transport in infected cells implies that these cells are unable to govern their carbon supply by sugar uptake from the surrounding apoplast. This hypothesis is supported by the ability of uninfected cells to take up glucose with high affinity and sucrose with biphasic kinetics and thus to deplete the apoplast of sugars. In line with their suggested task in uninfected nodule cells, plant monosaccharide transporters are frequently involved in the retrieval of leaked sugars within source tissue (Delrot, 1981; Maynard and Lucas, 1982b; Heineke et al., 1992). Similarly, plant sucrose transporters are involved in sucrose retrieval along transport pathways (Kühn et al., 1999; Lemoine, 2000). The existence of these retrieval systems underlines the suggested role of these cells in carbon distribution, as has been concluded from high plasmodesmatal densities between pairs of uninfected cells (Abd-Alla et al., 2000). These allow a symplastic transport of sugars to infected cells, where they are metabolized to dicarboxylic acids, the preferred carbon source for bacteroids (Streeter, 1995). However, the central tissue of V. faba nodules contains a relatively low number of uninfected cells (Abd-Alla et al., 2000). Further, expression and activity of enzymes involved in carbon metabolism (e.g. sucrose synthase) are specifically increased in uninfected cells (Gordon et al., 1995). This indicates that uninfected cells may metabolize sugars to organic anions to be released into the apoplast. Interestingly, organic anions are released in parallel with increased activities of sucrose synthase and PEP carboxylase from root cells under phosphorus starvation (Neumann et al., 1999). A low apoplastic pH, achieved by H+-ATPase activity (Fig. 1), would allow undissociated organic anions to enter the cytosol of infected cells in a passive way.
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It has been demonstrated that infected and uninfected protoplasts from the central tissue of root nodules differ in proton release and sugar uptake properties. Uninfected cells contain several sugar retrieval systems, while infected ones are not able to take up glucose or sucrose from the apoplast actively and therefore depend on uninfected cells for carbon supply.
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Acknowledgements |
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This work was supported by the Deutsche Forschungsgemeinschaft, priority programme 717. Radioactive work was carried out at the Central Biotechnical Unit, JLU Giessen. We thank Angela Gruber for her skilful technical assistance, Dr Gudrun Hoffmann-Thoma for advice in the uptake experiments, Dr Feng Yan for stimulating discussions, and Dr Frans JM Maathuis for correcting the English. We also thank the anonymous reviewers for valuable comments.
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References |
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