Journal of Experimental Botany, Vol. 51, No. 351, pp. 1663-1670,
October 2000
© 2000 Oxford University Press
Original Papers |
Transport of amino acids (L-valine, L-lysine, L-glutamic acid) and sucrose into plasma membrane vesicles isolated from cotyledons of developing pea seeds
Transport Physiology Research Group, Department of Plant Ecology and Evolutionary Biology, Utrecht University, Sorbonnelaan 16, NL-3584 CA Utrecht, The Netherlands
Received 22 May 2000; Accepted 24 May 2000
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Abstract |
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Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H+-gradient (
pH, inside alkaline) and/or an electrical gradient (
, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of pH. The imposition of stimulated the influx of valine by 20%, both in the absence and in the presence of pH. Uptake of L-lysine was more strongly stimulated by than by pH, and its pH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of . In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by pH or . Although the transport of sucrose was very low, a stimulating effect of pH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H+-symporters for the active uptake of sucrose and amino acids. Key words: Amino acids, cotyledons, plasma membrane, proton symport, sucrose.
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Introduction |
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Cotyledons are the bulkiest part of the legume seed in its later stages of development and account for most of the nutrient absorption and synthesis of storage compounds in the embryo. Developing seeds are supplied by a stream of phloem sap that carries along sucrose and amino acids serving as their main organic nutrients. The current view is that the contents of the terminal sieve elements pass symplastically to the seed coat parenchyma cells, from which they are released into the apoplastic space and are finally taken up by the cotyledons (for review: Patrick, 1997
). In contrast with sucrose, amino acids seem to undergo appreciable processing in the seed coat parenchyma, for the composition of the amino acid mixture released by seed coats is quite different from that in phloem exudates (Rochat and Boutin, 1991; Lanfermeijer et al., 1992).
Generally, the concentration dependence of the influx of solutes into plant cells can be analysed into one or more saturable components and a linear component (Borstlap, 1983). This also applies to the uptake of sucrose and amino acids into cotyledons of developing legume seeds. Cotyledons of soybean (Lichtner and Spanswick, 1981b; Thorne, 1982), pea (Lanfermeijer et al., 1991), and broad bean (McDonald et al., 1996) all take up sucrose by a saturable component (Km=5–15 mM; Vmax=3–9 µmol g-1 FW h-1) and a linear component (k=30–80 µmol g-1 FW h-1 M-1). Similarly, uptake of the neutral amino acid L-valine by pea cotyledons displayed a saturable component (Km=5 mM) and a linear component (k=100–200 µmol g-1 FW h-1 M-1). The saturable component of amino acid uptake did not appear before a rather advanced stage of development at which the water content of the cotyledons had decreased to about 65% (Lanfermeijer et al., 1990). At this stage the cotyledons are full-grown and about one-third of the final amount of storage proteins has already been deposited. In soybean cotyledons (water content 
80%), the uptake of 2-aminoisobutyric acid and L-glutamine is also dominated by a linear component. Nitrogen starvation of the isolated cotyledons apparently led to derepression of a saturable system with a Km for glutamine of 96 µM (Bennett and Spanswick, 1983). It is not known whether this system is comparable to that appearing during pea seed development.
Evidence has accumulated that saturable sucrose uptake by developing cotyledons is effected by a H+-symporter. The uptake is attended by a transient membrane depolarization (Lichtner and Spanswick, 1981a), depends strongly on the external pH and is sensitive to protonophores (Lichtner and Spanswick, 1981b; Thorne, 1982; Lanfermeijer et al., 1991; McDonald et al., 1996). In addition, more recent work with faba bean and pea has shown that transcripts of a gene encoding a H+/sucrose symporter localize to the outer cell layers of the cotyledons (Harrington et al., 1997; Weber et al., 1997; Tegeder et al., 1999). Proton symporters are probably also involved in amino acid uptake by cotyledons since the saturable uptake component is sensitive to protonophores and shows a distinct pH-dependency (Lanfermeijer et al., 1990). In developing seeds of Arabidopsis the H+/amino acid symporter AAP1 has been found to be expressed in embryo and endosperm (Hirner et al., 1998).
Aqueous polymer two-phase partitioning of the microsomal fraction from tissue homogenates can be used to obtain a fraction that is enriched in plasma membrane vesicles (Larsson et al., 1987). H+-gradients or membrane potentials can be imposed across the vesicle membranes to drive H+-coupled and/or electrogenic transport of solutes against their concentration gradients. In this way H+-symport of sucrose and/or amino acids has been demonstrated in plasma membrane vesicles from various plant tissues (for review: Bush, 1993). In the present paper the isolation and characterization of plasma membrane vesicles from cotyledons of developing pea seeds is described, and H+-symport of sucrose, L-valine and L-lysine in these vesicles is demonstrated. Transport studies with plasma membrane vesicles isolated from pea seed coats are presented in an accompanying paper (De Jong and Borstlap, 2000).
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Materials and methods |
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Plant material
Pea plants (Pisum sativum L. cv. Marzia) were grown from seeds (Nunhems Zaden B.V., Haelen, The Netherlands) in a growth chamber as described (De Jong and Wolswinkel, 1995
) but no flowers were removed. Seeds were harvested towards the end of seed filling (water content of the cotyledons 54–58%).
Isolation of plasma membrane vesicles
Isolation of microsomal membranes and plasma membrane purification were performed at 0–4 °C. Seeds were taken from the pods and after the removal of seed coat and embryonic axis the cotyledons (100–140 g FW) were blended with 200 ml of homogenization medium (50 mM MOPS-KOH, pH 7.5, 330 mM sucrose, 5 mM EDTA, 1 mM DTT, 0.75% (w/v) insoluble polyvinylpyrrolidone) in a Braun kitchen homogenizer. After three consecutive 20 s bursts at maximal speed, the resulting slurry was filtered through a layer of 250 µm mesh Perlon polyamide gauze and BSA was added to the filtrate to a final concentration of approximately 2 g l-1. To get rid of the large amounts of starch, the filtrate was first centrifuged at 4200 g for 10 min in a Sorvall RC-5 centrifuge with an SS 34 rotor. The pellet was discarded and the supernatant was centrifuged at 20 000 g for 15 min. The microsomal fraction was obtained by recentrifugation of the supernatant at 50 000 g for 90 min, and the microsomal pellet was taken up in resuspension medium (330 mM sucrose, 5 mM potassium phosphate, pH 7.8, 10 mM KCl) to a final volume of 10 ml. Isolation of plasma membrane vesicles was carried out by aqueous polymer two-phase partitioning (essentially as described by Larsson et al., 1987). Nine ml of the microsomal fraction was added to 27 g of phase mixture to form a 36 g phase system with a final concentration of 5.7% (w/w) dextran T500, 5.7% (w/w) polyethyleneglycol 4000, 330 mM sucrose, 5 mM potassium phosphate, pH 7.8, 10 mM KCl, 1 mM DTT, and 0.1 mM EDTA. After mixing thoroughly, the phases were separated by centrifugation at 1500 g for 5 min. The upper phase was then repartitioned twice with fresh lower phases giving U3 and the three lower phases were sequentially re-extracted with a fresh upper phase giving 
. The upper phases U3 and were each diluted 2-fold in washing medium (330 mM sorbitol, 50 mM HEPES-KOH, pH 7.0, 39 mM KCl, 1 mM DTT, 0.1 mM EDTA). After centrifugation at 100 000 g for 60 min in a Beckman L60 ultracentrifuge with an SW 28 rotor the supernatant was discarded and the combined pellets were resuspended in 40 ml of washing medium and centrifuged again at 100 000 g for 60 min. The final plasma membrane pellet was resuspended in 300–900 µl pH7K-medium (330 mM sorbitol, 50 mM HEPES-KOH, pH 7, 39 mM KCl, 0.1 mM DTT) to a final concentration of 0.3–1 mg protein ml-1, frozen in liquid nitrogen and stored at -80 °C until use.
Protein assay
Protein was assayed with the bicinchoninic acid reagent (Pierce) following the instructions of the manufacturer. Bovine serum albumin in 0.9% sodium chloride and 0.05% sodium azide was used as a standard, and the absorbance was read at 562 nm.
ATPase assay
ATPase activity was measured as the release of inorganic phosphate from ATP after 30 min of incubation at 30 °C. The reaction medium used was adopted from Larsson et al. (Larsson et al., 1988) with some minor modifications and contained 330 mM sucrose, 50 mM MES-TRIS, pH 6.5, 0.1 mM sodium molybdate, 1 mM sodium azide, 0.1 mM Na2EDTA, 25 mM K2SO4, 3 mM disodium ATP, 3 mM MgCl2, and 40 µl of vesicle suspension in a final volume of 500 µl. Molybdate and azide were included in the reaction medium to inhibit acid phosphatases and mitochondrial ATPase, respectively (Gallagher and Leonard, 1982). ATPase activity was determined in the absence and in the presence of 0.01% (w/v) Brij 58 ( polyoxyethylene 20 cethylether). This detergent exposes all latent ATP-binding sites without otherwise affecting the ATPase activity (Palmgren et al., 1990; Johansson et al., 1995). Accordingly, the percentage of latent ATPase activity was calculated as
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; Bowman et al., 1988). Inorganic phosphate was determined colorimetrically according to Coccuci and Marrè (Coccuci and Marrè, 1984). One ml of reagent consisting of 3% (w/v) (NH4)6Mo7O24.4H2O, 7.5% (v/v) of concentrated H2SO4, 3% (w/v) FeSO4.7H2O and 0.75% (w/v) sodium dodecyl sulphate was added to the ATPase incubation mixture. After 10 min the reaction was terminated by adding 0.5 ml of 7% (w/v) Na3-citrate. KH2PO4 in the presence of 3 mM Mg-ATP was used as a standard. The absorbance was measured at 750 nm.
Cytochrome c oxidase assay
Cytochrome c oxidase activity was determined according to Hodges and Leonard (Hodges and Leonard, 1974) with a few modifications: 0.01% (w/v) Triton X-100 was used as a detergent, the final concentration of cytochrome c was 45 µM, and the reference cuvet also contained 0.83 mM K3Fe(CN)6 to allow the complete oxidation of cytochrome c. Activities were calculated using a molar extinction of coefficient for the reduced cytochrome c of 18.5 mM-1 cm-1.
Glucan synthase II assay
The activity of glucan synthase II (1,3-ß-glucan synthase) was determined using UDP-[3H]-glucose as a substrate and measuring the incorporation of [3H]-glucose into polyglucan (essentially as described by Fredrikson and Larsson, 1989). The reaction mixture contained 0.012% (w/v) digitonine and DTT was omitted.
Transport assays
Uptake was measured after diluting the vesicle suspensions 15-fold into an uptake medium that contained about 10 kBq ml-1 of the 14C-labelled substrate and 0.1 µM valinomycin. To impose a transmembrane pH gradient (pH, inside alkaline) and a membrane potential (, inside negative) across the membrane, the vesicle suspension was diluted in pH5Na-medium (330 mM sorbitol, 50 mM MES-NaOH, pH 5.0, 47 mM NaCl, 0.1 mM DTT). Control assays, determining the uptake in the absence of gradients, were carried out by diluting the vesicles in pH7K-medium (330 mM sorbitol, 50 mM HEPES-KOH, pH 7.0, 39 mM KCl, 0.1 mM DTT). The pH5K-medium (330 mM sorbitol, 50 mM MES-KOH, pH 5.0, 47 mM KCl, 0.1 mM DTT) was used as the uptake medium to impose pH alone, and the pH7Na-medium (330 mM sorbitol, 50 mM HEPES-NaOH, pH 7.0, 39 mM NaCl, 0.1 mM DTT) to impose alone. The uptake experiments were started by adding the vesicle suspension to the uptake medium and were run at 20 °C in a water bath. At specified times, 200 µl aliquots of the incubation mixture were filtered through filter membranes (Schleicher & Schuell, ME25, pore size 0.45 µm), prewetted with uptake medium and placed on a filter holder (Schleicher & Schuell, type DN 025/4). The filters were washed four times, each time with 600 µl of uptake medium, transferred to scintillation vials, and air-dried. Radioactivity was determined after addition of 6 ml of Emulsifier Scintillator Plus (Packard) in a Tri-Carb 2200CA liquid scintillation analyser (Packard). In some cases initial rates of uptake were estimated by polynomial regression of the uptake-time curve (Dorando and Crane, 1984), and will be given with the fitting-derived standard errors.
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Results |
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Plasma membrane purity
About 0.6% of the amount of protein in the microsomal fraction was recovered in the plasma membrane fraction U3+
. Recovery of plasma membrane markers was very poor. As little as 3–4% of glucan synthase II and vanadate-inhibitable ATPase activity in the microsomal fraction was recovered in the plasma membrane fraction, whereas >60% of the activities remained in the first lower phase L1 (Table 1). Almost colourless plasma membrane preparations were obtained from the deep green microsomal fractions and the specific activity of the mitochondrial marker cytochrome c oxidase in U3+ was >50-fold lower than in the microsomal fraction (Table 2). About 90% of the ATPase activity in the plasma membrane fraction could be inhibited by vanadate. The specific activities of the plasma membrane markers were increased by 7-fold and 10-fold, respectively. A minor part (12%) of the ATPase activity in the microsomal fraction could be inhibited by bafilomycin and this activity was enriched 3.5 times in (U3+). About 50% of the plasma membrane vesicles had the right-side-out orientation, as judged from the latency of the vanadate-inhibitable ATPase activity (Table 2).
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Transport of L-valine
Uptake of L-valine by plasma membrane vesicles was determined under four conditions: after the simultaneous imposition of pH+, after imposition of pH alone or alone, and when no gradient was present at all. As can be seen in Fig. 1a, even in the absence of gradients a distinct, time-dependent uptake of valine was measured. This uptake could be largely removed by washing the vesicles with an osmoticum-free medium (Fig. 2a). The imposition of pH stimulated the valine influx about 2-fold, whereas the imposition of increased the influx by about 20%, both when imposed alone or in combination with pH. In the presence of pH+, valine uptake reached levels of about 230 pmol mg-1 protein after 10 min of incubation (Fig. 1a). Assuming an intravesicular volume of 5 µl mg-1 protein this uptake corresponds with an accumulation ratio of 40. The pH-stimulated uptake was abolished when CCCP was included in the uptake medium. However, CCCP also reduced the uptake in the absence of gradients (Fig. 2b).
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), 
) or no gradients (
). (a) Uptake of L-valine supplied at a concentration of 1.12 µM. Symbols represent the mean values ±SE of four preparations. Estimated initial influxes (pmol mg-1 protein min-1) were 46±2 (
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). (b) Effect of CCCP. Uptake was also determined when 10 µM CCCP was included in the pH5Na-medium (
), or pH7K-medium (
). Initial influxes: 34±3 (
), and vesicles from the L1-phase (
). Uptake was measured in pH5Na-medium. (d) Uptake by freshly prepared plasma membrane vesicles. Initial influxes: 87±4 (In microsomal vesicles and in vesicles from the first lower phase L1 uptake of valine was much lower than in the plasma membrane vesicles (Fig. 2c
). After 10 min of incubation the uptake was about 10-fold less, and the initial influxes were about 5-fold lower than in the plasma membrane vesicles.
Routinely, the uptake experiments were carried out with vesicles that had been stored at -80 °C, i.e. after one freeze/thaw treatment. In freshly prepared vesicles, the uptake of valine was about twice as high, both when pH+ were imposed and in the absence of gradients (Fig. 2d).
Transport of L-lysine and L-glutamic acid
The uptake-time curves for L-lysine were less regular than those for L-valine (Fig. 1b). Since extrapolation of the curves to time zero is problematic, no attempt was made to estimate the initial influxes. In the absence of gradients a time-dependent uptake was discernible, but it occurred at a lower rate than that of valine. The uptake was slightly enhanced by the imposition pH, and could be further increased by a factor of 6 when was imposed simultaneously. When imposed alone, had a greater effect than pH.
In the absence of gradients the uptake of glutamic acid was very pronounced, being about twice as high as that of valine. But in contrast to the uptake of valine and lysine that of glutamic acid was not significantly affected by the imposition of pH and/or (Fig. 1c).
Transport of sucrose
Uptake of sucrose by plasma membrane vesicles was very low (Fig. 3). When pH or pH+ were imposed, the uptake amounted to 6 pmol mg-1 protein after 10 min of incubation. After correction for the different substrate concentrations this uptake turns out to be about 25 times lower than for valine. A considerable part of the uptake appeared to be independent of the incubation time, as indicated by the intercepts of the uptake-time curves on the y-axis. Most likely, this time-independent uptake represents extravesicular label that was not removed during the washing of the vesicles on the filter membranes. It represents approximately 0.03% of the amount of label that was present in the 0.2 ml samples of the incubation mixture from which the vesicles were collected. Even though the uptake of sucrose could not be measured very accurately a significant increase in the uptake after the imposition of pH could be clearly demonstrated (Fig. 3). The imposition of , either alone or in combination with pH, had no detectable effect (not shown). After 10 min of incubation the pH-dependent uptake of sucrose amounted to 3 pmol mg-1 protein.
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Enrichment and recovery of plasma membranes by two-phase partitioning
Preliminary experiments had shown that the use of two-phase systems that contained 5 mM KCl and polymer concentrations of 5.3% or 5.5% resulted in plasma membrane fractions that were contaminated with chloroplast membranes. Yields of plasma membranes became extremely low when polymer concentrations were raised to 5.9%. Therefore, a two-phase system has been chosen with polymer concentrations of 5.7%, in which the KCl concentration was enhanced to 10 mM to reduce contamination with chloroplast membranes further.
The plasma membrane fraction consisted of the combined upper phases (U3+) that were obtained by the partitioning of a microsomal fraction against three lower phase (L1 to L3). As compared with the microsomal fraction, the two plasma membrane markers were enriched in this fraction by 7- and 10-fold, respectively (Table 2). This is close to the enrichment factors for these marker enzymes (10 and 8, respectively) in highly purified membrane vesicles from oat leaves (Larsson et al., 1987). Contamination of mitochondrial membranes in these preparations was very low. Only 0.01% of cytochrome c oxidase was recovered in (U3+), whereas Larsson et al. recovered 0.8% (Larsson et al., 1987).
Unfortunately, the recovery of plasma membranes in (U3+) was poor (Table 1). As little as 3–4% of the plasma membrane markers in the microsomal fraction was recovered in (U3+), whereas >60% was retained in the first lower phase L1. For comparison, Larsson et al. recovered 75% of the marker enzymes in (U3+), and 15% in L1 (Larsson et al., 1987).
It may be concluded that with a two-phase system containing 5.7% of the polymers and 10 mM KCl low yields of highly purified plasma membranes can be isolated from developing pea cotyledons.
H+/amino acid symport
During the development of the pea seed, a saturable system (Km
5 mM) for amino acid (L-valine) uptake appears in the cotyledons when their water content has decreased to 65%. The valine influx by this system has been found to be enhanced about four times when the external [H+] was raised from 0.1 to 10 µM and to be inhibited by CCCP (Lanfermeijer et al., 1990). It seems very likely, therefore, that the pH-dependent uptake of valine in the plasma membrane vesicles is effected by the same transporter as the saturable system identified in experiments with cotyledons. Probably, this transport is electrogenic, for it was enhanced by the imposition of (inside negative) across the vesicle membrane. Since was imposed as a K+-diffusion potential by a 15-fold dilution of a vesicle suspension containing 50 mM K+ into a potassium-free medium, its magnitude is expected to amount to -58x10log 15=-69 mV. The imposition of increased the valine influx by about 20%. This is less than has been observed for low-affinity amino acid transporters from Arabidopsis (AAP1/NAT2 and AAP5) expressed in Xenopus oocytes (Boorer et al., 1996a; Boorer and Fischer, 1997) which were stimulated about 2-fold when the membrane was hyperpolarized from 0 to -69 mV.
The cationic amino acid L-lysine was also transported into the vesicles by a H+-symport mechanism as evidenced by the stimulating effect of pH, particularly when imposed together with (Fig. 1b). It is possible that valine and lysine share the same transporter (Heremans et al., 1997). Low-affinity H+-symporters have been identified that accept neutral as well as cationic amino acids as substrates. (Fischer et al., 1995; Boorer and Fischer, 1997). This would imply that transport of lysine across the membrane is accompanied by the movement of two positive electrical charges, which might explain that it was much more strongly stimulated by than the transport of valine. However, the possibilty that lysine is partially transported by a uniporter cannot be excluded. Evidence that uniporters for cationic amino acids may occur in the plant plasma membrane has already been presented (Weston et al., 1995) when the uptake of lysine and arginine in vesicles from Ricinus roots was observed to be stimulated by but not by pH.
Amino acid transport under non-energized conditions
Even in the absence of pH or the uptake of L-valine in the vesicles was quite considerable amounting to 50% of that measured after the imposition of pH+. In freshly prepared vesicles the valine influx driven by the proton motive force as well as the influx under the non-energized condition was about twice as high as in vesicles after one freeze/thaw treatment. This may be taken as evidence that both fluxes are effected by the same transporter. Inhibition by CCCP of the valine influx under the non-energized condition indicates that the protonophore also had a direct inhibiting effect on the transporter as has been described for a H+/Cl--symporter (Alvarado and Vasseur, 1998).
The time-dependent uptake of labelled substrate in vesicles under non-energized conditions is generally thought to result from binding of the substrate to material inside the vesicles, or to the trapping of a product of enzymatic conversion (Dorando and Crane, 1984). An alternative explanation could be that the labelled substrate is exchanged against unlabelled substrate that was entrapped in the vesicles during their isolation. Developing pea cotyledons with a water content of 55% contain about 50 µmol g-1 FW of amino acids (Lanfermeijer et al., 1989). Since the cotyledons were homogenized in 2 vols of medium it is not unlikely that the total concentration of amino acids entrapped in the vesicles was in the order of 10 mM.
Transport of L-glutamic acid
Puzzling results were obtained in uptake experiments with glutamic acid (Fig. 1c). Under non-energized conditions, at an external pH 7, the uptake was very pronounced and approximately as high as the valine uptake after the imposition of pH+. Obviously, glutamic acid can be accumulated in the vesicles by a pH-independent transport mechanism. Glutamic acid is probably transported in its anionic form which, at pH 7, is by far the predominant ionic species with an abundance of 99.6%. But then a lower uptake is to be expected after the imposition of pH, where the external pH was 5 and, consequently, the abundance of the anionic species has decreased to 85%. Perhaps this decrease was compensated by some pH-dependent uptake. An attempt to test this supposition by using CCCP to eliminate possible pH-dependent uptake failed, however, because the pH-independent uptake of glutamate was also strongly inhibited by the protonophore (data not shown).
H+/sucrose symport
In isolated cotyledons (water content 55%) the influxes of L-valine and sucrose by the saturable systems, calculated for an external substrate concentration of 1 µM are approximately 30 and 15 pmol g-1 FW min-1, respectively. This contrasts with the results obtained with the plasma membrane vesicles, in which the sucrose influx was about 50-fold lower than that of valine. The sucrose influx into the vesicles may be so low because the media used in the first steps of their isolation contained a high concentration (0.33 M) of sucrose. It can be envisaged that sucrose entrapped in the vesicles transinhibits the influx of the labelled sucrose. Alternatively, the discrepancy between the valine- and sucrose influxes in the vesicles and those in the isolated cotyledons may be due to a different distribution of the transporters in the cotyledonary tissue. The sucrose transporter is known to be restricted to the outer cell layers of the cotyledon (McDonald et al., 1996; Weber et al., 1997; Tegeder et al., 1999). If uptake of labelled substrates by isolated cotyledons is mainly brought about by the outer cell layers, and if the amino acid transporter is also present in the storage parenchyma, the amino acid transport activity in vesicles may be much higher than anticipated from uptake experiments with cotyledons.
Because transport by a H+/sucrose symporter is electrogenic it will be more or less enhanced by . An increase in the activity of the H+/sucrose symporter from pea cotyledons in response to was probably too low to be detected in these experiments. This contrasts with sucrose transporters from leaves which have been clearly shown to be stimulated by (Lemoine and Delrot, 1989; Boorer et al., 1996b). Sucrose transporters in cotyledons of developing legume seeds could be functionally somewhat different from those in leaves. Another indication for this is that their Kms (5–15 mM) are considerably higher than the Kms (0.5–1 mM) of sucrose transporters from leaves.
Concluding remarks
Aqueous polymer two-phase partitioning was used to obtain plasma membrane vesicles from developing pea cotyledons. If slight contaminations with other membranes are tolerated, the very low recovery (3%) of plasma membranes can probably be greatly enhanced by using a two-phase system in which the polymer concentrations are lowered from 5.7% to 5.5%. The demonstration of the activity of H+-symporters for L-valine, L-lysine and sucrose provides additional evidence that cotyledonary cells take up sucrose and amino acids from the seed apoplasm by means of H+-symporters. Further work is required to clarify the relatively low activity of the H+/sucrose transporter, the specificity of the amino acid transporter(s), and the mechanism of glutamate transport.
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Acknowledgments |
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The authors wish to thank Jolanda Schuurmans for her support and for carrying out some of the uptake experiments.
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Notes |
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1 Present address: Academic Medical Center, Department of Cardiac Catherization B2-115, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
2 To whom correspondence should be addressed. Fax: +31 30 2518366. E-mail: a.c.borstlap{at}bio.uu.nl
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