Download Transport of amino acids (L-valine, L-lysine, L

Journal of Experimental Botany, Vol. 51, No. 351, pp. 1663±1670, October 2000
Transport of amino acids (L-valine, L-lysine,
L-glutamic acid) and sucrose into plasma membrane
vesicles isolated from cotyledons of developing pea seeds
A. de Jong1 and A.C. Borstlap2
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
Abstract
Introduction
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 twophase partitioning of a microsomal fraction and the
uptake was determined after the imposition of a
Hq-gradient (DpH, inside alkaline) anduor an electrical
gradient (Dy, 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 DpH. The imposition of Dy stimulated the influx
of valine by 20%, both in the absence and in the
presence of DpH. Uptake of L-lysine was more
strongly stimulated by Dy than by DpH, and its DpHdependent uptake was enhanced about 6-fold by the
simultaneous imposition of Dy. 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 DpH or Dy. Although the
transport of sucrose was very low, a stimulating
effect of DpH could be clearly demonstrated. The
results lend further support to the contention that
during seed development cotyledonary cells employ
Hq-symporters for the active uptake of sucrose and
amino acids.
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
®nally 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 in¯ux
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 mmol g 1 FW h 1) and a linear component
(k~30 80 mmol 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 mmol g 1 FW h 1 M 1). The
saturable component of amino acid uptake did not
Key words: Amino acids, cotyledons, plasma membrane,
proton symport, sucrose.
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: q31 30 2518366. E-mail: [email protected]
ß Society for Experimental Biology 2000
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de Jong and Borstlap
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 ®nal
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 mM
(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 Hq-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 Hqusucrose
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
Hquamino 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). Hq-gradients or
membrane potentials can be imposed across the vesicle
membranes to drive Hq-coupled anduor electrogenic
transport of solutes against their concentration gradients.
In this way Hq-symport of sucrose anduor 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 Hq-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).
Materials and methods
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
¯owers were removed. Seeds were harvested towards the end of
seed ®lling (water content of the cotyledons 54±58%).
Isolation of plasma membrane vesicles
Isolation of microsomal membranes and plasma membrane
puri®cation were performed at 0±4 8C. 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% (wuv)
insoluble polyvinylpyrrolidone) in a Braun kitchen homogenizer. After three consecutive 20 s bursts at maximal speed, the
resulting slurry was ®ltered through a layer of 250 mm mesh
Perlon polyamide gauze and BSA was added to the ®ltrate to
a ®nal concentration of approximately 2 g l 1. To get rid of
the large amounts of starch, the ®ltrate was ®rst 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 ®nal 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
®nal concentration of 5.7% (wuw) dextran T500, 5.7% (wuw)
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 U39. The upper phases U3 and U39 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 ®nal plasma membrane pellet was
resuspended in 300±900 ml pH7K-medium (330 mM sorbitol,
50 mM HEPES-KOH, pH 7, 39 mM KCl, 0.1 mM DTT) to a
®nal concentration of 0.3±1 mg protein ml 1, frozen in liquid
nitrogen and stored at 80 8C 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 8C. The
reaction medium used was adopted from Larsson et al. (Larsson
et al., 1988) with some minor modi®cations 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 ml of vesicle
suspension in a ®nal volume of 500 ml. Molybdate and azide
Plasma membrane vesicles from cotyledons
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% (wuv) 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
…activity with Brij 58 activity without Brij 58†
100%3
activity with Brij 58
Orthovanadate (1 mM) and ba®lomycin A1 (0.02 mmol mg 1
protein) were used to inhibit P-type and V-type ATPases,
respectively (Gallagher and Leonard, 1982; Bowman et al.,
1988). Inorganic phosphate was determined colorimetrically
according to Coccuci and MarreÁ (Coccuci and MarreÁ, 1984).
One ml of reagent consisting of 3% (wuv) (NH4)6Mo7O24.4H2O,
7.5% (vuv) of concentrated H2SO4, 3% (wuv) FeSO4.7H2O and
0.75% (wuv) sodium dodecyl sulphate was added to the ATPase
incubation mixture. After 10 min the reaction was terminated by
adding 0.5 ml of 7% (wuv) 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
modi®cations: 0.01% (wuv) Triton X-100 was used as a
detergent, the ®nal concentration of cytochrome c was 45 mM,
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 coef®cient for the reduced
cytochrome c of 18.5 mM 1 cm 1.
Glucan synthase II assay
The activity of glucan synthase II (1,3-b-glucan synthase) was
determined using UDP-w3Hx-glucose as a substrate and measuring the incorporation of w3Hx-glucose into polyglucan (essentially as described by Fredrikson and Larsson, 1989). The
reaction mixture contained 0.012% (wuv) 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 mM valinomycin. To
impose a transmembrane pH gradient (DpH, inside alkaline)
and a membrane potential (Dy, inside negative) across the
membrane, the vesicle suspension was diluted in pH5Namedium (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 DpH alone, and the pH7Na-medium (330 mM sorbitol,
50 mM HEPES-NaOH, pH 7.0, 39 mM NaCl, 0.1 mM DTT) to
impose Dy alone. The uptake experiments were started by
adding the vesicle suspension to the uptake medium and were
run at 20 8C in a water bath. At speci®ed times, 200 ml aliquots
of the incubation mixture were ®ltered through ®lter membranes
(Schleicher & Schuell, ME25, pore size 0.45 mm), prewetted
1665
with uptake medium and placed on a ®lter holder (Schleicher &
Schuell, type DN 025u4). The ®lters were washed four times,
each time with 600 ml of uptake medium, transferred to
scintillation vials, and air-dried. Radioactivity was determined
after addition of 6 ml of Emulsi®er 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 ®tting-derived standard errors.
Results
Plasma membrane purity
About 0.6% of the amount of protein in the microsomal
fraction was recovered in the plasma membrane fraction
U3qU39. 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 ®rst lower
phase L1 (Table 1). Almost colourless plasma membrane
preparations were obtained from the deep green microsomal fractions and the speci®c activity of the mitochondrial marker cytochrome c oxidase in U3qU39 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
speci®c 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 ba®lomycin and this
activity was enriched 3.5 times in (U3qU39). About 50%
of the plasma membrane vesicles had the right-side-out
orientation, as judged from the latency of the vanadateinhibitable ATPase activity (Table 2).
Transport of L-valine
Uptake of L-valine by plasma membrane vesicles was
determined under four conditions: after the simultaneous
imposition of DpHqDy, after imposition of DpH alone
or Dy 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 DpH stimulated the valine in¯ux about
2-fold, whereas the imposition of Dy increased the in¯ux
by about 20%, both when imposed alone or in combination
with DpH. In the presence of DpHqDy, valine uptake
reached levels of about 230 pmol mg 1 protein after
10 min of incubation (Fig. 1a). Assuming an intravesicular volume of 5 ml mg 1 protein this uptake corresponds
with an accumulation ratio of ;40. The DpH-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).
1666
de Jong and Borstlap
Table 1. Protein and activities of marker enzymes in the microsomal fraction, the first lower phase (L1), and the combined upper phases
(U3qU39)
Unless indicated otherwise, the data represent the mean"SE of four preparations, and the enzyme activities are expressed in mmol min 1.
Protein (mg)
Cytochrome c oxidase
Glucan synthase II a
ATPase, total activity b
Vanadate-inhibitable
Ba®lomycin-inhibitable
a
b
(U3qU39)
Microsomal
fraction
L1
73"7
86"11
79
5.86"0.36
4.38"0.37
0.71"0.15
55"7
70"9
82
3.76"0.27
2.73"0.10
0.61"0.04
0.42"0.11
0.009"0.002
2.4
0.21"0.08
0.19"0.07
0.02"0.01
Recovery (%)
L1
(U3qU39)
75
81
103
64
62
86
0.57
0.01
3.1
3.6
4.3
2.4
Activities (nmol min 1) of a single preparation.
Determined in the presence of molybdate and azide.
Table 2. Specific activities (nmol mg 1protein min 1) of marker enzymes in the microsomal fraction, the first lower phase (L1), and the
combined upper phases (U3qU39)
Unless indicated otherwise, the data represent the mean"SE of four preparations.
Cytochrome c oxidase
Glucan synthase GSII a
ATPase, total activity b
Vanadate-inhibitable
Ba®lomycin-inhibitable
Vanadate inhibition (%)
Ba®lomycin inhibition (%)
Latency (%) d
Microsomal fraction
L1
(U3qU39)
Enrichment in (U3qU39)
1244"24
0.86
82"9
62"10
10"1
75"4 c
12"2
33"2 c
1346"232
1.12
70"7
51"5
12"2
73"3
17"2
27"2
23"6
8.67
474"47
429"39
35"7
91"1
7"1
51"2
0.018
10.1
5.78
6.92
3.5
a
Single preparations.
Determined in the presence of molybdate and azide.
c
Mean"SE of five preparations.
d
Latency of the vanadate-inhibitable ATPase activity.
b
Fig. 1. Uptake of amino acids by plasma membrane vesicles from pea cotyledons after the imposition of DpHqDy (k), DpH (m), Dy (I) or no
gradients (j). (a) Uptake of L-valine supplied at a concentration of 1.12 mM. Symbols represent the mean values "SE of four preparations. Estimated
initial in¯uxes (pmol mg 1 protein min 1) were 46"2 (k), 38"2 (m), 21"2 (I), and 17"1 (j). (b) Uptake of L-lysine suplied at a concentration of
0.85 mM. Symbols represent the mean values"SE of three (k;j) or two (m;I) preparations. (c) Uptake of L-glutamic acid supplied at a concentration of 1.04 mM. Symbols represent the mean values of three preparations. For clarity the standard errors are only shown for the upper most and
the lower most symbols.
In microsomal vesicles and in vesicles from the ®rst
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 in¯uxes 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 8C, i.e. after one
Plasma membrane vesicles from cotyledons
Fig. 2. Uptake of L-valine by plasma membrane vesicles from pea
cotyledons. Uptake was determined after the imposition of DpHqDy
by diluting the vesicle suspension in pH5Na-medium (k), or in the
absence of gradients by diluting the vesicle suspension in pH7K-medium
(j). L-valine was supplied at a concentration of 1.12 mM. Initial in¯uxes
are expressed in pmol mg 1 protein min 1. (a) Effect of osmotic shock.
Aliquots of the incubation mixture were washed on the ®lter membrane
with pH7K-medium (j), or pH7K-medium without sorbitol (e). (b)
Effect of CCCP. Uptake was also determined when 10 mM CCCP was
included in the pH5Na-medium (\), or pH7K-medium (%). Initial
in¯uxes: 34"3 (k), 13.0"0.6 (j), 8.9"0.7 (\), and 5.3"1.0 (%). (c)
Valine uptake in plasma membrane vesicles (k), microsomal vesicles
(m), and vesicles from the L1-phase (^). Uptake was measured in
pH5Na-medium. (d) Uptake by freshly prepared plasma membrane
vesicles. Initial in¯uxes: 87"4 (k), and 37"4 (j).
freezeuthaw treatment. In freshly prepared vesicles, the
uptake of valine was about twice as high, both when
DpHqDy 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 in¯uxes. 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 DpH, and could be further
increased by a factor of ;6 when Dy was imposed
simultaneously. When imposed alone, Dy had a greater
effect than DpH.
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
1667
Fig. 3. Transport of sucrose into plasma membrane vesicles from pea
cotyledons. The uptake of sucrose, supplied at a concentration of 0.74
mM, was determined after the imposition of DpHqDy, DpH alone, Dy
alone, or in the absence of gradients by diluting vesicle suspensions in
pH5Na-medium, pH5K-medium, pH7Na-medium or pH7K-medium,
respectively. For clarity, and because Dy had no detectable effect, the
data for the pH5-media were combined (k) as were the data for the
pH7-media (j). Symbols represent mean values"SE of two measurements with ®ve vesicle preparations. The lines drawn resulted from a
quadratic (k) or linear regression analysis (j), giving intercepts on
the ordinate of 3.24"0.93 and 2.78"0.58, respectively. The estimated
initial in¯uxes (pmol mg 1 protein min 1) were: 0.61"0.42 (k) and
0.091"0.087 (j).
that of glutamic acid was not signi®cantly affected by the
imposition of DpH anduor Dy (Fig. 1c).
Transport of sucrose
Uptake of sucrose by plasma membrane vesicles was very
low (Fig. 3). When DpH or DpHqDy 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 ®lter 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 signi®cant increase in the uptake after
the imposition of DpH could be clearly demonstrated
(Fig. 3). The imposition of Dy, either alone or in combination with DpH, had no detectable effect (not shown).
After 10 min of incubation the DpH-dependent uptake of
sucrose amounted to ;3 pmol mg 1 protein.
1668
de Jong and Borstlap
Discussion
Enrichment and recovery of plasma membranes by
two-phase partitioning
Preliminary experiments had shown that the use of twophase 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 (U3qU39) 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 puri®ed
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 (U3qU39), whereas Larsson et al.
recovered 0.8% (Larsson et al., 1987).
Unfortunately, the recovery of plasma membranes in
(U3qU39) was poor (Table 1). As little as 3±4% of the
plasma membrane markers in the microsomal fraction
was recovered in (U3qU39), whereas >60% was retained
in the ®rst lower phase L1. For comparison, Larsson et al.
recovered ;75% of the marker enzymes in (U3qU39),
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 puri®ed plasma membranes can be
isolated from developing pea cotyledons.
Hquamino acid symport
During the development of the pea seed, a saturable
system (Kmf5 mM) for amino acid (L-valine) uptake
appears in the cotyledons when their water content has
decreased to ;65%. The valine in¯ux by this system has
been found to be enhanced about four times when the
external wHqx was raised from 0.1 to 10 mM and to be
inhibited by CCCP (Lanfermeijer et al., 1990). It seems
very likely, therefore, that the DpH-dependent uptake of
valine in the plasma membrane vesicles is effected by the
same transporter as the saturable system identi®ed in
experiments with cotyledons. Probably, this transport is
electrogenic, for it was enhanced by the imposition of Dy
(inside negative) across the vesicle membrane. Since Dy
was imposed as a Kq-diffusion potential by a 15-fold
dilution of a vesicle suspension containing 50 mM Kq
into a potassium-free medium, its magnitude is expected
to amount to ±58310log 15~ 69 mV. The imposition of
Dy increased the valine in¯ux by about 20%. This is less
than has been observed for low-af®nity amino acid
transporters from Arabidopsis (AAP1uNAT2 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 Hq-symport mechanism as
evidenced by the stimulating effect of DpH, particularly
when imposed together with Dy (Fig. 1b). It is possible
that valine and lysine share the same transporter
(Heremans et al., 1997). Low-af®nity Hq-symporters
have been identi®ed 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 Dy
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 Dy but not
by DpH.
Amino acid transport under non-energized conditions
Even in the absence of DpH or Dy the uptake of L-valine
in the vesicles was quite considerable amounting to
;50% of that measured after the imposition of
DpHqDy. In freshly prepared vesicles the valine in¯ux
driven by the proton motive force as well as the in¯ux
under the non-energized condition was about twice as
high as in vesicles after one freezeuthaw treatment. This
may be taken as evidence that both ¯uxes are effected by
the same transporter. Inhibition by CCCP of the valine
in¯ux under the non-energized condition indicates that
the protonophore also had a direct inhibiting effect on the
transporter as has been described for a HquCl -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
Plasma membrane vesicles from cotyledons
1
;55% contain about 50 mmol g 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 DpHqDy. Obviously, glutamic acid can be
accumulated in the vesicles by a DpH-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 DpH, 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 DpH-dependent uptake. An attempt to test
this supposition by using CCCP to eliminate possible
DpH-dependent uptake failed, however, because the
DpH-independent uptake of glutamate was also strongly
inhibited by the protonophore (data not shown).
Hqusucrose symport
In isolated cotyledons (water content ;55%) the in¯uxes
of L-valine and sucrose by the saturable systems,
calculated for an external substrate concentration of
1 mM 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 in¯ux
was about 50-fold lower than that of valine. The sucrose
in¯ux into the vesicles may be so low because the media
used in the ®rst 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
in¯ux of the labelled sucrose. Alternatively, the discrepancy between the valine- and sucrose in¯uxes 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 Hqusucrose symporter is
electrogenic it will be more or less enhanced by Dy. An
increase in the activity of the Hqusucrose symporter from
1669
pea cotyledons in response to Dy 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 Dy (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 Hq-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 Hq-symporters. Further
work is required to clarify the relatively low activity of the
Hqusucrose transporter, the speci®city of the amino acid
transporter(s), and the mechanism of glutamate transport.
Acknowledgements
The authors wish to thank Jolanda Schuurmans for her support
and for carrying out some of the uptake experiments.
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