Download Dehydroascorbate Uptake Activity Correlates with

Dehydroascorbate Uptake Activity Correlates with Cell
Growth and Cell Division of Tobacco Bright Yellow-2
Cell Cultures
Nele Horemans*, Geert Potters, Leen De Wilde, and Roland J. Caubergs
University of Antwerp, Department of Biology, Plant Physiology Groenenborgerlaan 171, B-2020, Antwerp,
Belgium
Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been
proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the
activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a
tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA
transport was tested with 14C-labeled ASC. Ferricyanide (1 mm) or dithiothreitol (1 mm) was included in the test to keep the
external 14C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with
growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in
the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are
normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake.
Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mm DHA. In
addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An
increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show
that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC
in cell growth will be discussed.
Ascorbate (ASC) is one of the best known antioxidants present in plants, protecting plant cells from
oxidative challenges (Noctor and Foyer, 1998). However, ASC is more than just a defense molecule. A
clear role in a number of plant physiological processes has been attributed to this molecule, for example, electron donation in different biosynthetic pathways (Arrigoni and De Tullio, 2000), regulation of
cell elongation (González-Reyes et al., 1998), and cell
proliferation (De Gara and Tommasi, 1999).
The last two decades of research has focused on the
functioning of ASC in the regulation of plant cell
growth (Arrigoni et al., 1992; Wang and Faust, 1992;
De Gara et al., 1996; Tommasi et al., 1999). An ASCdeficient mutant vtc1 that contains only 30% of wildtype ASC levels has also been shown to have a significant reduction in growth (Veljovic-Jovanovic et
al., 2001). Recent research has indicated that ASC has
effects on cell elongation as well as cell division (for
review, see Horemans et al., 2000a). Moreover, ASC
has been indicated as one of the possible factors
important in regulating the transition of cells through
the cell cycle (for review, see Potters et al., 2002).
The mechanism underlying the action of ASC in
growth regulation is still a question of debate. Several studies have indicated the redox status of the
* Corresponding author; e-mail [email protected]; fax: 32-3218-04-17.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.022673.
ASC molecules (expressing the percentage of the total ASC pool that is in the reduced state) as an
important factor in this respect. For example, if reduced ASC is added to Allium sp. or maize (Zea mays)
root cells of the quiescent center, it induces these
normally nondividing cells to pass form G1 into the
S-phase (Liso et al., 1988; Kerk and Feldman, 1995).
Moreover, as shown in the Allium sp. roots, ASC also
promotes cell proliferation in the entire root meristem and the pericycle (Arrigoni et al., 1992; Paciolla
et al., 2001). In strong contrast to the growthpromoting role of ASC, its oxidized form, dehydroascorbate (DHA), seems to inhibit cell proliferation (De Cabo et al., 1993; Kerk and Feldman, 1995;
De Pinto et al., 1999; Potters et al., 2000). Moreover,
the activity of the extracellular enzyme ASC oxidase
in Bright Yellow (BY)-2 cells depends on the cell cycle
phase (Kato and Esaka, 1999). More specifically, it
increases during G2 and M phase. Apparently, the
redox status of ASC in the apoplast is an important
factor in cell cycle progression.
At a subcellular level, the concentration and redox
status of ASC seems to differ between the different
cell compartments (Horemans et al., 2000b). At the
moment, it seems that the compartment in which
the ASC redox status is most readily influenced is the
apoplast. The data of Kato and Esaka (1999), as well
as of De Pinto et al. (1999) indicate an intimate connection between apoplastic and cytoplasmic ASC metabolism and cell proliferation, even up to the level of
the actual cell cycle. Therefore, it was postulated that
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361
Horemans et al.
understanding the mechanisms regulating the subcellular distribution of ASC or controlling its redox
status between the apoplast and the cytoplasm will
provide interesting information concerning the role
and mechanism of ASC on this cell cycle control. In
this respect, recent research has indicated two interesting proteins present in the plant plasma membrane. One of them is a high-potential ASC-reducible
cytochrome b561, which has been shown to mediate
transmembrane electron transport, which can, at
least in vitro, use monodehydroascorbate as an external electron acceptor (Horemans et al., 1994; for
review, see Asard et al., 2001). In addition, the plant
plasma membrane contains a high-affinity DHA
transporter. This transporter has been shown to mediate the uptake of DHA into protoplasts of barley
(Hordeum vulgare; Rautenkranz et al., 1994), tobacco
(Nicotiana tabacum) BY-2 cell culture (Horemans et al.,
1998b) or into isolated plasma membrane vesicles of
Phaseolus vulgaris (Horemans et al., 1996, 1997,
1998a). Upon the discovery that the DHA uptake
activity is enhanced in ASC-loaded plasma membrane vesicles, it was postulated that the carrier operates with an exchange mechanism in which uptake
of DHA coincides with a simultaneous export of ASC
(Horemans et al., 1998a). In this paper, it is shown
that in a tobacco BY-2 cell line the activity of the DHA
transporter changes throughout the growth cycle and
the cell cycle. However, the physiological significance remains to be discussed.
RESULTS
DHA Uptake Activity Changes during Cell Culture
A first experiment focused on the activity of the
DHA transporter throughout the growth stage of the
BY-2 cell line. Therefore, uptake activity was measured in protoplasts isolated at different culture ages.
Figure 1A describes the growth curve of the BY-2 cell
line. It needs to be stated that a specific lag phase
could not be observed. After their transfer to fresh
medium, cells grow exponentially from d 1 to 5, and
afterward, net growth decreases to zero and the cells
enter into the stationary phase. Protoplasts were isolated at different time points and 50 ␮m 14C-labeled
ASC was added to monitor its uptake (Fig. 1B). When
no reductant or oxidant was added (control protoplasts), the uptake was seen to increase until it reached
a maximum rate of 30 pmol 10⫺6 cells min⫺1. This
maximum was reached in protoplasts isolated 4 d
after culture initiation, i.e. in the middle of the exponential growth phase. As previously reported, the addition of 1 mm ferricyanide (FC) is sufficient to oxidize
all externally present ASC immediately. In this test
situation, all radioactive molecules are present as 14CDHA. The curve in Figure 1B showing DHA uptake
(in the presence of FC) throughout the culture of the
BY-2 cells follows quite closely the uptake under control conditions: an increase of the uptake rate until d 4,
and a steady activity of 25 pmol 10⫺6 cells min⫺1
afterward. When uptake was measured in the presence of 1 mm dithiothreitol (DTT), the uptake rate was
around 10 pmol 10⫺6 cells min⫺1 and did not change
throughout the growth cycle. DTT normally keeps
14
C-ASC in the reduced form. In the presence of 1 mm
DTT, the amount of radioactive molecules taken up
into the cells was lower than under control conditions.
At d 4, a statistically significant difference was detected between the uptake in the presence of DTT on
the one hand, and the uptake into control protoplasts
or in the presence of FC on the other hand (P ⬍ 0.05).
Statistical difference could not be demonstrated at the
other time points.
DHA Uptake Rates Were Not Affected by Elevated
Internal ASC Levels
As shown in other reports, the endogenous ASC
levels change significantly in BY-2 cells as these cells
progress through the exponential growth phase and
into the stationary phase (De Pinto et al., 1999; Kato
and Esaka, 1999). On the other hand, it was shown
that the DHA carrier possibly takes up DHA and at
the same time exports ASC (Horemans et al., 1998a).
Therefore, it was conceivable that an increased internal ASC level could trigger the observed increased
DHA uptake throughout the growth cycle.
To test this hypothesis, a method was devised to
obtain freshly isolated protoplasts containing more
ASC than normal protoplasts. As described before,
when culturing BY-2 cells in the presence of 1 mm
ASC or 1 mm DHA for 4 h, these cells will contain 10to 20-fold more ASC than control cells (Potters et al.,
2000). Here, protoplasts were prepared from cells
that had been incubated with 1 mm DHA for 4 h (Fig.
2, 4 h, treated), as well as from cells that had been
subjected to a mock treatment (Fig. 2, 4 h, nontreated). ASC levels in treated protoplasts were
found to be three to four times higher than nontreated protoplasts. This effect was independent of
the age of the cell culture as it was observed in cells
from a 4- or 7-d-old culture (Fig. 2A). Uptake into
control protoplasts and ASC-loaded protoplasts did
not differ significantly (P ⬍ 0.05) in protoplasts isolated from a 4-d-old culture (Fig. 2B) or in those from
a 7-d-old culture (Fig. 2C). An elevated ASC concentration inside the protoplasts did not change uptake
rates in the presence of FC or DTT (Fig. 2, B and C) as
compared with protoplasts that had normal ASC levels. As expected, transport into freshly harvested
cells did not differ from that into cells undergoing a
mock treatment for 4 h.
DHA Uptake throughout the Cell Cycle
The observation of the differential activity of the
DHA transporter throughout the growth of the BY-2
cells prompted a further analysis of this connection.
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Dehydroascorbate Uptake Activity and Cell Growth
Figure 1. DHA uptake activity throughout the
growth cycle of a BY-2 cell culture. A, Growth
curve of a BY-2 cell suspension. Error bars denote SE, with n ⫽ 3 for all data points. B, Variation in DHA uptake at different time points
after culture medium refreshment. F, Control;
E, ⫹FC; Œ, ⫹DTT. Error bars denote SE; n ⫽ 3
for d 1 and 2, n ⫽ 6 for d 3, n ⫽ 4 for d 4, n ⫽
12 for d 5, n ⫽ 6 for d 6, and n ⫽ 7 for d 7, for
all three treatments.
Therefore, we used the ability of the BY-2 cells to be
synchronized in their cell division to study a possible
connection between DHA uptake activity and the cell
cycle. A BY-2 culture was synchronized with aphidicolin. Upon release of the aphidicolin block, the cells
are in S phase and were permitted to fulfill their cell
cycle from that point onward, until M-phase was
reached (Fig. 3A). Furthermore, one extra synchronization was performed, using aphidicolin and propyzamide, to observe the cell cycle from M to S phase
(Fig. 3B). Samples were taken every 2 h and were
subjected to protoplastation. The time noted in the x
axis of Figure 3, A and B, denotes the complete time
elapsed between the release from the aphidicolin or
propyzamide cell cycle block and the eventual transport test. The actual harvesting of the cells from the
synchronized culture occurred 2.5 h before this point,
which is the time necessary for protoplast isolation
and the actual transport test. All results from these
graphs will be described, with the moment of the
transport test taken as a reference. Transport rates
were established in cells without addition of external
redox compounds, or in the presence of 1 mm FC or
1 mm DTT. Uptake in the presence of FC or into the
control cells was most affected during the progression
of cells through the cell cycle. A 3-fold increase (P ⬍
0.05) was observed for the transport rate of DHA in
the presence of FC around 8.5 h after aphidicolin
release (Fig. 3A) as compared with the transport rates
before (4.5 and 6.5 h). After this time point, transport
activity decreased again. A significant difference (P ⬍
0.025) was also found at the 8.5-h time point between
uptake in the presence of FC compared with that in
the presence of DTT. It is noteworthy that compared
with nonsynchronized cultures, DHA uptake activity
in control conditions (i.e. without FC or DTT) differed
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Horemans et al.
Figure 3. DHA uptake activity in different phases of the cell cycle. A,
Variation in DHA uptake activity at different time points ranging from
S to M phase. F, Control; E, 1 mM FC added during the uptake assay;
closed triangles, 1 mM DTT added during the uptake assay. Average
and SE of three independent experiments shown. B, Variation in DHA
uptake at different time points, ranging from M to S phase. Symbols
identical to A. One experiment with three replicates is shown. Bars
indicate SE of these replicates.
Figure 2. Internal ASC levels and DHA uptake activity of protoplasts
loaded with ASC. A, Internal ASC levels in protoplasts isolated from
BY-2 cells that have or have not been incubated for 4 h in medium,
enriched with 1 mM DHA. Dark gray bars, Four-day-old culture;
dashed bars, 7-d-old culture. B, DHA uptake activity measured as the
amount of 14C-labeled molecules taken up after 20 min of incubation
into protoplasts prepared from a 4-d-old BY-2 culture treated or not
with 1 mM DHA for 4 h before protoplastation. 0 h indicates that
protoplasts are prepared out of a culture without any pretreatment.
White bars, Fifty micromolar 14C-ASC; gray bars, 14C-ASC and 1 mM
FC added during the uptake assay; black bars, 14C-ASC and 1 mM
DTT during the uptake assay. C, DHA uptake activity measured as
the amount of 14C-labeled molecules taken up after 20 min of
incubation into protoplasts prepared of a 7-d-old BY-2 culture treated
or not with 1 mM DHA for 4 h before protoplastation. 0 h indicates
that protoplasts are prepared out of a culture without any pretreatment. White bars, Fifty micromolar 14C-ASC; gray bars, 14C-ASC and
1 mM FC added during the uptake assay; black bars, 14C-ASC and 1
mM DTT during the uptake assay. Error bars represent SE in all figures.
For all cases, n ⫽ 2 for protoplasts isolated after 4 h of incubation
with DHA or of mock incubation; n ⫽ 4 for protoplasts, isolated
without any pretreatment of the cell culture.
slightly although not significantly from the uptake
measured in the presence of FC.
When cells were synchronized by the consecutive
addition of aphidicolin and propyzamide, a similar
3-fold increase in transport capacity was observed
2.5 h after release from the cell cycle block (Fig. 3B).
Again, it was the transport in the presence of FC or
into control protoplasts that was significantly (P ⬍
0.05) affected, whereas no change could be detected
in the transport activity in the presence of DTT. Later
time points in the experiment uptake stayed at a
basal level of 50 pmol 10⫺6 ppl and did not show any
dependence on the presence of FC or DTT.
DISCUSSION
In this work, evidence has been presented on the
correlation between growth phase and activity of the
DHA transporter, described previously (Horemans et
al., 1998b). It has been established before that it is the
oxidized form, DHA, that is preferentially taken up
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Dehydroascorbate Uptake Activity and Cell Growth
by plant cells (Horemans et al., 1997, 1998b). In other
words, the amount of radioactive label taken up is
representative of the amount of DHA that has been
relocated from the external medium to the cytoplasm. In this paper, the attempt was made to investigate whether this DHA uptake activity is in some
way changing throughout growth and cell cycle. Apparently, cells that exhibit a high rate of growth (cells
in the middle of the exponential growth phase, Fig.
1A) also exhibit a high capacity for DHA uptake (Fig.
1B). The average size of the BY-2 protoplasts did not
differ between different time points (data not
shown), indicating that the observed higher DHA
uptake capacity is not a consequence of cell volume.
Previously, it was shown that BY-2 protoplasts possess the capacity of oxidizing any external ASC
added to their medium (Horemans et al., 1998b; Potters et al., 2000). Therefore, one may suspect that
rather than a regulation of the transport mechanism,
it is merely this oxidizing capacity that is upregulated. However, uptake in the presence of FC
(which causes complete oxidation of the 14C-ASC
within seconds) is never different from the uptake
into control cells (Fig. 1B). This indicates that the
oxidation of 14C-ASC in control conditions is not a
limiting factor influencing the uptake rate.
De Pinto et al. (1999) as well as Kato and Esaka
(1999) demonstrated a 3- to 4-fold increase in the
internal ASC concentration during the exponential
growth phase. Horemans et al. (1998a) showed that
the DHA carrier as measured in isolated P. vulgaris
plasma membrane vesicles possibly uses an ASC/
DHA exchange mechanism whereby uptake of external DHA coincides with the export of internal ASC.
The same phenomenon can be demonstrated in isolated plasma membranes of BY-2 cells (N. Horemans,
unpublished data). In theory, the increased uptake
may thus be caused by a stimulation of the exchange
mechanism due to the increased ASC concentration.
However, an altered internal ASC concentration in
the protoplasts (Fig. 2A), achieved by loading the
cells before protoplast isolation, did not change the
DHA uptake rate of the protoplasts in 4- or 7-d-old
cells (Fig. 2, B and C). Therefore, the difference in the
rate of DHA uptake is not dependent on the internal
ASC concentration. However, this mechanism is not
excluded as a possible driving force for the DHA
carrier. The cytosolic concentration of ASC is in the
millimolar range (Foyer and Lelandais, 1996) and it is
probably high enough to provide maximal stimulation of the DHA uptake process. Therefore the
amount taken up by the cells is probably only dependent on the external DHA concentration, which was
evidently identical for every measurement in the different tests. Therefore, the observed differences are
reflecting the overall capacity for DHA transport of
the cells.
It would be interesting to support this conclusion by
testing ASC-depleted protoplasts. Two treatments
have been suggested in the literature: administration
of lycorine, an inhibitor of ASC biosynthesis (Arrigoni
et al., 1975; De Gara et al., 1994), and the addition of
4-hydroxy-(3, 3, 5, 5)-tetramethylpiperidine-N-oxyl,
which oxidizes the internal ASC pool in animal cells
within 30 min (Njus and Kelley, 1993; Winkler et al.,
1994). However, lycorine needs several hours of incubation before the desired effect can be demonstrated,
and in such a long time period, cell growth itself is
affected by the slowly declining cytoplasmic ASC
content. 4-Hydroxy-(3, 3, 5, 5)-tetramethylpiperidineN-oxyl has been tested (data not shown), but in contrast to animal cells, we could not observe any effect
on the ASC levels in our system. It would be interesting in this respect to test DHA uptake in the ASC
biosynthesis mutant vtc1 that contains only 30% of
wild-type ASC levels (Conklin et al., 1996; VeljovicJovanovic et al., 2001).
As mentioned above, a large increase in cellular
ASC concentration with a peak during the exponential growth phase (De Pinto et al., 1999; Kato and
Esaka, 1999) has been observed. In addition, several
enzymes are transiently active when cells are most
actively dividing: the enzyme catalyzing the last step
in the ASC biosynthesis, l-galactono-␥-lactone dehydrogenase (Smirnoff et al., 2001), as well as the enzymes active in the regeneration of ASC (such as
DHA reductase or MDHA reductase; Noctor and
Foyer, 1998), which are significantly more active during the exponential growth phase than before or
after, in the stationary phase (De Pinto et al., 2000).
Intriguingly, at least the DHA reductase and the
MDHA reductase activity change throughout the cell
cycle as well, and peak around G1 (Kato and Esaka,
1999). Moreover, these authors also demonstrated
changes in the internal ASC redox status throughout
the cell cycle. Whereas the ASC concentration did not
change, the DHA concentration increased significantly, exactly at the M/G1 transition (Kato and
Esaka, 1999). In this paper, we have investigated a
possible relation between DHA transporter activity
and the cell cycle. It was found that DHA uptake
activity was up-regulated 2- to 3-fold in protoplasts
made from cells collected 6 to 8 h after aphidicolin
release or immediately after propyzamide release.
This means that at the moment of harvesting, these
cells are in M phase. The actual DHA uptake assay is
2.5 h after the harvesting in each instance (time needed
for protoplastion). Therefore, the question remains
whether these cells will complete their actual cytokinesis during the protoplastation process or whether
they will remain in M-phase until they regenerate the
cell wall. However, it is reasonably safe to propose
that the increase in transport capacity occurs during M
phase and the transition from M to G1.
The sudden and large increase in transporter activity around the M/G1 transition indicates for the first
time that the activity of the DHA transporter on the
plant plasma membrane may be highly regulated. In
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Horemans et al.
animal cells, compared with plant cells, vitamin C
transport has been studied more extensively. However, in animal studies, information on the regulation
of the different transport systems is fragmentary. The
sodium-dependent ASC carrier can be inhibited by
Tyr kinase inhibitors like AG18 and genistein or
ouabain (Diliberto et al., 1983; Behrman et al., 1996;
Spielholz et al., 1997) or protein kinase activator
phorbol 12-myristate 13-acetate (Liang et al., 2002).
These observations indicate the presence of a phosphorylation site on the animal ASC carrier. On the
other hand, the DHA uptake that is mediated by
GLUT transporters in animal cells has been shown to
be rapidly enhanced under stress conditions (Rivas et
al., 1997; Laggner and Goldenberg, 2000). Apparently, an independent stress signal or an elevated
DHA concentration outside were sufficient to have
an increased expression of the transporter protein or
to recruit additional transporter molecules to the cell
surface. Which mechanisms are responsible for the
fairly large and instantaneous increase in DHA transporter activity during the cell cycle that is reported
here is still to be investigated. Similarly, it remains to
be seen whether other defense-related phenomena
are able to change DHA transport capacity as well.
MATERIALS AND METHODS
Plant Material
Tobacco (Nicotiana tabacum cv BY-2) cell suspension was propagated as
described by Nagata et al. (1992) in Murashige and Skoog medium supplemented with 0.2 g L⫺1 KH2PO4, 30 g L⫺1 Suc, 0.2 mg L⫺1 2,4dichlorophenoxyacetic acid, 0.01 g L⫺1 thiamine-HCl, and 0.1 g L⫺1 myoinositol, set at pH 5.8 with KOH. Cells were cultured in a rotary shaker at
130 rpm and at 27°C in the dark. Weekly subculturing was initiated by
transferring 4 mL of a 7-d-old stationary culture to 100 mL of fresh medium.
Cell Synchronization
lected through a short centrifugation step (150g) and were washed two times
in a so-called protoplast medium containing Murashige and Skoog salts,
0.02% (w/v) KH2PO4, and 0.4 m mannitol. After the final centrifugation
step, the concentration of protoplasts was determined with a Burker-grid
hematocytometer and the protoplasts were diluted to an average concentration of 106 protoplasts mL⫺1. Viability of the protoplasts was checked
before and after every experiment by estimating their capacity to exclude
the dye Evans Blue (0.05% [w/v] dissolved in the protoplast medium to
avoid osmotic shock) and normally amounted 90% (w/v).
For preparation of ASC-loaded protoplasts (i.e. protoplasts containing
significantly more ASC than their control counterparts), cells were incubated for 4 h before protoplastation in medium containing 1 mm ASC or 1
mm DHA. As previously shown, this treatment leads to an intracellular raise
in ASC concentration up to 20-fold of the control cells (Potters et al., 2000).
After 4 h, cells were collected by filtration over a Buchner filter and were
converted into protoplasts as described above.
ASC Measurements
For ASC determination, protoplasts were diluted (1:1) in 6% (w/v)
m-phosphoric acid ⫹ 1 mm EDTA ⫹ 1% (w/v) polyvinylpyrrolidone, and
snap-frozen in liquid nitrogen. ASC and DHA were subsequently extracted
through three cycles of freezing and thawing; the homogenate was centrifuged at 50,000g for 15 min at 4°C. The supernatant was collected and kept
in ice and in the dark until use, or ws stored at ⫺20°C until HPLC analysis.
ASC determination was carried out on reverse-phase HPLC. The chromatographic system consisted of a reverse-phase type C-18 column (3-␮m particle diameter, 150 mm, internal diameter of 4.6 mm, LiChroSpher; Alltech,
Deerfield, IL) and an isocratic pump (LC-10ADVP; Shimadzu
S-Hertogenbosch, The Netherlands) coupled to a home-made amperometric
detection system (glassy carbon working electrode, calomel reference electrode, reference potential 1,000 mV). The electrochemical detector was connected to a personal computer via an SS420 board (Shimadzu). Chromatogram analysis was performed with the ClassVP software package
(Shimadzu). Total ASC (ASC ⫹ DHA) was determined by reducing 100 ␮L
of each sample with 100 ␮L of a 200 mm DTT/400 mm Tris solution. The
high Tris concentration ensures an increase in the pH of the solution to 6
(checked for every sample), which is needed for an efficient reduction of
DHA to ASC. After 30 min of incubation, the reduction reaction was
stopped by addition of 600 ␮L of the eluent, thus lowering the pH significantly to pH 3. The DHA concentration was estimated as the difference
between the reduced and total ASC concentration.
ASC/DHA Transport
Synchronization was performed according to Nagata et al. (1992). Briefly,
13 mL of a stationary culture was transferred to 100 mL fresh medium
supplemented with the S-phase blocker aphidicolin (5 ␮g mL⫺1). After 24 h,
cells were washed thoroughly and returned to the shaker; at this moment,
cells were synchronized at the beginning of S phase. An expanded version
of this protocol was used to study M and G1 phases. To do so, propyzamide
(6 ␮m) was added to block cells in prometaphase 3 h after the release form
the aphidicolin block. After another 6 h, cells were washed again. A synchronization degree of 95% and more could be obtained (i.e. the mitotic
index immediately after propyzamide release, data not shown). After one
cycle, the synchrony had fallen already to a maximum of 15%. However,
compared with the mitotic index in a nonsynchronized culture (3%), or
during the rest of the experiment (1%), this level of synchrony was still high
enough to be used as a marker for cell cycle progression.
Preparation of Protoplasts and ASC-Loaded Protoplasts
Protoplasts were prepared as described by Nagata (1992) with minor
modifications as described by Horemans (1998b). Cells were collected on a
Buchner filter and were incubated in a cell wall-degrading solution (Murashige and Skoog salts, 0.02% [w/v] KH2PO4, and 0.4 m mannitol, containing 1% [w/v] cellulase Onozuka R-10 [Yakult Pharmaceutical, Tokyo] and
0.1% [w/v] pectolyase Y-23 [Kyowa Chemical Products Co Ltd, Osaka,
Japan]) at 30°C in the dark. Normally, 1 g of cells was diluted in 8 mL of this
cell wall-degrading solution. After a 2-h incubation, protoplast were col-
Uptake of ASC or DHA was measured according to Horemans et al.
(1998b) by adding 50 ␮m l-[14C]ascorbic acid (Amersham, Gent, Belgium) to
75.103 freshly prepared protoplasts (concentration generally around 2,500
protoplasts per ␮L). The volume was adjusted to 100 ␮L with protoplast
medium. The addition of 1 mm FC assured that the 14C-labeled ASC was
completely oxidized to 14C-DHA; the addition of 1 mm DTT ensures that at
least a part of the 14C-ASC is present in the reduced form. After a 20-min
incubation, the cells were diluted 50-fold with ice-cold washing medium (10
mm nonlabeled ASC and 0.4 m mannitol, pH 5.5 [KOH]), collected on a
cellulose nitrate filter (0.45 ␮m; Sartorius, Göttingen, Germany), and rinsed
by the further addition of 15 mL of washing buffer. The filters were dissolved in scintillation cocktail (Filter count; Packard, Brussels, Belgium). For
blank experiments, samples were washed immediately after the addition of
the radioactive labeled molecules to the protoplasts. In a typical ASC uptake
experiment, three replicas were made for each condition tested together
with two blanks. The number (n) of separate experiments is added in the
figure legends.
Received February 26, 2003; returned for revision April 21, 2003; accepted
June 14, 2003.
ACKNOWLEDGMENTS
N.H. and G.P. are both postdoctoral workers at FWO-Vlaanderen.
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Dehydroascorbate Uptake Activity and Cell Growth
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