Dehydroascorbate Influences the Plant Cell
Cycle through a Glutathione-Independent
Reduction Mechanism
Geert Potters*, Nele Horemans, Silvia Bellone, Roland J. Caubergs, Paolo Trost, Yves Guisez,
and Han Asard
Laboratory of Plant Physiology, Department of Biology, University of Antwerp, B–2020 Antwerp, Belgium
(G.P., N.H., R.J.C., Y.G.); Laboratory of Plant Physiology, Department of Biology, University of Bologna,
I–40126 Bologna, Italy (S.B., P.T.); and Department of Biochemistry, University of Nebraska, Lincoln,
Nebraska 68588 (H.A.)
Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both
glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been
proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione
concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright
Yellow-2 (BY-2) cells. Addition of 1 mM DHA did not change the endogenous glutathione concentration. Total glutathione
depletion of BY-2 cells was achieved after 24-h incubation with 1 mM of the glutathione biosynthesis inhibitor L-buthionine
sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mM DHA was observed
within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting
its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione
concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with
a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both
compounds act independently in growth control.
Ascorbate (ASC) and glutathione (GSH) are well
known antioxidants, participating in the cellular defense of plants against oxidative stress. Biotic and
abiotic stress conditions therefore generally affect cellular ASC levels and/or the ASC redox status (defined
here as the percentage of reduced ASC upon the total
pool of ASC and the oxidized dehydroascorbate
[DHA]). For example, the ASC content and redox
status of shoots and roots of Phaseolus vulgaris is
sensitive to zinc stress (Cuypers et al., 2001), and ASC
is oxidized in tomato (Lycopersicon esculentum) plants
infected with Botrytis cinerea (Kuźniak and Sk1odowska, 1999, 2001). Especially in the apoplast, the ASC
redox status is quite sensitive to stress conditions.
Different types of environmental stress result in an
increased oxidation of the apoplastic ASC pool (Castillo and Greppin, 1988; Luwe et al., 1993; Takahama,
1993; Gossett et al., 1994; Plöchl et al., 2000). In turn,
GSH has been implicated in herbicide and heavy metal
defense (Kömives et al., 1998). For example, GSH is
oxidized after copper treatment in Lemna minor
(Teisseire and Vernet, 2000) or under ozone fumigation
in Spinacia oleracea (Luwe et al., 1993), and GSH
metabolism is enhanced under different forms of
stress (May and Leaver, 1993; Xiang and Olivier, 1998).
* Corresponding author; e-mail [email protected]; fax 32–3–
265–3417.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.033548.
In any case, ASC and GSH are thought to be
intimately connected. In the chloroplast, GSH functions as an electron donor for DHA reductase, regenerating ASC through DHA reduction (the so-called
ascorbate-glutathione cycle, Foyer and Halliwell, 1976;
Noctor and Foyer, 1998). The oxidized form of GSH
(designated GSSG) that results from this reaction
is subsequently reduced by an NADPH-dependent
GSSG reductase. The components of this ASC regenerating pathway have also been demonstrated in the
cytoplasm (Borracino et al., 1986; Noctor and Foyer,
1998). Moreover, transformed plants overexpressing
GSSG reductase show higher foliar ASC levels and
improved tolerance to oxidative stress. Conversely,
reduced GSSG reductase activity resulted in increased
stress sensitivity (Noctor and Foyer, 1998). Furthermore, the cellular GSH pool is drained upon addition
of exogenous DHA, at least in roots of white lupin
(Lupinus albus) and onion (Allium cepa; Paciolla et al.,
2001). Last, recent experiments from Chen et al. (2003)
show that enhanced expression of a GSH-dependent
DHA reductase increases tissue concentrations of ASC.
In addition to their functioning as antioxidants, ASC
and GSH are essential in different physiological phenomena in plant cells (May et al., 1998; Noctor et al.,
1998; Arrigoni and De Tullio, 2000). For example, an
ever-increasing body of evidence demonstrates the
role of ASC and DHA in the regulation of cell growth
and division (Potters et al., 2002). GSH has also been
implicated in the regulation of plant and animal cell
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Potters et al.
growth and division. GSH metabolism is intimately
linked to the control of root hair development and
root cell growth (Sánchez-Fernández et al., 1997).
Normal cell cycle progression in a tobacco (Nicotiana
tabacum) cell suspension was blocked after addition of
L-buthionine sulfoximine (BSO). This compound is
a nontoxic, highly specific inhibitor of g-glutamylcysteine synthase, which is the first step in GSH biosynthesis (Griffith and Meister, 1979; May and Leaver,
1993; May et al., 1998; Vernoux et al., 2000). Similar
findings have been reported in animal systems (Shaw
and Chou, 1986; Atzori et al., 1994; Poot et al., 1995).
Moreover, in exemplary cases ASC and GSH even are
involved in cell cycle and growth control in similar
ways; the size of the ASC pool in the Zea mays
quiescent center is markedly decreased, compared to
the pool in the meristematic cells close by (Kerk and
Feldman, 1995), but also the level of GSH in the
Arabidopsis quiescent center is markedly lower, again
compared to the surrounding tissues (May et al., 1998).
This suggests that ASC and GSH may be closely linked
when it comes to growth control.
We have previously demonstrated that exogenous
DHA, but not ASC, when added during the G1 phase
of the tobacco L. cv Bright Yellow-2 (BY-2) cell cycle, is
able to delay normal cell cycle progression (Potters
et al., 2000). However, internal DHA concentrations
were not affected by this treatment, whereas ASC levels increased, suggesting a rapid reduction of DHA to
ASC. The DHA-mediated increase of cellular ASC and
effect on cell cycle progression shows an interesting
specificity. A mere increase of cellular ASC may result
in faster cell proliferation rates (Liso et al., 1988;
Arrigoni et al., 1989; Innocenti et al., 1990; Kerk and
Feldman, 1995; Davey et al., 1999; De Pinto et al., 1999)
but has never been reported to delay the cell cycle. We
therefore suggested that the fact that the cell’s need to
invest in DHA reduction, rather than the increased
levels of ASC, may influence cell cycle progression.
This system opens a way to assess the role of the
GSH/GSSG redox pair in the DHA-mediated halt in
cell cycle progression in BY-2 suspension cells. Hopefully, it provides information on the extent of the
intimate entanglement of ASC and GSH, in terms of
DHA reduction and in terms of growth control. The
aim of this work, therefore, is to challenge the
hypothesis that GSH is the first and foremost reductant for DHA, and that the action of either ASC or
GSH on a given physiological phenomenon should
always involve changes in the other component’s
concentration or redox status.
increase in intracellular ASC levels. Although only the
oxidized form was taken up, no increase in intracellular DHA concentration could be observed, suggesting a rapid uptake and internal reduction (Potters
et al., 2000). To study the contribution of GSH in this
DHA reduction, changes in intracellular ASC, DHA,
GSH, and GSSG levels were followed after addition of
1 mM DHA to an exponentially growing BY-2 culture.
Samples were collected with 1-h intervals, with the
‘‘0-h’’ sample taken immediately before DHA addition. A control culture, where extra medium was
added instead of DHA, was sampled as well. Although the internal ASC concentration increased
20-fold (data not shown for this set of experiments;
see Fig. 4), no significant change could be observed in
the intracellular GSH pool of control cells and DHAtreated cells up to 6 h after treatment (P \ 0.05; Fig. 1).
In addition, no changes were observed in the total
amount of glutathione (GSH plus GSSG), or in its
redox status (data not shown). The high variability of
our data is surprising. However, this variation is for
the most part due to variation between different
cultures, since sampling the same culture flask produced fairly comparable data. Since the GSH content
in BY-2 cells is drastically decreased during this
period, as is GSSG reductase (de Pinto et al., 2000),
this may cause the huge differences between cell
cultures.
Nitrosourea Fails to Inhibit the Plant GSSG Reductase
But Causes Cell Death
The above-presented results possibly indicate that
GSH may not be involved in the rapid reduction of
DHA taken up by the cells. An alternative explanation
is that GSSG may itself have been rapidly re-reduced
to GSH masking changes in its redox status, even
after the first hour of sampling. To evaluate this possibility, we applied 1,3-bis(2-chloroethyl)-1-nitrosourea
(BCNU), a known inhibitor of the animal GSSG
RESULTS
Effect of DHA Addition on GSH Redox Status
in BY-2 Cells
Addition of 1 mM DHA to the medium of an
exponentially growing BY-2 culture led to a strong
Figure 1. DHA addition has no effect on the intracellular GSH
concentration. Intracellular GSH content in control conditions (black
diamonds) or after addition of 1 mM DHA (white squares). Average of
seven independent culture flasks (except for time [t] ¼ 3 h, where
n ¼ 3). Bars indicate SE; t0 ¼ immediately after DHA addition.
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Glutathione-Independent Dehydroascorbate Reduction
reductase (for example, Kehrer, 1983; Harlan et al.,
1984; Kaneko et al., 2002) and its plant counterpart
(Gullner and Dodge, 2000; Piquery et al., 2002).
Concentrations ranging from 50 to 500 mM of BCNU
were added to the BY-2 culture medium in the absence
or presence of 1 mM DHA, and samples were collected
every hour after addition. In animal cells, concentrations of 500 mM BCNU were effective in GSSG reductase inhibition (Riley, 1984; Ueda-Kawamitsu et al.,
2002). In our BY-2 culture, even after addition of 500
mM NU, no changes were observed in the cellular GSH
or GSSG content, as compared to the control cells, even
after addition of 500 mM BCNU (data not shown).
Unfortunately, the cells started to die after 6 h of exposure to the compound (Fig. 2), which is just after the
time interval during which the culture was sampled.
The rate at which the cells died was concentration
dependent (percentages of viable cells after addition
of both 500 and 400 mM BCNU are shown in Fig. 2).
Cell viability was indistinguishable from the control
culture even after 24 h of incubation with 250 mM
BCNU (or all concentrations below). As an aside,
addition of 1 mM DHA seemed to alleviate the cell
death rates after application of 500 mM BCNU.
However, this trend could not be confirmed statistically. Furthermore, DHA has no effect on cell death
upon application together with lower BCNU concentrations (400 mM). As a control experiment, we have
run a native gel confirming that BCNU does inhibit
GSSG reductase, but only slightly, even at the 500-mM
concentration (data not shown).
Uptake and Internal Reduction of DHA Proceeds
Even in GSH-Depleted Cells
As an alternative approach to investigate the role of
GSH in the rapid reduction of intracellular DHA, we
Figure 2. BCNU affects plant cell viability. Cell viability, as measured
in Evans blue-stained cell aliquots, of cells under control conditions
(white diamonds), or after addition of 400 mM (circles) or 500 mM
(squares) BCNU (black symbols) or BCNU plus 1 mM DHA (white
symbols) to the cell population. Values (measured in four independent
cultures) are all represented on the chart to properly indicate trends in
the data. Abscissa represents time after the start of the experiment.
Figure 3. Depletion of the GSH pool in BY-2 cells with DEM and BSO.
Intracellular GSH content in control cultures (black circles), or in
cultures treated with 1 mM DEM (white squares) or with 1 mM BSO
(white triangles; dashed line). The t ¼ 0 point is the start of sample
analysis, as in Figure 4, to permit comparison. BSO has been added
24 h before, DEM 2 h before t ¼ 0. Both compounds remained in
the medium for the remainder of the experiment. Results are the average
of two independent culture flasks; bars show SE.
attempted to decrease the cellular GSH content.
Diethylmaleate (DEM) covalently binds GSH, thereby
lowering the physiologically active intracellular levels
(Mulder and Ouwerkerk-Mahadevan, 1997; Nagai
et al., 2002). Pretreatment of BY-2 cells for 2 h with
1 mM DEM decreases the intracellular GSH content
in BY-2 cells to one tenth of the control value (Fig. 3;
0 h meaning 2 h after addition of DEM, to permit
comparison with Fig. 4).
An alternative method to decrease the intracellular
GSH content involves the addition of the GSH
biosynthesis inhibitor BSO. Addition of 1 mM BSO,
24 h before the start of the actual measurements,
resulted in the complete depletion of cellular GSH (Fig
3; 0 h meaning 24 h after addition of BSO, to permit
comparison with Fig. 4). Cell viability was not affected, neither by BSO nor by DEM treatments, up
to 36 h and 14 h after the addition, respectively (all
values in treated and control cultures between 90%
and 95%).
The effect of both compounds on ASC metabolism
was checked as a control experiment. Apparently,
endogenous ASC concentrations in BY-2 cells were not
affected by DEM or BSO addition only (Fig. 4). The
effect of DHA addition was tested on DEM- and BSOtreated cells. DEM and BSO were kept in the culture
medium after DHA addition to ensure continuously
low or zero levels of GSH in the cells (compared with
Fig. 3). Addition of DHA to control cells led to a 20fold increase of the intracellular ASC concentration
within 3 h (Fig. 4). No changes were observed in the
internal DHA concentration, confirming the intracellular capacity to efficiently reduce DHA (as described
before, Potters et al., 2000). The accumulation of intracellular ASC was significantly lower 1 h after DHA
treatment of cells pretreated with DEM and BSO
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Potters et al.
sion (Fig. 6). The combined addition of 1 mM GSH and
1 mM DHA resulted in a delay similar to that with
DHA alone. Surprisingly, however, addition of extra
GSH alone also caused a delay in the cell cycle progression, though not as large as that caused by DHA
addition.
DISCUSSION
Without GSH, BY-2 Cells Still Possess DHA
Reduction Capacity
Figure 4. ASC generation in GSH-depleted cells. Intracellular ASC
content (black symbols, no DHA added; white symbols, after addition
of 1 mM DHA) in control cultures (squares), in cultures treated with
BSO (triangles) or DEM (circles). The T ¼ 0 point is the point at which
DHA was added and represents also the start of sampling, as in Figure 3,
to permit comparison. BSO has been added 24 h before, DEM 2 h
before t ¼ 0. Both compounds remained in the medium during the
remainder of the experiment. Results presented here are the average of
two independent results; bars indicate SE.
(P \ 0.05, Fig. 4). However, over the course of the
experiment, all DHA was effectively taken up into
the cells and reduced to ASC. Six hours after DHA
treatment the ASC content of the DEM- or BSO-treated
cells was not significantly different from the ASC
content in the control cells (again, P \ 0.05). These
results demonstrated that cells effectively depleted
from GSH levels by DEM or BSO treatment were still
capable of reducing high intracellular DHA levels,
although at different initial rates when compared to
control cells. Measurements of the DHA uptake
(transport) rates were performed, showing that neither
BSO nor DEM did affect the DHA uptake capacity of
the cells (data not shown).
GSH is a well-known antioxidant, implicated in the
enzymatic regeneration of ASC through the reduction
of DHA (Foyer and Halliwell, 1976, 1977; Jiménez
et al., 1997; Noctor and Foyer, 1998). This role is, for
example, illustrated by the decrease of GSH levels in
onion and white lupin roots upon DHA treatment
(Paciolla et al., 2001), and by the identification of
different GSH-consuming DHA reductases (for review, see Potters et al., 2002). On the other hand, different proteins have been proposed to carry out DHA
reduction without using GSH in animals (for review,
see Potters et al., 2002), and plant thioredoxins apparently show DHA reducing activity in a native gel assay
(Morell et al., 1997; Foyer and Mullineaux, 1998). In
general, it is supposed that thiol-containing proteins,
and most likely those containing a dicysteinyl motif,
are capable of fulfilling this function. (Paciolla et al.,
2001; De Tullio et al., 2002; Wilson, 2002; Banhegyi
et al., 2003). This raised the questions about the
identity of the possible other reductant(s) of DHA
and about the extent of the contribution of GSH in this
set of DHA reducing agents.
The first part of this work involved the verification
of GSH as an electron donor for DHA. In previous
work, we have demonstrated that upon addition of
a large amount of DHA (100 mmol in 100 mL BY-2
Effect of GSH Depletion or Addition on
Cell Cycle Progression
To investigate the effect of altered intracellular GSH
levels on the DHA-mediated arrest in cell cycle
progression, synchronized BY-2 cells were treated
with DHA and BSO. DHA treatment (1 mM) of cells
synchronously passing through the G1 phase resulted
in a strong delay in cell division, confirming our
previous results (Fig. 5; Potters et al., 2000). Interestingly, treatment of synchronized cells with 1 mM BSO,
effectively lowering the intracellular GSH concentrations, delayed cell cycle progression to a similar extent
(Fig. 5). Simultaneous application of DHA and BSO
resulted in the complete block of mitotic activity.
To further determine the role of intracellular GSH in
cell cycle progression, 1 mM of GSH was added to
M-phase synchronized cells, in the absence and presence of DHA. GSH is readily taken up by the cell
(Vernoux et al., 2000). As observed before, addition of
1 mM DHA caused a delay in the cell cycle progres-
Figure 5. Effect of DHA and BSO on the cell cycle. Changes in cell
cycle progression after addition of 1 mM DHA (black squares), 1 mM
BSO (white triangles), or both (crosses) versus control culture (black
diamonds). Abscissa represents the time after release from the
propyzamide-induced cell cycle block (at which time point cells are
restrained in M phase). The average of two synchronized cultures is
presented here; bars indicate SE.
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Glutathione-Independent Dehydroascorbate Reduction
Figure 6. Effect of DHA and GSH on the cell cycle. Cell cycle progression in synchronized cells, under control circumstances (diamonds) or after addition of 1 mM DHA (squares), 1 mM GSH (triangles)
or 1 mM DHA plus 1 mM GSH (crosses). Abscissa represents the time
after release from the propyzamide-induced cell cycle block (at which
time point cells are restrained in M phase). Values are the average of
three independent cultures; bars indicate SE.
suspension), all DHA is taken up and internally
reduced. Remarkably (and contrary to the results of
Paciolla et al., 2001), this treatment did not affect
the GSH pool in our BY-2 system (Fig. 1). Several
possibilities could explain this discrepancy: a rapid
turnover of cellular GSH, either by reduction of GSSG
or by an increased biosynthesis, or because GSH has
a limited role in DHA reduction in these cells.
To assess the role of GSH in DHA reduction, we
have tried to influence cellular GSH levels by
‘‘scavenging’’ GSH with DEM, by inhibiting GSH
biosynthesis with BSO, and by blocking GSSG reductase activity by BCNU. BCNU is a good inhibitor
for the animal GSSG reductase and has even some
effect on the plant enzyme, but, unfortunately, prolonged exposure of BY-2 cells caused the cells to die
(Fig. 2), and a concentration of 500 mM, which is quite
effective in animal cells, does not affect GSH levels in
BY-2 cells. It should therefore be questioned whether
this compound is as useful in plants as in animal
systems. Perhaps BCNU should only be used for longterm events (as performed by Piquery et al., 2002),
which was impossible in our system. The cell death we
encountered seems a rather unspecific event.
Scavenging of cellular GSH levels by DEM was
effective in the BY-2 cells and reduced free GSH
concentrations to 10% of that in control cells. Since
only redox active molecules are detected in our HPLC
analysis, our measurements indicate the effective reduction of the reducing capacity of GSH in the cells.
Although the reduction rate of DHA to ASC was
lowered by the DEM treatment, similar levels of
reduction were reached as in the untreated cells at
the end of the experiment (Fig. 4). The possibility that
even small GSH concentrations might have been
enough to reduce DHA due to a fast re-reduction of
GSSG cannot be ruled out completely.
Experiments using the GSH biosynthesis inhibitor,
BSO, however, provided further evidence that GSH is
not the primary reductant for the observed DHA
reduction. After a 24-h BSO treatment, cells were
completely devoid of GSH (Fig. 3). Similar to the DEMtreated cells, BSO treatment did not affect the uptake
rates but resulted in slower reduction of intracellular
DHA to ASC (Fig. 4). The observations that addition of
DHA did not change intracellular GSH levels and that
depletion of intracellular GSH did not affect the
reduction of DHA to ASC strongly support the idea
that GSH is not the sole, and maybe even not the
primary, reductant for DHA in BY-2 cells.
For example, the observation that the appearance of
ASC was slower in GSH-depleted BY-2 cells (Fig. 4)
can be explained in two ways (which are not mutually
exclusive). It is possible that the observed DHA
reduction involves different pathways, including one
which is GSH dependent. Both in the case of DEMand BSO-treated cells, DHA reduction proceeds
significantly slower than under control conditions
(Fig. 4). It might be argued that this difference is due to
the lack of GSH in these cells. In that case we estimate
that, based on the comparison of reduction rates,
around 50% of the incoming DHA is being reduced
with the aid of GSH. One may even speculate that the
GSH-independent reduction of DHA is proportional
to the area under the BSO-plus-DHA curve in Fig. 4,
and the area between the control curve (1 mM DHA)
and the BSO-plus-DHA curve proportional to the
GSH-dependent DHA reduction. The fact that there
seems to be a difference (not statistically proven)
between the DEM-plus-DHA and the BSO-plus-DHA
curve might then be attributed to the fact that DEM
also impacts on other thiol groups.
On the other hand, it is also possible that depleting
the GSH pool forces the reductants involved in the
reduction of DHA to be used in other physiological
processes slowing down the DHA reduction. Although our data do not allow us to distinguish between these two possibilities, this does not affect
the major conclusion of the involvement of a GSHindependent pathway in cellular DHA reduction. Any
distinction between both explanations requires a definitive identification of the components of the DHA
reduction pathways.
Similar experiments performed on different animal
cell types before have demonstrated the existence of
different mechanisms for DHA reduction. For example, DHA uptake and internal reduction were severely
hampered in GSH-depleted human (HepG2) and rat
(H4IIE) liver cells (Li et al., 2001). DHA reduction was
also shown to be impaired in GSH-depleted erythrocytes (May et al., 2001b) or endothelial cells (May
et al., 2001a), suggesting the involvement of GSH in
DHA reduction. However, GSH did apparently not
play any role in ASC recycling in human HaCaT
keratinocytes (Savini et al., 2000), or in HL-60 cells
(Guaiquil et al., 1997). It should therefore be interesting to check the involvement of GSH in DHA
reduction in different plant cell types. Indicative for
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Potters et al.
the presence of different mechanisms in different
tissues are the results on onion and white lupin root
tips, demonstrating that GSH is involved in DHA
reduction (Paciolla et al., 2001). It must be noted,
however, that in BY-2 cells, this effect on thiol groups
could not be duplicated (G. Potters and N. Horemans,
unpublished results).
ASC and GSH Are Not Linked for Cell Cycle Control
Using the tools to affect cellular GSH levels, we also
explored the connection between the effect of DHA on
cell cycle progression and the possible connection to
GSH. GSH depletion as a result of a BSO treatment
blocks the cell cycle in BY-2 cells (Potters et al., 2000).
Therefore, we wanted to check whether the observed
DHA-mediated delay of the cell cycle was in fact
mediated by a change in GSH content (or redox
status). However, an increase in internal GSH did not
prevent the observed DHA-mediated cell cycle delay
(Fig. 6). Moreover, GSH depletion and DHA addition
were shown to have an additive effect, strongly
suggesting that both compounds act in a different
pathway, each ultimately influencing the cell cycle
in its own fashion. Therefore, previous observations
demonstrating the involvement of ASC and GSH in
cell cycle progression (Liso et al., 1988; De Gara and
Tommasi, 1999; De Pinto et al., 1999, 2000; Kato and
Esaka, 1999; Potters et al., 2000; Vernoux et al., 2000,
Paciolla et al., 2001) might probably also be the result
of independent pathways. Also, the cells are completely devoid of GSH—there is no GSH left to be
influenced by the exogenous DHA. That DHA still
affects the cell cycle progression under these conditions is an additional indication for the general independence of both compounds.
Surprisingly, we also noted that the mere addition of
GSH did influence the cell cycle slightly, although in
an inhibitory way. This is in apparent contradiction
to the results of Vernoux et al. (2000) or Xiang et al.
(2001)—where in both instances a lower GSH content
in the cell is linked to an inhibition of cell division—as
well as to our own results (Fig. 5). This indicates that,
more than the mere presence of a compound, its
proper concentration is crucial for optimal functioning
of the cell’s physiology. If this equilibrium is shifted in
either direction, the cell loses its balance and normal
functioning is disturbed or even disrupted. In the
actual case of GSH, Creissen et al. (1999) already
demonstrated that plants overproducing GSH are
surprisingly more sensitive to oxidative stress.
Possible Alternative Mechanisms for DHA Reduction
and Growth Control?
Our hypothesis is—albeit indirectly—also supported by other results; plants with 20% of the control
GSH levels did not show altered tolerance to oxidative
stress (May et al., 1998). Apparently, defense against
stress situations sometimes occurs irrespective of the
GSH concentration. In addition, plants with only 5% of
the wild-type GSH level grew normally (Xiang et al.,
2001). On the other hand, in the Arabidopsis vtc-1
mutant (which contains only 30% of a wild-type
plant’s ASC level), both stress resistance and growth
are affected (Veljovic-Jovanovic et al., 2001). This also
suggests that GSH and ASC are not necessarily linked
to one another and that they rather have distinct
functions to fulfill.
Of course, the question remains as to which
compound(s) will assist in GSSG-independent DHA
reduction. Both in animal and plant cells, the thioredoxin/thioredoxin reductase system, glutaredoxin,
protein disulfide isomerase, and even less-known proteins all have been suggested to possess DHA-reduction activity (Wells et al., 1990; Del Bello et al., 1994;
Trümper et al., 1994; Hou and Lin, 1997; May et al., 1997;
Morell et al., 1997; Hou et al., 1999; Potters et al., 2002).
Therefore different sources of electrons for DHA
reduction should be considered in future work concerning the effects of ASC and DHA on cell physiology.
In any case, the involvement of the thioredoxin/
thioredoxin reductase system may be a suitable working hypothesis, explaining the inhibition of cell cycle
progression by DHA. Apparently, DHA is only capable
of slowing down cell cycle progression when added in
G1 phase. Addition of 1 mM DHA in G2 phase does not
affect the cell cycle at all (G. Potters and N. Horemans,
unpublished results). This suggests that DHA influences the cell cycle through processes which are specific
for either G1 or S phase. Interestingly, the thioredoxin/
thioredoxin reductase system is involved in the deliverance of deoxyribonucleotides (obviously necessary for the passage through S phase). A competition
for reducing equivalents between DHA and ribonucleotides, which both need to be reduced, might slow
down the supply for deoxyribonucleotides, and therefore effectively halts S phase progression. Unfortunately, up to now no known inhibitor of the animal
thioredoxin/thioredoxin reductase system has been
proven to inhibit DHA reduction in our BY-2 cells, and
this hypothesis remains highly uncertain.
On the other hand, the thioredoxin/thioredoxin
reductase system has even in plants been shown to
interact directly or indirectly with DHA, both on
a protein level and on an mRNA level. For example, both DHA and GSSG deactivated thioredoxin
f-activated enzymes (Nishizawa and Buchanan, 1981),
and ASC influences the expression of genes, coding for
thioredoxin-activated or -repressed proteins (Kiddle
et al., 2003). Further research is thus needed to look
for the proper integration of ASC/DHA metabolism
in thiol-mediated redox signaling and regulation.
CONCLUSION
Our results demonstrate that, similar to animal cells,
different pathways for DHA reduction exist in plant
cells, the nature of which remains unknown. It is true
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Glutathione-Independent Dehydroascorbate Reduction
that one cannot ignore the many examples where
a functional link between GSH and ASC metabolism
has been crucial in understanding what happens (see
the many examples listed in the introduction). Indeed,
GSH is probably a team player in the whole network
of DHA reducing reactions. Nevertheless, we feel
that the question about DHA reduction should be
broadened; whereas GSH and DHA are most likely
connected, for example, in stress resistance, their
influence on growth regulation may be mediated by
other, distinct pathways. As an aside, we also suggest
that GSH and ASC have apparently a distinct function
in the regulation of cell cycle progression and may
each be part of a different oxidative stress-sensitive
pathway (Reichheld et al., 1999). Further research will
have to identify and specify the different components
on which each molecule acts.
release, during G1 phase, 1 mM DHA, 1 mM GSH and/or 1 mM BSO were
added to the cell culture. The concentrations of these compounds were chosen
in accordance to existing literature (Potters et al., 2000; Vernoux et al., 2000).
Determination of Mitotic Index and Viability
Every hour after synchronization, a 500-mL aliquot of the cell suspension
was taken from the culture and put on ice to allow the cells to sediment. The
remaining medium was removed 10 min after harvesting. The cells were then
fixed in a 3:1 ethanol:acetic acid mixture and stored at 48C until further
analysis. Mitotic indices were determined by staining the chromatin with
orcein (2% w/v in a 1:1 lactic acid:propionic acid mixture) and determining
the percentage of cells displaying one of the mitotic phases under a bright field
microscope. At least 500 cells were counted in every sample.
To assess the viability of the cells after different treatments, Evans blue was
added (in a final concentration of 0.5%) to 500-mL aliquots of cell suspension.
Stained cells were considered dead; 500 cells were counted in each sample.
Uptake of DHA
Uptake of radiolabeled [14C]ASC and [14C]DHA into protoplasts of BY-2
cells was performed as described by Horemans et al. (1998).
MATERIALS AND METHODS
Plant Material
A tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cell suspension was propagated as described by Nagata et al. (1992), in Murashige and
Skoog (1962) medium, supplemented with 0.2 g L1 KH2PO4, 30 g L1 sucrose,
0.2 mg L1 2,4-dichlorophenoxyacetic acid, 0.01 g L1 thiamine-HCl, and
0.1 g L1 myoinositol, set at pH 5.8 with KOH. Cells were cultured in a rotary
shaker at 130 rpm and at 278C, in the dark. Weekly subculturing was initiated by transferring 4 mL of a 7-d-old stationary culture to 100 mL of fresh
medium. DHA uptake experiments were performed with cells in full exponential growth (4 d after inoculation) to ensure a valid comparison with a
synchronized (dividing) culture, as used in the cell cycle experiments. Treatments were either added exactly at the beginning of the measurements (1 mM
DHA, 1 mM ASC, BCNU) or at the time specified in the ‘‘Results’’ section
(1 mM BSO, 1 mM DEM).
HPLC Measurement of ASC and GSH
Intracellular and extracellular ASC and GSH content was determined by
reversed phase HPLC separation, followed by amperometric detection. Cells
were collected on a Büchner filter (Haldenwanger, Berlin) at intervals of 1 h.
Aliquots of around 0.3 g fresh weight were resuspended in 1 mL of extraction
medium, consisting of 6% m-phosphoric acid and 1 mM EDTA. Insoluble
polyvinylpyrridoline (1%) was added, and the cells were subsequently snapfrozen in liquid nitrogen. ASC, DHA, GSH, and GSSG were subsequently
extracted through three cycles of freezing and thawing; the homogenate was
centrifuged at 50,000g for 15 min at 48C. ASC determination in the supernatant
was carried out by reverse phase HPLC (RP type C-18 column, LiChroSpher,
Alltech, Deerfield, IL; isocratic pump, 0.8 mL min1, LC-10ADVP, Shimadzu,
Columbia, MD) coupled to an electrochemical detection system (reference
potential 1,000 mV; supplied by Prof. Dr. L. Nagels, University of Antwerp,
Belgium). Chromatogram analysis was performed with the Class VP software
package (ClassVP 5.0, Shimadzu). Samples were diluted 1:4 in mobile phase
(2 mM KCl, pH set at 2.5 with H3PO4) prior to injection. Total ASC (ASC plus
DHA) was determined by reducing 100 mL of each sample with 100 mL of
a 200-mM dithiothreitol plus 400-mM Tris solution (at a final pH of 6, checked
in random samples). After 1-h incubation at room temperature, samples were
acidified again by addition of 300-mL mobile phase and kept at 48C until
injection. The DHA concentration was estimated as the difference between the
reduced and total ASC concentration.
Synchronization of BY-2 Cells
BY-2 cell cultures were blocked in their cell cycle with aphidicolin and
propyzamide (Nagata et al., 1992; Samuels et al., 1998) and released when
synchronized at the beginning of M phase. Two hours after propyzamide
Native PAGE GSSG Reductase Assay
Soluble proteins were extracted from 2 g of a 3-d-old BY-2 culture with
20 mL of the buffer described in De Gara et al. (1997). Native gel electrophoresis and subsequent staining for GSSG reductase activity was performed
according to Foyer et al. (1991). Every gel was cut in three pieces; before the
actual activity stain, each gel strip was soaked for 15 min in buffer, buffer plus
250 mM BCNU, or buffer plus 500 mM BCNU. These concentrations of BCNU
were also added to the staining mixture afterwards.
ACKNOWLEDGMENTS
G.P. and N.H. are postdoctoral researchers at the Fund for Scientific
Research, Flanders (F.W.O.-Vlaanderen). Mr. Eddy Biebaut is gratefully
acknowledged for his technical assistance. Mrs. Inge Van Dyck was a great
help in preparing the figures. Dr. J. Kapila is acknowledged for helpful
discussion. This paper is a contribution of the University of Nebraska
Agricultural Research Division, Lincoln, Nebraska.
Received September 17, 2003; returned for revision December 8, 2003;
accepted January 4, 2004.
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