The Plant Journal (2002) 31(6), 729±740
Flux control of sulphate assimilation in Arabidopsis
thaliana: adenosine 5¢-phosphosulphate reductase is more
susceptible than ATP sulphurylase to negative control by
thiols
Pierre Vauclare1,², Stanislav Kopriva1,³, David Fell2, Marianne Suter1, Liliane Sticher3, Peter von Ballmoos1,
Urs KraÈhenbuÈhl4, Roel Op den Camp1,§ and Christian Brunold1,*
1
Institute of Plant Sciences, University of Berne, Altenbergrain 21, CH-3013 Berne, Switzerland,
2
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX30BP, UK,
3
Departement de Biologie, Unite de Biologie veÂgeÂtale, Universite de Fribourg, 3 Rue Albert Gockel, CH- 1700 Fribourg,
Switzerland, and
4
Departement fuÈr Chemie und Biochemie, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
Received 3 January 2002; revised 26 April 2002; accepted 21 May 2002.
*
For correspondence (fax +41 31 332 20 59; e-mail chbrunold@ips.unibe.ch).
²
Present address: IE-BPV, University of Lausanne, CH-1015 Lausanne, Switzerland.
³
Present address: IFB, UniversitaÈt Freiburg, D-79085 Freiburg, Germany.
§
Present address: IPW, ETH ZuÈrich, CH-8092 ZuÈrich, Switzerland.
Summary
The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5¢phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined
in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased
internal cysteine, g-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and
extractable activity. An effect on APR could already be detected at 0.2 mM L-cysteine, whereas ATP
sulphurylase was signi®cantly affected only at 2 mM L-cysteine. APR mRNA, protein and activity were
also decreased by GSH at 0.2 mM and higher concentrations. In the presence of L-buthionine-S, Rsulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mM L-cysteine had no effect on APR activity,
indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO
and 0.5 mM GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP
sulphurylase activity was not affected by this treatment. Tracer experiments using 35SO42± in the
presence of 0.5 mM L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity
and the ¯ux of label into cysteine, GSH and protein, but had no effect on the activity of all other
enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent
with the hypothesis that thiols regulate the ¯ux through sulphate assimilation at the uptake and the
APR step. Analysis of radioactive labelling indicates that the ¯ux control coef®cient of APR is more than
0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of
external sulphate is inhibited by GSH to a greater extent than the ¯ux through the pathway, and that
the ¯ux control coef®cient of APR for the pathway, including the transport step, is proportionately less,
with a signi®cant share of the control exerted by the transport step.
Keywords: APS reductase, ATP sulphurylase, Arabidopsis, ¯ux control, roots, sulphate assimilation.
Introduction
Higher plants and many microorganisms growing with
sulphate as sulphur source reduce it to the level of
sulphide for the synthesis of cysteine, methionine, coenã 2002 Blackwell Science Ltd
zymes and iron sulphur clusters of enzymes (Brunold,
1993; HoÈfgen et al., 2001; Leustek and Saito, 1999; Schmidt
and JaÈger, 1992). The reaction sequence from sulphate to
729
730 Pierre Vauclare et al.
Figure 1. Sulphate assimilation and glutathione synthesis in plants.
The products and enzymes involved, cofactors used and incorporation of
cysteine and methionine into proteins are presented. O-acetyl-L-serine
(OAS) is formed from acetyl-CoA and L-serine by serine acetyltransferase
(SAT). Buthionine sulphoximine (BSO) is an inhibitor of gglutamylcysteine (g-EC) synthetase.
cysteine is called assimilatory sulphate reduction (Figure
1). The ®rst step is the activation of sulphate catalysed by
ATP sulphurylase (EC 2.7.7.4). The adenosine 5¢-phosphosulphate (APS) thus formed is the sulphonyl donor for the
®rst reduction step in sulphate assimilation of plants and
algae (Brunold, 1993; Leustek and Saito, 1999; Schmidt,
1972; Suter et al., 2000). The enzyme involved has previously been named APS sulphotransferase (Schmidt, 1972).
It has recently been demonstrated (Suter et al., 2000) that
this enzyme is identical to APS reductase (APR) obtained
by functional complementation of Escherichia coli mutants
(Gutierrez-Marcos et al., 1996; Setya et al., 1996). The
product of APR, SO32±, is reduced to S2± via sulphite
reductase using electrons from ferredoxin. Finally,
cysteine is formed by incorporation of S2± into O-acetyl-Lserine (OAS) via OAS sulphydrylase. OAS is synthesized
from L-serine and acetyl-coenzyme A by serine acetyltransferase (SAT). Sanda et al. (2001) have proposed that
SO32± produced in the APR reaction is used not only for
cysteine synthesis, but also for the formation of sulpholipids (Figure 1).
Because of its strategic position at the beginning of the
pathway (Figure 1), ATP sulphurylase has often been
examined with respect to its regulatory function (for
reviews see Brunold, 1990; Brunold, 1993; HoÈfgen et al.,
2001; Leustek and Saito, 1999; Schmidt and JaÈger, 1992). In
general, activity and steady-state mRNA levels increased
when plants were starved for sulphur or had a high need of
cysteine for glutathione (GSH) synthesis (Figure 1), and
decreased when plants were fed reduced forms of sulphur
such as H2S, cysteine or GSH. Recently, it was shown that
in Arabidopsis thaliana ATP sulphurylase activity was
decreased by GSH treatment (Lappartient et al., 1999).
These authors had shown previously that the rate of SO42±
uptake and the activity of ATP sulphurylase were increased
simultaneously in Brassica napus plants following SO42±
withdrawal from the culture solution, and decreased after
the restoration of SO42± supply (Lappartient and Touraine,
1996). Because external GSH supply resulted in increased
accumulation of cysteine and GSH, both compounds
might be responsible for the control of ATP sulphurylase
activity and SO42± uptake. Phloem sap analysis indicated,
however, that GSH rather than cysteine was the signal
acting in B. napus (Lappartient and Touraine, 1996). This
hypothesis was corroborated by demonstrating that the
repression of ATP sulphurylase mRNA accumulation by
external cysteine was relieved by L-buthionine-S, Rsulphoximine (BSO), an inhibitor of GSH formation
(Lappartient et al., 1999). A regulatory function of ATP
sulphurylase in sulphate assimilation was also demonstrated using Indian mustard lines, which overexpressed
an ATP sulphurylase from A. thaliana and accumulated
GSH (Pilon-Smits et al., 1999). Using tobacco cells
(Hatzfeld et al., 1998) or tobacco plants (Hatzfeld et al.,
2002) overexpressing ATP sulphurylase, it was shown that
ATP sulphurylase abundance was not limiting for cell
metabolism. These results are in agreement with recently
published results obtained with Brassica oleracea cultivated with H2S as sulphur source (Westerman et al., 2001),
and from A. thaliana cultivated under nitrogen de®ciency
(Koprivova et al., 2000), which showed that ATP sulphurylase was less susceptible than APR to regulatory signals.
Corresponding results were with Brassica, which showed
that cysteine and GSH repress APR mRNA and activity
more than ATP sulphurylase, reversing the accumulation
in APR caused by cadmium treatment (Lee and Leustek,
1999). When analysing regulatory phenomena in assimilatory sulphate reduction, it seems appropriate to study
other components of the pathway besides ATP sulphurylase. Good candidates are sulphate transporters and APR,
which change their activity according to the needs of
plants for reduced sulphur (Schmidt and JaÈger, 1992;
Smith et al., 1997; Smith et al., 2000; Takahashi et al., 1997).
In addition, serine acetyltransferase, which forms OAS,
thus linking assimilatory sulphate and nitrate reduction
(Figure 1), should be included (Neuenschwander et al.,
1991).
In this paper we present evidence that sulphate uptake
and APR are more susceptible to regulation by thiols than
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
Regulation of APS reductase by thiols
731
4-week-old Arabidopsis plants. A signi®cant decrease of
40% of extractable APR activity was detected in the roots
after 24 h with 2 mM cysteine. This decrease was paralleled by an accumulation of L-cysteine, g-EC and GSH
(Figure 2b±d). In shoots, however, no signi®cant change of
these parameters could be detected. As the cysteine
treatment affected neither APR activity nor the level of
thiols in shoots, we decided to use root cultures for the
subsequent experiments. This had the advantage that the
plant material could easily be grown aseptically, circumventing problems with bacterial growth due to application
of cysteine.
Effects of L-cysteine on ATP sulphurylase and APR in
Arabidopsis root culture
Figure 2. APR activity and thiols in roots and shoots of Arabidopsis
thaliana cultivated with cysteine.
APR activity (a); and contents of cysteine (b); g-glutamylcysteine (g-EC)
(c); and GSH (d) in roots (hatched bars) and shoots (open bars) were
measured in 4-week-old plants cultivated for 24 h with 0, 0.2 or 2 mM Lcysteine in the nutrient solution. Mean values 6 SD of four
measurements are presented. Bars with different letters indicate values
signi®cantly different at P = 0.01.
ATP sulphurylase and the other enzymes of sulphate
assimilation, and that APR is regulated by GSH rather than
by cysteine, and present an estimation of the ¯ux control
coef®cient of APR.
Results
Effects of L-cysteine on APR in Arabidopsis roots and
shoots
Figure 2 illustrates the effect of exogenously applied Lcysteine on extractable APR activity in shoots and roots of
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
Using extracts from A. thaliana root cultures, we examined
the effect of concentrations of cysteine, cystine, oxidized
glutathione (GSSG), GSH and g-EC up to 10 mM on in vitro
APR and ATP sulphurylase activity. None of these thiols
and dithiols affected the activity of the two enzymes (data
not shown).
Roots cultivated with different concentrations of Lcysteine for 24 h had decreased extractable APR activity
(Figure 3a). A loss of 50 and 90% APR activity was detected
in the roots cultivated with 0.2 and 1 mM L-cysteine,
respectively. ATP sulphurylase activity, however, was not
signi®cantly affected by cysteine concentrations up to
1 mM (Figure 3b). Only at 2 mM L-cysteine was there a
decrease of 32% (Figure 3b). At this cysteine concentration
APR activity had decreased by 95%, indicating that this
enzyme activity was more susceptible than ATP sulphurylase to thiol control. Consistent with the effect on APR
activity, cysteine also decreased the level of APR protein
(Figure 3a, W). There was also a decrease in the accumulation of mRNA of the three APR isoforms, with APR2 and
APR3 mRNA showing a faster decrease than APR1 mRNA
(Figure 3a, N). ATP sulphurylase protein and mRNA levels
were signi®cantly lower than controls only when 2 mM Lcysteine was added to the root culture medium (Figure 3b,
W, N). As already detected in the roots of whole plants,
thiol analysis revealed that the decrease in APS reductase
activity (Figure 3a) correlated with an increase in content of
total cysteine, g-EC and GSH (Figure 3c). This increase in
thiol content was exclusively due to an increase in reduced
thiols. The oxidized thiols cystine and GSSG were not
different between controls and L-cysteine-treated roots
(data not shown). This result indicated that it was the
increase in reduced thiols which was involved in decreasing APR activity, protein and mRNA.
When the activity of APR of A. thaliana roots was
measured at different times after the addition of 0.2 mM Lcysteine to the nutrient solution, a loss of »55% of initial
activity was detected after 1 h, and after 8 h a level
732 Pierre Vauclare et al.
corresponding to 20% of the control level was reached
(Figure 4a). The decrease in APR activity induced by
cysteine was paralleled by a corresponding decrease in
APR protein (Figure 4a, W, cys) and APR2 mRNA (Figure
4a, N), which was measured here and in the following
experiments as a representative APR isoform (Figure 4a, N,
APR2, cys). When L-cysteine was removed after 24 h by
changing the cultivation medium, initial APR activity was
restored within 2 h, indicating a reversible effect of Lcysteine (data not shown). L-cysteine induced an increase
in acid-soluble thiols (Figure 4c). GSH content more than
doubled during the 8 h period, but the biggest effect of Lcysteine was detected in cysteine and g-EC, which reached
levels of 500 and 1100%, respectively, of the controls. All
three thiols increased during the ®rst 2±4 h with cysteine,
and a constant high level was maintained for the remaining 4±6 h.
Determination of the thiol responsible for APR regulation
Figure 3. Activity (A), protein level (W) and accumulated mRNA (N) of
APR (a); ATP sulphurylase (ATPS) (b); and thiols (c)of isolated roots of
Arabidopsis thaliana cultivated for 24 h with different cysteine
concentrations.
For activity, mean values 6 SD of four measurements are presented.
Bars indicated with different letters represent values signi®cantly
different at P = 0.01. Ethidium bromide-stained RNA was included as
control for RNA loading and intactness (RNA).
From Figure 4(c) it is evident that extracellular application of L-cysteine caused an increase not only in the
internal cysteine level, but also in the levels of g-EC and
GSH. It was not clear which of these thiols was
involved in regulating APR. With the aim of identifying
the thiol involved, root cultures were incubated with
various GSH concentrations (Figure 5). This treatment
induced a signi®cant decrease in APR activity at 0.2 mM
GSH; higher GSH concentrations gradually decreased
APR activity to lower levels (Figure 5a). APR protein and
APR2 mRNA also decreased with increasing GSH levels
(Figure 5a, W, N, APR2). As the GSH treatment also
induced a signi®cant increase in the cysteine and g-EC
contents of root cultures (Figure 5b), it was not possible
to assign the regulation of APR to either of the three
thiols. Therefore BSO, an inhibitor of GSH synthesis
(Figure 1), was introduced. The addition of 0.5 mM BSO
alone or together with 0.2 mM L-cysteine decreased the
GSH level in the roots (Figure 6b). Under both conditions, APR activity was comparable to that of the control
cultivated without cysteine (Figure 6a). APR protein and
mRNA accumulated to higher levels in BSO-treated
roots compared to controls (Figure 6a, W, N), indicating
that the low GSH levels (Figure 6b) induced APR protein
and mRNA accumulation without affecting APR activity.
Simultaneous addition of BSO and GSH in the presence or
absence of cysteine increased GSH to high levels (Figure
6b) and decreased APS reductase activity to »25% of the
controls cultivated without cysteine (Figure 6a).
The results from Figure 6, taken together, lead to the
conclusion that GSH levels above control levels consistently led to decreased APR activity, protein and APR2
mRNA. High levels of cysteine or g-glutamylcysteine,
however, were not consistently correlated with decreased
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
Regulation of APS reductase by thiols
APR activity, protein and APR2 mRNA, indicating that GSH
rather than cysteine or g-glutamylcysteine was the regulatory thiol. Figure 6(a) shows that ATP sulphurylase activity,
protein and mRNA were not signi®cantly affected by the
various treatments.
In vivo ¯ux through the sulphate assimilation pathway
In order to analyse the effects of decreased APR
activities on the ¯ux through assimilatory sulphate
reduction, roots that had been cultivated with 0.5 mM
35
SO42± for
L-cysteine or GSH for 20 h were fed with
4 h, and the amount of radioactivity incorporated into
sulphate, thiols and proteins was determined. Similarly
to the results given above (Figures 3a, 5a, 6a), APR
activity decreased to 10 and 25% of control levels in
cultures supplemented with cysteine and GSH, respect-
733
ively (Figure 7). Cysteine treatment signi®cantly
increased the levels of all thiols, whereas GSH feeding
only increased internal cysteine and GSH signi®cantly
(Figure 7b). The labelling of [35S]sulphate reached 31
and 53% of that in control roots (Figure 7c). Cysteine
treatment reduced the labelling of cysteine, GSH and
protein to 12.5, 9.0 and 6.5% of control levels, respectively (Figure 7d±f). In roots incubated with GSH,
labelling of these compounds was 31.5, 17.0 and
12.8% of the controls for cysteine, GSH and protein,
respectively. In the control roots, 34% of total 35S was
detected in reduced form in proteins, cysteine or GSH,
whereas in the roots treated with cysteine or GSH the
portion of reduced labelled compounds reached only 8.9
and 10.2%, respectively.
None of the other enzymes of assimilatory sulphate
reduction ± ATP sulphurylase (Figures 3b, 6a), sulphite
Figure 4. Activity (A, cys), protein level (W, cys) and accumulated mRNA (N, cys) of APR (a)and contents of cysteine, gEC and GSH (b, cys) of Arabidopsis
thaliana root cultures during 8 h after addition of 0.2 mM cysteine to the medium.
Controls (C) were cultivated without cysteine during the experimental period. Mean values 6 SD of four independent experiments are presented for APR
activity and thiol content. Values indicated with different letters are signi®cantly different at P = 0.01. Ethidium bromide-stained RNA was included as
control for RNA loading and intactness (RNA, cys; RNA C).
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
734 Pierre Vauclare et al.
reductase, OAS sulphhydrylase and serine acetyltransferase ± was affected by 0.5 mM L-cysteine or GSH (data not
shown).
Discussion
Figure 5. Activity (A), protein amount (W) and accumulated mRNA (N) of
APR (a) and levels of acid-soluble thiols (b) of Arabidopsis thaliana root
cultures 24 h after addition of various GSH concentrations to the culture
medium.
The accumulated mRNA is shown for APR2. Ethidium bromide-stained
RNA was included as control for RNA loading and intactness (RNA).
Mean values 6 SD of four measurements are presented for APR activity
and thiol level. Bars indicated with different letters represent values
signi®cantly different at P = 0.01.
The decrease of APR activity in the presence of 0.2 mM Lcysteine was fast, leading to a level of 45% of controls after
1 h, and to 20% of controls after 8 h (Figure 4a).
Comparison with the doubling time of root material of
>200 h indicates that this fast decrease was based not only
on a decrease of enzyme synthesis and dilution of the
existing enzyme molecules by growth, but also on degradation of APR molecules. As the whole pathway of
assimilatory sulphate reduction is restricted to plastids
(Brunold, 1993; Frankhauser and Brunold, 1978; Hell et al.,
1997; Leustek and Saito, 1999; Rotte and Leustek, 2000;
Schmidt, 1972), this degradation took place in this cell
compartment. The three isoforms of APR mRNA were
differentially affected by externally applied cysteine: APR2
and APR3 mRNAs decreased faster than APR1 mRNA. This
is in contrast to the effect of nitrogen de®ciency, where
APR3 mRNA decreased more slowly than the two other
isoforms (Koprivova et al., 2000). These contrasting results
demonstrate that the three APR isoforms are differentially
regulated by increased thiol levels and nitrogen de®ciency.
It is tempting to speculate that this differential regulation is
related to the fact that APR has a function not only in
cysteine synthesis, but also in sulpholipid formation
(Figure 1; Sanda et al., 2001). It is possible that one of the
APR isoforms and SQD1 protein directly and speci®cally
interact. This would allow sulpholipid synthesis without
build-up of toxic SO32± levels (Sanda et al., 2001).
It has been shown in vitro with spinach leaves that
the plastid-localized serine acetyltransferase was inhibited by low cysteine concentrations (Brunold and Suter,
1982). In vivo, such a feedback regulation of serine
acetyltransferase could lead to a decreased level of Oacetyl-L-serine, the acceptor of S2± produced in assimilatory sulphate reduction (Figure 1). O-acetyl-L-serine is
also an inducer of APS reductase (Koprivova et al., 2000;
Leustek and Saito, 1999; Neuenschwander et al., 1991).
A decrease in O-acetyl-L-serine induced by increased
cysteine levels could therefore negatively affect APR
synthesis and sulphate assimilation. Arabidopsis thaliana contains three isoforms of serine acetyltransferase
(Noji et al., 1998). The isoform localized in the chloroplast, where sulphate assimilation is localized, is
insensitive to cysteine and GSH (Noji et al., 1998).
Therefore O-acetyl-L-serine production in chloroplasts
should proceed uninhibited, even in the presence of
increased levels of both thiols. The cytoplasmic isoform
of serine acetyltransferase from A. thaliana, however, is
sensitive to cysteine inhibition. Increased cytoplasmic
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
Regulation of APS reductase by thiols
cysteine could decrease O-acetyl-L-serine levels, which
in turn could decrease APR expression. But Figure 6
shows that the increased cysteine levels alone
(Figure 6a,b, BSO + cys) did not affect APR activity,
indicating that O-acetyl-L-serine was not involved in
the regulatory effects presented here.
As APR was the only enzyme involved in cysteine
synthesis that was affected by cysteine and GSH at low
concentrations, feeding with radioactive sulphate in the
presence or absence of these thiols represented a good
possibility for estimating the effect of decreased APR
activity on the ¯ux through assimilatory sulphate reduction, and for putting bounds on the value of the ¯ux
control coef®cient of APR (CJAPR) (Fell, 1997):
APR
DlnAPS
CJAPR = DlnJ / (DlnEAPR + eAPS
735
where DJ = change in ¯ux; DE = change in enzyme;
APR
= elasticity of APR to a change
DAPS = change in APS; eAPS
in APS. The estimation of CJAPR is preferentially done with
the results obtained after GSH addition, which directly
affects APR activity. Addition of cysteine is more complicated because it requires conversion to GSH to cause
effects, and the raised internal concentration of cysteine
may also cause product inhibition of the pathway (Figure
7b).
For this estimation it is assumed that (i) there are no
other routes that mediate the effect of GSH, other than its
effects on the activity of APR; (ii) the pathway starts at
internal sulphate; (iii) ATP sulphurylase is near equilibrium
and exerts little control, so that the internal sulphate
re¯ects the APS concentration; and (iv) GSH concentration
in the proplastids was not affected by external GSH
Figure 6. Activity (A), enzyme protein (W) and accumulated mRNA (N) of APR and ATP sulphurylase (ATPS) (a) and cysteine, gEC and GSH (b) of
Arabidopsis thaliana root cultures 24 h after addition of 0.5 mM BSO, 0.2 mM L-cysteine and 0.5 mM GSH to the culture medium as indicated.
Mean values 6 SD of four independent measurements are presented. Different letters above the bars indicate values signi®cantly different at P = 0.01.
Ethidium bromide-stained RNA was included as control for RNA loading and intactness.
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
736 Pierre Vauclare et al.
treatment. Starting from the ¯ux response coef®cient
(Kacser and Burns, 1973) to GSH, and taking account of
the relevant elasticities (Fell, 1997), a ¯ux control coef®cient for APR of 0.92 can be estimated (for details of the
estimation see Supplementary Material). This estimation is
based on a change in ¯ux calculated from label incorporated into GSH and protein of 135.1 to 21.4 pmol g±1 (Figure
7e,f) in the presence of GSH, a decrease of APR activity to
25% of control (Figure 7a), and a decrease of APS
concentration assumed to be proportional to internal
sulphate, which decreased from 408.0 to 217.8 pmol g±1
(Figure 7c). The change in ¯ux may be overestimated
because the label in the cysteine pool may be diluted by
breakdown of exogenous GSH and as a result of protein
turnover, which may alter during GSH treatment. This
might well be so, as the ¯ux is reduced to 15.8% whereas
the cysteine labelling is only reduced to 31.5% of control,
while the total cysteine content actually increased by 50%.
If the decrease in labelling of the cysteine pool is used for
calculating the ¯ux control coef®cient of APR, a value of
Figure 7. Activity of APR (a); acid-soluble thiols (b); radioactivity in SO42± (c); cysteine (d); GSH (e); and proteins (f) in Arabidopsis thaliana root cultures
24 h after addition of 0.5 mM L-cysteine (L-Cys) or 0.5 mM GSH to the culture medium, as indicated.
Controls were cultivated without additions. Labelling with 35SO42± was 20±24 h after the additions. Mean values 6 SD of four independent experiments are
presented. Bars indicated with different letters represent values signi®cantly different at P = 0.01.
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
Regulation of APS reductase by thiols
0.57 is obtained. Considering the fact that the ¯ux control
coef®cients of all enzymes involved in sulphate reduction
add up to 1, the more realistic lower value of 0.57 for APR
indicates that this enzyme is not rate-limiting, but plays an
important role in controlling the ¯ux through assimilatory
sulphate reduction. It should be stressed, however, that
the transport of external sulphate into roots was inhibited
by GSH to a greater extent than the ¯ux through the
pathway of assimilatory sulphate reduction, as internal
sulphate decreased (Figure 7c) even though sulphate
assimilation was inhibited. The change in the internal
sulphate pool permits calculation of the proportional
inhibition (activation) constant de®ned by Korzeniewski
et al. (1995), which represents the degree of inhibition
(activation) of the set of reactions synthesizing a metabolite relative to that of the set of reactions utilizing it. In this
way, the relative contributions of upstream and downstream control mechanisms are assessed. In the
Supplementary Material it is suggested that the inhibition
of APR is possibly 50±65% of the inhibition of the uptake
step, although at present we have no evidence at molecular level of this inhibition. The coupled inhibition or
activation of more than one step in a pathway is a
mechanism that allows changes in ¯ux to occur in
response to changed circumstances without large changes
in metabolite levels. If only the activity of the transport
step, or only that of the APR, were changed, then there
would be much larger changes in internal sulphate and
APS as sulphur uptake varies. There are examples in many
other biochemical systems of this mechanism of `multisite modulation' (Fell and Thomas, 1995; Thomas and Fell,
1998) or `parallel activation (inhibition)' (Korzeniewski,
1998; Korzeniewski et al., 1995; Shulman et al., 1995)
limiting the changes in levels of intermediary metabolites.
The results of Lappartient et al. (1999) and those presented here indicate that GSH, rather than cysteine, is the
thiol compound used as a signal for regulating ATP
sulphurylase and APR. This contrasts with maize, where
L-cysteine was postulated as the regulating thiol (Bolchi
et al., 1999). This contradiction might be explained by the
fact that in maize, a C4 plant, sulphate assimilation and
GSH synthesis are exclusively or almost exclusively
localized in bundle sheath and mesophyll cells, respectively (Burgener et al., 1998). A feedback mechanism in the
bundle sheath cells that requires GSH synthesis would
thus be ineffective.
By taking the ®ndings reported here together with
previously published results (Brunold and Schmidt,
1978; Hatzfeld et al., 1998; Lappartient and Touraine,
1996; Lappartient et al., 1999; Noji et al., 1998; Rotte and
Leustek, 2000; Saito et al., 2000; Smith et al., 1997; Smith
et al., 2000; Takahashi et al., 1997; Westerman et al.,
2001) the following sequence emerges of regulatory
effects induced by increased thiol levels. At moderately
ã Blackwell Science Ltd, The Plant Journal, (2002), 31, 729±740
737
increased cysteine concentrations, GSH levels are
increased and the uptake of sulphate as well as the
levels of APR transcript, protein and, consequently
activity are decreased (Figure 7a±c) (Brunold and
Schmidt, 1978; Westerman et al., 2001). When the
cysteine concentration is further increased, the additionally accumulating GSH causes a decrease in ATP
sulphurylase mRNA, protein and enzyme activity
(Lappartient et al., 1999), resulting in decreased APS
levels and decreased substrate availability of APR.
These regulatory mechanisms combined lead to a
continuous downregulation of sulphate assimilation;
they are different from those recently described by
Bick et al. (2001) where APR was post-translationally
regulated by oxidative stress.
The regulatory mechanisms described here contribute
to the demand-driven control of sulphate assimilation,
and may be important for plants in general for maintaining optimal cysteine concentration. They are especially important when plants grow in environments that
induce low or high internal GSH levels, such as
exposure to very low external sulphate concentrations
(Brunold et al., 1987; Saito et al., 2000) or to high
external sulphate (Brunold et al., 1987) or H2S
(Westerman et al., 2001).
Experimental procedures
Plant cultivation and treatment
Arabidopsis thaliana var. Columbia plants were cultivated as
described (Kopriva et al., 1999). Experiments with intact plants
were performed after a cultivation period of 4 weeks. One day
before measurements, plants were incubated in Hentschel (1970)
nutrient solution containing 0, 0.2 or 2 mM L-cysteine.
Root cultures were maintained in ARC medium (Czako et al.,
1993) in 250 ml Erlenmeyer ¯asks containing 50 ml medium, and
cultivated in the dark at 25°C on a rotary shaker at 70 rpm. New
cultures were started from existing cultures after 2±3 weeks by
transferring 25±33% of the root material to new medium. The
doubling time of root fresh weight was 231 6 22 h. All experiments were performed with 12±13-day-old root cultures.
Two days before the experiments, roots were routinely transferred into fresh medium.
Enzyme assays
The root material was extracted 1 : 20 (w/v) in 0.1 M Tris±HCl
(pH 8) containing 30 mM Na2SO3, 0.5 mM 5¢-AMP and 10 mM
dithioerythritol (DTE), using a glass homogenizer (Heidolph,
Schwabach, Germany). Adenosine 5¢-phosphosulphate reductase
(APR) was measured according to Brunold and Suter (1990) by
measuring the acid volatile radioactivity formed in the presence
of [35S]APS and DTE (Brunold and Suter, 1990). ATP sulphurylase
activity was determined by measuring the production of ATP from
adenosine 5¢-phosphosulphate (APS) and pyrophosphat (PPi)
with a luciferase system (Schmutz and Brunold, 1982), using a
Lumag/3M biocounter (model M 2010, Lumag, Basel,
738 Pierre Vauclare et al.
Switzerland). ATP sulphurylase activity was stable in crude
extracts up to at least 6 h and was routinely measured 2±3 h
after homogenization of the plant material. Sulphite reductase
activity was determined in a coupled assay system using Oacetyl-L-serine sulphhydrylase to measure the sulphide formed
(von Arb and Brunold, 1983). The activity of O-acetyl-L-serine
sulphhydrylase was routinely determined according to Pieniacek
et al. (1973) by measuring the cysteine formed from O-acetyl-Lserine and S2±. Serine acetyltransferase activity was measured
according to Harms et al. (2000). The protein concentrations in the
extracts were determined according to Bradford (1976), with
bovine serum albumin as a standard.
Determination of cysteine, g-EC and GSH
Reduced thiols were measured as monobromobimane derivatives
(Newton et al., 1981) after reduction using bis-(2-mercapto
ethylsulphone) (BSO) according to Kopriva et al. (1999). Oxidized
thiols were determined by masking the reduced thiols with 50 mM
N-ethylmaleinimide (NEM) for 15 min and removing excess NEM
by extracting ®ve times with equal volumes of toluene. NEM
extract (300 ml) was reduced with 9 mM BSO in 0.2 M Tris±HCl,
5 mM EDTA pH 8. The samples were analysed by reverse-phase
HPLC with ¯uorescence detection, as described by Schupp and
Rennenberg (1988) and modi®ed by RuÈegsegger and Brunold
(1992).
Isolation of total RNA and Northern blotting
Root material was pulverized using mortar and pestle in liquid
nitrogen, and RNA was isolated by phenol extraction and
selective precipitation with LiCl. Electrophoresis of RNA was
performed on formaldehyde±agarose gels at 120 V. RNA was
transferred onto Hybond-N nylon membranes (AmershamPharmacia Biotech, Uppsala, Sweden) and hybridized with 32Plabelled cDNA probes for chloroplastic ATP sulphurylase (ATPS1)
and the three APR isoforms. The membranes were washed four
times at different concentrations of SSC in 0.1% SDS (w/v) for
20 min, the ®nal washing step was 0.5 3 SSC, 0.1% SDS at 65°C.
Exposure to a X-ray ®lm (Medical RX, Fuji, Tokyo) was for 3±
8 days at ±80°C. These hybridization and washing conditions
allowed no cross-hybridization of the APR isoforms when tested
with RNA in vitro transcribed from APR cDNA clones obtained
from Dr T. Leustek (Center for Agricultural Molecular Biology,
Rutgers University, New Brunswick, NJ, USA). The ATPS1 cDNA
corresponding to accession number U05218was ampli®ed by RT±
PCR from Arabidopsis total RNA, and the identity of PCR
fragments was veri®ed by sequencing (Koprivova et al., 2000).
The Northern analysis was performed on two independent RNA
preparations with the same results.
was performed on two independent protein preparations with the
same results.
Labelling experiments using
35
SO42±
Carrier-free 35SO42± with an activity of 1 mCi was added to the
root cultures 20 h after they had been supplied with fresh ARC
medium containing 0.5 mM L-cysteine or GSH. The speci®c
radioactivity of the nutrient solutions was estimated after the
addition of 35SO42± by measuring the radioactivity of an aliquot,
using a betamatic V liquid scintillation counter (Kontron, ZuÈrich,
Switzerland) and quantifying the sulphate after dilution by a
factor of 100 with Milli-Q water, using a NaOH gradient on an ion
chromatographic system Dionex DX-500. After a 4 h feeding
period, roots were extracted and extracts processed as described
by Koprivova et al., 2000). The radioactivity of cysteine and GSH
was determined using the liquid scintillation counter speci®ed
above. The 35S incorporated into protein was measured after
precipitation from 200 ml extract with 10% TCA, washing twice
with 1% TCA and once with 96% ethanol, and redissolving in
400 ml 0.2 M NaOH. Radioactivity in an aliquot of the protein
solution was determined using the scintillation counter speci®ed
above.
Statistical analysis
The Student Newmann±Keuls method (SIGMA STAT for Windows,
Version 1.0, 1992±94, SPSS, Chicago, IL, USA) was routinely used
to determine the signi®cance of differences between treatments.
The results presented in Figure 5(a) were analysed using the t-test
of SIGMA STAT for Windows; those in Figure 7(b) using the Mann±
Whitney Rank Sum Test of SIGMA STAT for Windows.
Acknowledgements
We thank Dr Leustek from the Center for Agricultural Molecular
Biology, Rutgers University, New Brunswick, USA for cDNA
probes of the three APR isoforms; R. Hintermann for excellent
secretarial work; and W. Tanner, E. Bhend and L. Grainger for
technical assistance. This work was supported by the KoÈrberFoundation, the Swiss National Science Foundation, Grant No.
3149246-96 to C.B. and Grant No. 31-395.93 to L.S., and by the
Swiss Agency for Science and Education (B.B.W.).
Supplementary Material
The following material is available from http://www.blackwellscience.com/products/journals/suppmat/TPJ/TPJ1391/TPJ1391sm.htm
Control analysis of sulfur assimilation
Western blot analysis
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