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188

Regulation of sulfate transport and synthesis of sulfur-containing
amino acids
Kazuki Saito
Recent research indicates that several sulfate transporters —
exhibiting different tissue specificities and modes of
expression — may play distinct roles in sulfate uptake within
specific tissues and in long-distance sulfate translocation.
The transcription levels of particular genes and feedback
inhibition of serine acetyltransferase play major roles in
regulating sulfur assimilation and cysteine synthesis.
O-acetylserine and glutathione presumably act within the
cysteine synthesis pathway as derepressor and repressor,
respectively. A unique autoregulatory mechanism that
stabilizes mRNA levels has recently been proposed for the
regulation of methionine synthesis.
Addresses
Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology
and Biotechnology, Research Center of Medicinal Resources, Chiba
University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan;

e-mail: ksaito@p.chiba-u.ac.jp

Figure 1
GSH,Cys

Abbreviations
APS
adenosine 5′-phosphosulfate
GSH
glutathione
Km
Michalis–Menten constant
OAS
O-acetylserine
Sultr
sulfate transporter

Introduction
Sulfur, a macronutrient that is essential for plant growth, is
found in two sulfur-containing amino acids, cysteine (Cys)

and methionine (Met). In addition, sulfur is contained in a
variety of cellular components and plays critical biochemical roles in a number of cellular processes, such as redox
cycles, detoxification of heavy metals and xenobiotics, and
metabolism of secondary products [1••,2••,3].
In the past few years, remarkable progress has been made
in understanding the mechanism and regulation of sulfate
transport, sulfate assimilation and the synthesis of sulfurcontaining amino acids. This progress has been achieved
using molecular cloning and transgenic technology
[1••,2••,3,4]. In this review, I describe what is known about
the molecular regulation of the sulfur-assimilation system
with particular emphasis on recent progress.

Sulfate transport
Sulfur is taken up by plants in its inorganic sulfate form
(Figure 1). Sulfate transporters that are localized in the
root plasma membrane mediate uptake from external
environments (i.e. the soil or apoplasts) into the symplastic system [5]. Plasma-membrane transport systems
for xylem loading in roots and unloading in leaves are

Sulfur

starvation

(a)

mRNA
(isoform specific)
SO42– (Intra)

(b)
APS

(c)
SO32–

(d)
Acetyl-CoA

Current Opinion in Plant Biology 2000, 3:188–195
1369-5266/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.

SO42– (Extra)

Ser

Aspartate

S2–

(f)
OAS

Activity inhibition
(isoform specific)

(e)

Cys

O-PHS

(g)

Threonine

Cystathione

(h)
Homocysteine

(i)
Met
Current Opinion in Plant Biology

Biosynthetic pathway for the sulfur-containing amino acids, Cys and
Met. The enzymes involved in the pathway are: (a) sulfate transporter;
(b) ATP sulfurylase; (c) APS reductase; (d) sulfite reductase;
(e) OAS(thiol)-lyase [cysteine synthase]; (f) Ser acetyltransferase;
(g) cystathione γ-synthase; (h) cystathione β-lyase; and (i) Met
synthase. O-PHS, O-phosphohomoserine. Arrow indicates induction

(derepression). Bar indicates inhibition (repression).

also necessary. Once inside plant cells, sulfate is transported by organelle-membrane transport systems within
the chloroplast envelopes and tonoplast membranes. In
each membrane system, transporters or channels that
mediate the movement of sulfate in both directions are
presumably needed [6].
The cDNA clones encoding plant plasma-membrane sulfate
transporters were first isolated from a tropical forage legume,
Stylosanthes hamata [7]. Three cDNAs were isolated from this

Regulation of sulfate transport and synthesis of sulfur-containing amino acids Saito

legume each of which encoded a H+/sulfate symporter that
exhibited either high or low affinity for sulfate ions when
functionally expressed in yeast. The predicted secondary
structures of the sulfate transporter proteins encoded by these
cDNAs included 12 membrane-spanning domains, which are
typical of cation/solute symporters [7]. Seven cDNAs encoding putative sulfate transporters have been isolated and
characterized from Arabidopsis thaliana [8–10,11•,12,13••].

Transgenic expression of three of these sulfate transporters
(Sultr) cDNAs, Sultr1;1 (AST101), Sultr2;1 (AST68) and
Sultr2;2 (AST56), was able to rescue the growth of a yeast
mutant that lacked sulfate transporters, suggesting that the
proteins encoded by these cDNAs function in the uptake of
sulfate. Like the sulfate transporters isolated from S. hamata,
these Arabidopsis transporters have variable affinity for sulfate:
Sultr1;1 exhibited a high affinity towards the sulfate ion
(Km = 3.6 µM; where Km is the Michalis–Menten constant,
i.e. the substrate concentration that allows the reaction to proceeed at one-half its maximum rate), whereas Sultr2;1 and
Sultr2;2 are low-affinity sulfate transporters (Km = 0.41 mM
and >1.2 mM, respectively) [13••].
The levels of mRNAs that encode the high-affinity sulfate
transporters from S. hamata [7], barley [14,15•], A. thaliana
[13••] and maize [16] increased in response to sulfate starvation in roots. The low-affinity transporter mRNA levels were
also increased by sulfate deprivation [7,9]. The expression of
Sultr1;1 (a high-affinity transporter in A. thaliana) reached its
maximum earlier than that of Sultr2;1 (a low-affinity transporter in A. thaliana), indicating that the responses of these
two transporters to sulfur deficiency are regulated differently [13••]. The Sultr1;1 gene was expressed in the epidermal
and cortical cells of roots but not in above-ground tissues;

whereas the Sultr2;1 was found to be expressed in the stele
of roots and the vascular tissues of shoots and leaves [9,13••].
These results suggest that Sultr1;1 is primarily responsible
for the initial uptake of sulfate ions from the rhizosphere in
the outermost cell layers, and that its expression is sensitive
to the sulfate concentration outside and/or inside of cells.
The function of Sultr2;1 in roots is presumably to transport
apoplastic sulfate into symplasts in the stele, where sulfate
leaked from xylem vessels may accumulate. In shoot vascular bundle tissues, Sultr2;1 is probably involved in unloading
sulfate from xylem vessel into the symplastic system for
translocation to the mesophyll cells.
Besides the plasma membrane transporters that mediate
the uptake and long-distance transport of sulfate, an intracellular transport system for sulfate is also thought to be
present. A system that transports sulfate into chloroplasts
is necessary because chloroplasts are the almost exclusive
sites for the reduction of sulfate into sulfide. The analysis
of the transient expression of a fusion protein made up of
Sultr4;1 (AST82) and jellyfish green fluorescent protein
revealed that Sultr4;1 is a chloroplast-localized protein
[11•]. In addition to the H+/sulfate symporter, a bacterialtype ATP-dependent sulfate permease is also suspected

to be present in chloroplasts of higher plants. Although

189

the vacuole is presumed to be the major compartment for
sulfate storage within cells, little information is available
on sulfate transporters in tonoplasts [17]. Molecular information on such intracellular sulfate-transport systems and
channels in the plasma membrane [18] is also limited.

Activation and reduction of sulfate
ATP sulfurylase activates sulfate by converting it to adenosine 5′-phosphosulfate (APS) (Figure 1b). ATP-sulfurylase
activity has been detected in chloroplasts and the cytosol.
Indeed, two cDNAs encoding both a chloroplastic and a
cytosolic isoform of ATP sulfurylase have been isolated
from potato [19]. In A. thaliana, four cDNAs have been isolated that all seem to encode chloroplastic forms of ATP
sulfurylase [20,21,22••]. The cytosolic isoform of ATP sulfurylase is presumably produced from one of these four
genes by the use of a different translational start codon.
Two reports have described the over-expression of ATP
sulfurylase in plants [23•,24•]. Its over-expression in tobacco cells had no effect on sulfate influx and sulfate content,
although the ATP-sulfurylase activity in transgenic cells

was eight-times higher than that in the cells of wild-type
plants [23•]. In contrast, transgenic Brassica juncea that
over-expressed ATP sulfurylase accumulated more glutathione (GSH) than normal and exhibited resistance to
selenate, which is a toxic analog of sulfate [24•].
The enzymatic step involved in the conversion of APS to
sulfite (Figure 1c) has been the subject of debate for a long
time. Recent data confirm that this reaction is catalyzed by
a single enzyme called APS reductase [25•]. This enzyme is
a GSH-dependent reductase [26•], which used to be called
APS sulfotransferase [27•]. In B. juncea, both ATP sulfurylase and APS reductase are known to be induced upon
exposure to cadmium [28]. The expression of APS reductase is also regulated by light and diurnal rhythm [29•]. In
maize, ATP-sulfurylase and APS-reductase activities take
place almost exclusively in the bundle-sheath cells [30].
Ferredoxin-dependent sulfite reductase catalyzes the sixelectron reduction of sulfite to sulfide [31,32] (Figure 1d).
Also in maize, distinct electron transfer systems using different isoforms of ferredoxin and ferredoxin-NADP+
reductase have been shown to operate in the leaves and
roots [33•]. It is interesting to note how the supply of electrons from the photosystems or NADPH to sulfur or
nitrogen assimilation is regulated via feredoxin.

Cys synthesis

The final step in cysteine synthesis is the reaction that
incorporates a sulfide moiety into the β-position of alanine [1••] (Figure 1e). The carbon skeleton is derived
from serine (Ser) via O-acetylserine (OAS) (Figure 1f).
Two enzymes, Ser acetyltransferase and OAS(thiol)-lyase
[Cys synthase], are involved in this step [1••]. Although
the reduction of sulfate into sulfide takes place almost
exclusively in chloroplasts, Ser acetyltransferase
and OAS(thiol)-lyase are localized in three major

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Physiology and metabolism

compartments of plant cells, that is the cytosol, chloroplasts and mitochondria [34–36,37••,38]. In mitochondria,
there is an additional β-cyanoalanine synthase that
exhibits similar catalytic activity to that of OAS(thiol)lyase [39,40 ••], although cytosolic β-cyanoalanine
synthase activity is subscribed to side activity of
OAS(thiol)-lyase (Cys synthase) [41•]. Mitochondrial
β-cyanoalanine synthase was previously identified as the
mitochondrial form of OAS(thiol)-lyase [35,40••,42].
It has been shown that cytosolic Ser acetyltransferase is
feedback inhibited by Cys at a physiological concentration
(2–10 µM), but the plastidic and mitochondrial forms are
not subject to this regulation [37••]. These results suggest
that feedback inhibition is an important regulatory mechanism for OAS levels in the cytosol. Because OAS is
presumably a positive regulatory factor that derepresses the
genes that encode enzymes involved in sulfur assimilation
(see below), the feedback regulation of cytosolic Ser acetyltransferase by Cys is especially important. The specific
residues of Ser acetyltransferase that are responsible for
feedback regulation have been identified recently [43•].
Ser acetyltransferase and OAS(thiol)-lyase [44,45] form a
bienzyme complex in the chloroplast. OAS(thiol)-lyase concentrations in the chloroplast are, however, far in excess of
Ser-acetyltransferase concentrations indicating that only a
fraction of OAS(thiol)-lyase associates with Ser acetyltransferase [46••]. A large amount of OAS(thiol)-lyase is therefore
present in a free form. The bound form of OAS(thiol)-lyase,
which has dramatically reduced catalytic activity, seems to
modulate the activity of Ser acetyltransferase in the enzyme
complex. The free form of OAS(thiol)-lyase is presumably
responsible for the actual catalytic function of this enzyme.
This is a special mechanism that is responsible for maintaining Cys biosynthesis at full capacity [46••]. In
A. thaliana, the expression of cytosolic OAS(thiol)-lyase is
induced by exposure to salt and heavy-metal stress, via a
mechanism that probably involves mediation by abscisic
acid [47•]. Under these stress conditions, cytosolic
OAS(thiol)-lyase is expressed in the leaf trichomes, suggesting a possible connection between this enzyme and
detoxification of heavy metals in trichomes [48].
Overexpression of OAS(thiol)-lyase or Ser acetyltransferase in transgenic tobacco conferred increased
tolerance of oxidative stress caused by sulfite [49],
paraquat (M Noji et al., personal communication) or
hydrogen peroxide [50•]. These results suggest that it
may become possible to engineer stress-resistant plants
by manipulating Cys synthesis.

Regulatory circuit for sulfate transport,
assimilation and Cys synthesis
It is well known that sulfate uptake and assimilation activities are derepressed under sulfur-deficient conditions. In
Arabidopsis, this derepression is correlated with the
inducible expression of a particular set of genes that

encode sulfate transporter isoforms, Sultr1;1 and Sultr2;1,
and APS reductase [9,13••]. As yet, we do not know which
signals are involved in the induction of these genes and
how they operate.
OAS is thought to be a positive regulator (i.e. derepressor or
inducer) of gene expression in sulfate-starved plants, as it is
in bacteria [51]. In fact, OAS has also been shown to induce
the expression of genes encoding a high-affinity sulfate
transporter in barley, thereby overriding the repressive effect
of sulfur-rich nutritional conditions [14]. Similar results have
been obtained using Arabidopsis, in which only Sultr1;1 and
Sultr2;1 of seven sulfate-transporter genes were induced by
the addition of OAS (Y Hatzfeld et al., personal communication). The expression of mRNA encoding APS-reductase
was also induced by OAS in Arabidopsis [52]. In the cotyledons of Arabidopsis and soybean, cellular OAS concentration
increases under sulfur-starved conditions. The application of
OAS to immature soybean cotyledons resulted in the elevated expression of mRNA encoding a sulfur-poor seed storage
protein; this response is similar to that caused by sulfur starvation [53•]. In maize cells, addition of OAS resulted in
increased sulfate uptake and ATP-sulfurylase activity [54].
In contrast to the role of OAS as a positive regulator, GSH
and Cys are thought to be negative regulators (i.e. repressors)
of genes whose expression is regulated by sulfur status
[16,55]. Using ‘split-root’ experiments, it was shown that
GSH is a phloem-translocated signal molecule that represses
the expression of the genes that encode the sulfate transporter Sultr2;1 and ATP sulfurylase [56,57••].
The role of OAS in regulating the entire sulfur assimilation
pathway takes us back to the its role in the control of Ser
acetyltransferase activity. Given the fact that cytosolic Ser
acetyltransferase is regulated by Cys feedback inhibition,
this feedback inhibition may be controlled further by
cytosolic signal(s) that are induced by a change of sulfur status. Recent investigations have clearly demonstrated that
transient increases in cytosolic Ca2+ concentration are
caused by sulfate-deprivation in Arabidopsis (K Inoue et al.,
personal communication). In addition, Ca2+ can partially
increase the activity of cytosolic Ser acetyltransferase even
in the presence of inhibitory cysteine concentrations.
These results suggest a previously unknown role for Ca2+ in
desensitizing Ser acetyltransferase to cysteine inhibition.
They suggest that cytosolic Ca2+ finely modulates the feedback regulation of Ser acetyltransferase and ultimately
controls cytosolic OAS levels. In soybean, Ca2+-dependent
protein kinase phosphorylates a putative cytosolic Ser
acetyltransferase, which is a feedback-inhibited isoform
[58]. The resulting phosphorylated enzyme is still active
but no longer subject to feedback inhibition by Cys.
Taking all this information together, it is possible to form a
hypothetical model of signal transduction and the regulatory circuit for sulfate transport and assimilation (Figure 2).
Under sulfur-sufficient conditions, steady-state levels of Cys
inhibit the activity of Ser acetyltransferase; thus, the OAS

Regulation of sulfate transport and synthesis of sulfur-containing amino acids Saito

191

Figure 2
Hypothetical model for signal transduction of
sulfate transport and assimilation. CDPK,
Ca2+-dependent protein kinase; SAT-c,
cytosolic Ser acetyltransferase.

Plasma membrane

Nutritional stress

(S-starvation, GSH, etc)

Ca2+
CDPK

activation

ATP
SAT-c

(Cys sensitive)

Ser
SAT-c*

(Desensitized)

Acetyl-CoA
OAS

Induction

Nucleus
Gene expression
(Sulfate transporter,
APS reductase)

Feedback
inhibition

Cys
Met GSH Sulfur
compounds

levels are kept low, resulting in the repression of the expression of the genes that encode sulfate transporters and APS
reducatse. External or internal stimuli such as sulfate deficiency or decreasing GSH levels trigger Ca2+ mobilization
into the cytosol. Ca2+ partially desensitizes cytosolic Ser
acetyltransferase to Cys inhibition, and Ca2+-dependent
protein kinase phosphorylates this isoform; both of these
changes result in increased Ser-acetyltransferase activity and
subsequently OAS production. The elevated OAS concentration activates the expression of the repressed genes that
encode proteins involved in sulfate transport and assmilation. This hypothetical mechanism explains the fine
modulation of sulfate transport and assimilation that ensures
that Cys levels remain constant even prior to a decrease in
Cys level caused by sulfate starvation. The molecules that
are known to be involved in early signal perception and
transduction have been isolated from the unicellualar green
algae Chlamydomonas reinhardtii by mutant analysis [59,60•];
these molecules might also be involved in sulfate sensing in
higher plants.

Met synthesis
As shown in Figure 1, Met is synthesized in three steps from
Cys and O-phosphohomoserine (which is derived from
aspartic acid) [61,62•]. Cystathione γ-synthase catalyzes the
first step (Figure 1g) that involves the β-replacement of the
phosphate group of O-phosphohomoserine with Cys to form
cystathione. The second enzyme in the pathway is cystathione β-lyase (Figure 1h), which catalyzes the cleavage of
a β-C-S bond to produce homocysteine. The final step
(Figure 1i) is catalyzed by Met synthase, which transfers the
methyl group from methyltetrahydrofolate to homocysteine.
This reaction is important not only in de novo Met synthesis
but also in the recycling of S-adenosylmethionine, which is

H2S

SO42–
Current Opinion in Plant Biology

a key compound serving as a methyl donor in a variety of
methy-transfer reactions.
Plastids contain all of the enzymes involved in the conversion of aspartate to homocysteine. Nevertheless, the steps
from homocysteine to Met and the recycling of S-adenosylmethionine are localized in the cytosol. Cystathione
γ-synthase has been shown to be localized in the plastids of
spinach leaves [63] and A. thaliana [64•]. The predicted cystathione γ-synthase structures deduced from its cDNAs
from A. thaliana [65] and potato [66•] also suggest that this
enzyme is localized in the plastids. In spinach, there are two
distinct forms of cystathione β-lyase, one localized in the
plastids and one in the cytosol [67]. The cytosolic isoform
catalyzes the β-cleavage of cystine more efficiently than that
of cystathione, indicating that the proper substrate for the
cytosolic cystathione β-lyase is cystine rather than cystathione [68••]. In contrast, the plastidic isoform efficiently
catalyzes the β-cleavage of cystathione. These findings were
confirmed in A. thaliana [69] and Echinochloa colonum [70], in
which cystathione β-lyase is only detected in the plastids. A
cDNA encoding cystathione β-lyase was cloned from
A. thaliana [69]. The predicted polypeptide encoded by this
cDNA contained a putative plastid-transit peptide. The
plant Met synthase is localized in the cytosol, and its cDNAs
have been cloned from several plant species [68••,71,72].
Multiple regulatory mechanisms control the synthesis of
amino acids from the aspartate family (i.e. Met, lysine and
threonine). The control of cystathione γ-synthase activity
primarily regulates the Met-biosynthetic enzymes. The
cellular activity of cystathione γ-synthase is regulated negatively by the presence of Met, and positively by lysine and
threonine [62•]. A recent analysis of a Met over-producing

192

Physiology and metabolism

A. thaliana mutant revealed that the stability of the mRNA
that encodes cystathione γ-synthase is negatively auto-regulated by an amino-acid stretch in its own translational
product (i.e. cystathione γ-synthase) that takes place when
Met or its metabolites are present [73••]. This amino-acid
stretch is highly conserved amongst several cystathione γsynthases from different species, and hence, this unique
regulatory mechanism is likely to be functionally conserved
in plant cells. The antisense suppression of the cystathione
γ-synthase encoding gene resulted in severe growth reduction, which is restored by the exogenous addition of Met
[68••,74]. This finding suggests an essential role for cystathione γ-synthase in controlling plant growth.

3.

Hell R: Molecular physiology of plant sulfur metabolism. Planta
1997, 202:138-148.

4.

Saito K, Takahashi H, Noji M, Inoue K, Hatzfeld Y: Molecular
regulation of sulfur assimilation and cysteine synthesis. In Sulfur
Nutrition and Assimilation in Higher Plants. Edited by Brunold C,
Rennenberg H, De Kok LJ, Stulen I, Davidian J-C. Berne: Paul Haupt;
2000: in press.

5.

Clarkson DT, Hawkesford MJ, Davidian J-C: Membrane and longdistance transport of sulfate. In Sulfur Nutrition and Assimilation in
Higher Plants — Regulatory Agricultural and Environmental Aspects.
Edited by De Kok LJ, Stulen I, Rennenberg H, Brunold C, Rauser WE.
The Hague: SPB Academic Publishing; 1993:3-20.

6.

Davidian J-C, Hatzfeld Y, Cathala N, Tagmount A, Vidmar JJ: Sulfate
uptake and transport in plants. In Sulfur Nutrition and Assimilation
in Higher Plants. Edited by Brunold C, Rennenberg H, De Kok LJ,
Stulen I, Davidian J-C. Berne: Paul Haupt; 2000: in press.

7.

Smith FW, Ealing PM, Hawkesford MJ, Clarkson DT: Plant members
of a family of sulfate transporters reveal functional subtypes. Proc
Natl Acad Sci USA 1995, 92:9373-9377.

8.

Takahashi H, Sasakura N, Noji M, Saito K: Isolation and
chracterization of a cDNA encoding a sulfate transporter from
Arabidopsis thaliana. FEBS Lett 1996, 392:95-99.

9.

Takahashi H, Yamazaki M, Sasakura N, Watanabe A, Leustek T,
de Almeida Engler J, Engler G, Van Montagu M, Saito K: Regulation of
cysteine biosynthesis in higher plants: a sulfate transporter
induced in sulfate-starved roots plays a central role in Arabidopsis
thaliana. Proc Natl Acad Sci USA 1997, 94:11102-11107.

Conclusions
Although events in the sulfur assimilation pathway in plants
have been confirmed only in the past few years, progress in
understanding the mechanism and molecular regulation of
this pathway has been remarkably rapid. Nevertheless, the
available information on sulfur allocation [30,75•] and signal
transduction is still limited. We can expect that further molecular analysis of sulfur transport and assimilation will
provide significant insights into the pathways involved in
the biosynthesis of sulfur-containing amino acids and will
ultimatley lead to the molecular engineering of these pathways. The biosynthesis pathways for sulfur-containing
amino acids are of particular interest because of a variety of
cellular functions (e.g. redox cycling, detoxification etc.) are
ascribed to sulfur-containing compounds.

Note added in proof
Detailed functional characterisation and tissue localization
of three distinct sulfate transporters from A. thaliana has
recently been provided [13••]. A fourth ATP sulfurylase
has been isolated from A. thaliana [22••]. Evidence is provided for the identity of β-cyanoalanine synthase with a
mitochondrial OAS(thiol)-lyase (cysteine synthase) in
spinach and A. thaliana [40••].

Acknowledgements
I would like to thank all of those colleagues in plant sulfur research, both
inside and outside of my laboratory, who provided unpublished results and
preprints of papers. The research in my group was supported in part by
grants-in-aid for scientific research from the Ministry of Education, Science,
Sports and Culture, Japan, by the Research for the Future Program
(96I00302) of the Japan Society for the Promotion of Science, and by the
Asahi Glass Foundation.

References and recommended reading

Papers of particular interest, published within the annual period of review,
have been highlighted as:

• of special interest
•• of outstanding interest
Saito K: Biosynthesis of cysteine. In Plant Amino Acids
Biochemistry and Biotechnology. Edited by Singh BK. New York:
Marcel Dekker, Inc.; 1999:267-291.
One of the most recent reviews describing the mechanism and regulation of
Cys biosynthesis.

10. Takahashi H, Sasakura N, Kimura A, Watanabe A, Saito K:
Identification of two leaf-specific sulfate transporters in
Arabidopsis thaliana (Accession No. AB012048 and AB004060).
Plant Physiol 1999, 121:686.
11. Takahashi H, Asanuma W, Saito K: Cloning of an Arabidopsis cDNA
•
encoding a chloroplast localizing sulphate transporter isoform.
J Exp Botany 1999, 50:1713-1714.
This paper provides evidence of the in vivo translocation of a putative
Arabidopsis sulfate transporter into chloroplasts. The investigation that is
described used a fusion protein in which a sulfate transporter isoform is
bound to a green fluorescent protein.
12. Yamaguchi Y, Nakamura T, Harada E, Koizumi N, Sano H: Isolation
and characterization of a cDNA encoding a sulfate transporter
from Arabidopsis thaliana (Accession No. D89631). Plant Physiol
1997, 113:1463.
13
••

Takahashi H, Watanabe-Takahashi A, Smith FW, Blake-Kalff M,
Hawkesford MJ, Saito K: The roles of three functional sulfate
transporters involved in uptake and translocation of sulfate in
Arabidopsis thaliana. Plant J 2000, in press.
This study provides the first detailed characterization of three functional sulfate transporters. The different tissue localizations of these three transporters is emphasized and suggests that these transporters have distinct
roles as high- and low-affinity transporters.
14. Smith FK, Hawkesford MJ, Ealing PM, Clarkson DT, Berg PJV,
Belcher AR, Warrilow AGS: Regulation of exprression of a cDNA
from barley roots encoding a high affinity sulphate transporter.
Plant J 1997, 12:875-884.
15. Vidmar JJ, Schjoerring JK, Touraine B, Glass ADM: Regulation of the
•
hvst1 gene encoding a high-affinity sulfate transporter from
Hordeum vulgare. Plant Mol Biol 1999, 40:883-892.
The authors of this paper and [14] describe the cloning of a high-affinity sulfate
transporter from barley. This transporter is induced by sulfate depletion. GSH
down-regulated the concentration of mRNA encoding this sulfate transporter
even in sulfate-starved plants in which the transporter is usually derepressed.
16. Bolchi A, Petrucco S, Tenca PL, Foroni C, Ottonello S: Coordinate
modulation of maize sulfate permease and ATP sulfurylase
mRNAs in response to variations in sulfur nutritional status:
stereospecific down-regulation by L-cysteine. Plant Mol Biol 1999
39:527-537.

1.
••

17.

2. Leustek T, Saito K: Sulfate transport and assimilation in plants.
•• Plant Physiol 1999, 120:637-643.
One of the most recent reviews on sulfate transport and assimilation in
plants.

18. Frachisse J-M, Thomine S, Colcombet J, Guern J, Barbier-Brygoo H:
Sulfate is both a substrate and an activator of the voltage-

Mornet C, Tommasini R, Hörtensteiner S, Martinoia E: Transport of
sulphate and reduced sulphur compounds at the tonoplast
membrane. In Sulphur Metabolism in Higher Plants Molecular,
Ecophysiological and Nutritional Aspects. Edited by Cram WJ,
De Kok LJ, Stulen I, Brunold C, Rennenberg H. Leiden: Backhuys
Publishers; 1997:1-11.

Regulation of sulfate transport and synthesis of sulfur-containing amino acids Saito

dependent anion channel of Arabidopsis hypocotyl cells. Plant
Physiol 1999, 121:253-261.
19. Klonus D, Höfgen R, Willmitzer L, Riesmeier JW: Isolation and
characterization of two cDNA clones encoding ATP-sulfurylase
from potato by complementation of a yeast mutant. Plant J 1994,
6:105-112.
20. Leustek T, Murillo M, Cervantes M: Cloning of a cDNA encoding ATP
sulfurylase from Arabidopsis thaliana by functional expression in
Saccharomyces cerevisiae. Plant Physiol 1994, 105:897-902.
21. Logan HM, Cathala N, Grignon C, Davidian J-C: Cloning of a cDNA
encoded by a member of the Arabidopsis thaliana ATP sulfurylase
multigene family. J Biol Chem 1996, 271:12227-12233.
22. Hatzfeld Y, Lee S, Lee M, Leustek T, Saito K: Functional
•• characterization of a gene encoding a fourth ATP sulfurylase
isoform from Arabidopsis thaliana. Gene 2000, in press.
In this study, the gene encoding fourth chloroplastic ATP sulfurylase was isolated. The authors discuss the possibility that the cytosolic ATP sulfurylase
is produced from one of the genes encoding chloroplastic isoforms by the
use of a different initiation Met codon.
23. Hatzfeld Y, Cathala N, Grignon C, Davidian J-C: Effect of ATP
•
sulfurylase overexpression in Bright Yellow 2 tobacco cells. Plant
Physiol 1998, 116:1307-1313.
This is the first report to describe the effects of overexpression of ATP sulfurylase. Although the ATP sulfurylase activity was increased 8-fold in the
BY-2 cells of transgenics tobacco, no effects on cell growth and sensitivity
to selenate were observed.
24. Pilon-Smit EAH, Hwang S, Lytle CM, Zhu Y, Tai JC, Bravo RC,
•
Chen Y, Leustek T, Terry N: Overexpression of ATP sulfurylase in
Indian mustard leads to increased selenate uptake, reduction,
and tolerance. Plant Physiol 1999, 119:123-132.
This paper describes similar work to that reported in [23•]. ATP sulfurylase
was overexpressed in transgenic Indian mustard (Brassica juncea). The
transformants showed increased selenate uptake, reduction and tolerance,
suggesting that ATP sulfurylase mediates the reduction of selenate and limits the rate of selenate assimilation.

193

32. Yonekura-Sakakibara K, Ashikari T, Tanaka Y, Kusumi T, Hase T:
Molecular characterization of tobacco sulfite reductase: enzyme
purification, gene cloning, and gene expression analysis.
J Biochem 1998, 124:615-621.
33. Yonekura-Sakakibara K, Onda Y, Ashikari T, Tanaka Y, Kusumi T,
•
Hase T: Analysis of reductant supply systems for ferredoxindependent sulfite reductase in photosynthetic and
nonphotosynthetic organs of maize. Plant Physiol 2000, 122:887-894.
In this paper, the authors reconstituted the sulfite-reduction system, which
involves ferredoxin, ferredoxin-NADP+ reductase and sulfite reductase, using
an appropriate combination of tissue-specific enzyme isoforms. This allowed
the authors to investigate the efficiency of electron transfer by different enzyme
isoforms in different tissues, and to examine the complementation of an E. coli
sulfite reductase-deficient mutant by genes encoding the various isoforms.
34. Lunn JE, Droux M, Martin J, Douce R: Localization of ATP sulfurylase
and O-acetylserine(thiol)lyase in spinach leaves. Plant Physiol
1990, 94:1345-1352.
35. Takahashi H, Saito K: Subcellular localization of spinach cysteine
synthase isoforms and regulation of their gene expression by
nitrogen and sulfur. Plant Physiol 1996, 112:273-280.
36. Ruffet M-L, Lebrun M, Droux M, Douce R: Subcellular distribution of
serine acetyltransferase from Pisum sativum and characterization
of an Arabidopsis thaliana putative cytosolic isoform. Eur J
Biochem 1995, 227:500-509.
37.
••

Noji M, Inoue K, Kimura N, Gouda A, Saito K: Isoform-dependent
differences in feedback regulation and subcellular localization of
serine acetyltransferase involved in cysteine biosynthesis from
Arabidopsis thaliana. J Biol Chem 1998, 273:32739-32745.
The subcellular localization of three Ser acetyltransferases from A. thaliana
was determined by transient expression of fusion proteins in which each of
these three enzymes were fused with green fluorescence protein.
Furthermore, it is shown that only the cytosolic isoform of Ser acetyltransferase (i.e. not the chloroplastic and mitochondrial isoforms) is regulated by
feedback inhibition by Cys. This work provides evidence of the important
regulatory role of cytosoic Ser acetyltransferase in Cys biosynthesis.

25. Bick JA, Leustek T: Plant sulfur metabolism — the reduction of
•
sulfate to sulfite. Curr Opin Plant Biol 1998, 1:240-244.
An up-to-date short review on the reduction of sulfate to sulfite and the
enzymes involved in this reaction.

38. Hesse H, Lipke J, Altmann T, Höfgen R: Molecular cloning and
expression analyses of mitochondrial and plastidic isoforms of
cysteine synthase (O-acetylserine (thiol)lyase) from Arabidopsis
thaliana. Amino Acids 1999, 16:113-131.

26. Bick J-A, Åslund F, Chen Y, Leustek T: Glutaredoxin function for the
•
carboxyl-terminal domain of the plant-type 5¢-adenylylsulfate
reductase. Proc Natl Acad Sci USA 1998, 95:8404-8409.
The authors show that APS reductase has two domains. They present
evidence showing that the carboxyl-terminal domain has a glutaredoxin-like
function and that APS reductase requires GSH for its catalytic activity.

39. Warrilow AGS, Hawkesford MJ: Separation, subcellular location
•
and influence of sulphur nutrition on isoforms of cysteine
synthase in spinach. J Exp Botany 1998, 49:1625-1636.
This paper and [41•] deal with the relation between β-cyanoalanine synthase
and OAS(thiol)-lyase [Cys synthase]. The authors of this paper present definitive evidence, which was obtained using purification techniques, to show that
β-cyanoalanine synthase is predominantly located in mitochondria of spinach.

27.
•

Suter M, von Ballmoos P, Kopriva S, Op den Camp R, Schaller J,
Kuhlemeier C, Schürmann P, Brunold C: Adenosine
5′′-phosphosulfate sulfotransferase and adenosine
5′′-phosphosulfate reducatse are identical enzymes. J Biol Chem
2000, 75:930-936.
This is a conclusive report that shows that APS reductase and APS sulfotransferase, which are thought to be involved in the carrier-bound pathway of
APS reduction, are identical enzymes. APS sulfotransferase was purified,
cloned and characterized using a recombinant form of the enzyme from
Lemna minor.
28. Heiss S, Schäfer HJ, Haag-Kerwer A, Rausch T: Cloning sulfur
assimilation genes of Brassica juncea L.: cadmium differentially
affects the expression of a putative low-affinity sulfate transporter
and isoforms of ATP sulfurylase and APS reductase. Plant Mol
Biol 1999, 39:847-857.
29. Kopriva S, Muheim R, Koprivova A, Trachsel N, Catalano C, Suter M,
•
Brunold C: Light regulation of assimilatory sulphate reduction in
Arabidopsis thaliana. Plant J 1999, 20:37-44.
This paper describes the evidence for the existence of a diurnal rhythm in the
expression of the mRNA that encodes APS reductase and for the activity of
this enzyme. This evidence suggests that APS reductase is regulated by a
similar mechanism to that controlling nitrate reductase. Sucrose is shown to
be a positive effector of the expression of mRNA encoding APS reductase
and of the activity of this enzyme.
30. Burgener M, Suter M, Jones S, Brunold C: Cyst(e)ine is the transport
metabolite of assimilated sulfur from bundle-sheath to mesophyll
cells in maize leaves. Plant Phsyiol 1998, 116:1315-1322.
31. Bork C, Schwenn JD, Hell R: Isolation and characterization of a
gene for assimilatory sulfite reductase from Arabidopsis thaliana.
Gene 1998, 212:147-153.

40. Hatzfeld Y, Maruyama A, Schmidt A, Noji M, Ishizawa K, Saito K:
•• β-Cyanoalanine synthase is a mitochondrial cysteine synthaselike protein in spinach and Arabidopsis thaliana. Plant Physiol
2000, in press.
Definitive evidence is provided for the identity of β-cyanoalanine synthase
with a mitochondrial OAS(thiol)-lyase [cysteine synthase], of which cDNA
was previously isolated from spinach [42].
41. Maruyama A, Ishizawa K, Takagi T, Esashi Y: Cytosolic β
•
cyanoalanine synthase activity attributed to cysteine synthases in
Cocklebur seeds. Purification and characterization of cytosolic
cysteine synthases. Plant Cell Physiol 1998, 39:671-680.
The authors used purification and a partial amino-acid sequence analysis of
the purified β-cyanoalanine synthase to show that the cytosolic activity of βcyanoalanine synthase is controlled by OAS(thiol)-lyase [Cys synthase].
42. Saito K, Tatsuguchi K, Takagi Y, Murakoshi I: Isolation and
characterization of cDNA that encodes a putative mitochondrionlocalizing isoform of cysteine synthase (O-acetylserine(thiol)-lyase)
from Spinacia oleracea. J Biol Chem 1994, 269:28187-28192.
43. Inoue K, Noji M, Saito K: Determination of the sites required for the
•
allosteric inhibition of serine acetyltransferase by L-cysteine in
plants. Eur J Biochem 1999, 266:220-227.
This study uses a series of recombinant mutant proteins to determine the
residues and domains of Ser acetyltransferase that are involved in the feedback inhibition of this enzyme by Cys.
44. Saito K, Yokoyama H, Noji M, Murakoshi I: Molecular cloning and
characterization of a plant serine acetyltransferase playing a
regulatory role in cysteine biosynthesis from watermelon. J Biol
Chem 1995, 270:16321-16326.

194

Physiology and metabolism

45. Bogdanova N, Hell R: Cysteine synthesis in plants: protein–protein
interactions of serine acetyltransferase from Arabidopsis thaliana.
Plant J 1997, 11:251-262.
46. Droux M, Ruffet M-L, Douce R, Job D: Interactions between serine
•• acetyltransferase and O-acetylserine (thiol) lyase in higher plants.
Structural and kinetic properties of the free and bound enzymes.
Eur J Biochem 1998, 255:235-245.
The significance of the bienzyme complex involving OAS(thiol)-lyase and Ser
acetyltransferase, and the importance of greater cellular concentrations of
uncomplexed OAS(thiol)-lyase Ser acetyltransferase relative to those of the
uncomplexed enzyme, is explained. An interesting regulatory mechanism is
proposed that involves both free and bound forms of the two enzymes. Within
the complex, the bound form of OAS(thiol)-lyase may act as a structural
and/or regulatory subunit of Ser acetyltransferase. Actual OAS(thiol)-lyase
activity is attributed to the free form, because the activity of bound form of
OAS(thiol)-lyase is dramatically decreased.
47.
•

Barroso C, Romero LC, Cejudo FJ, Vega JM, Gotor C: Salt-specific
regulation of the cytosolic O-acetylserine (thiol)lyase gene from
Arabidopsis thaliana is dependent on abscisic acid. Plant Mol Biol
1999, 40:729-736.
Salt and heavy metal stresses induced OAS(thiol)-lyase activity suggesting
a connection between these stresses and trichome-specific gene expression [48]. The induction of gene expression is presumably mediated by
abscisic acid.
48. Gotor C, Cejudo FJ, Barroso C: Tissue-specific expression of
ATCYS-3A, a gene encoding the cytosolic isoform of
O-acetylserine (thiol)lyase in Arabidopsis. Plant J 1997 11:347-352.
49. Saito K, Kurosawa M, Tatsuguchi K, Takagi Y, Murakoshi I: Modulation
of cysteine biosynthesis in chloroplasts of transgenic tobacco
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Plant Physiol 1994, 106:887-895.
50. Blaszczyk A, Brodzik R, Sirko A: Increased resistance to oxidative
•
stress in transgenic tobacco plants overexpressing bacterial
serine acetyltransferase. Plant J 1999, 20:237-243.
This paper describes the first study of overexpression of Ser acetyltransferase in plants. Although overexpression of this enzyme resulted in only two
or three times higher concentrations of Cys and GSH, much greater resistance to hydrogen peroxide was generated.
51. Kredich NM: Biosynthesis of cysteine. In Escherichia coli and
Salmonella typhimurium: Cellular and molecular biology, vol 1, edn 2.
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52. Koprivova A, Suter M, Op den Camp R, Brunold C, Kopriva S:
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thaliana. Plant Physiol 2000, 122:737-746.
53. Kim H, Hirai MY, Hayashi H, Chino M, Naito S, Fujiwara T: Role of
•
O-acetyl-L-serine in the coordinated regulation of the expression
of a soybean seed storage-protein gene by sulfur and nitrogen
nutrition. Planta 1999, 209:282-289.
In conditions in which sulfur is deficient (or a low ratio of sulfur to nitrogen
availability exists), the β-subunit of β-conglycinin (i.e. the sulfur-poor soybean
seed storage protein) is accumulated. This paper provides evidence that
OAS accumulates to higher concentrations in A. thaliana and soybean
cotyledons under sulfate-deficient conditions. The addition OAS results in an
elevated accumulation of the mRNA encoding the β-subunit and of the protein itself. These findings suggest that OAS is a positive signal molecule for
gene expression in sulfur-starved plants.
54. Clarkson D, Diogo E, Amâncio S: Uptake and assimilation of
sulphate by sulphur deficient Zea mays cells: the role of O-acetylL-serine in the interaction between nitrogen and sulphur
assimilatory pathways. Plant Physiol Biochem 1999, 37:283-290.
55. Herschbach C, Rennenberg H: Influence of glutathione (GSH) on
net uptake of sulphate and sulphate transport to tobacco plants.
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56. Lappartient AG, Touraine B: Demand-driven control of root ATP
sulfurylase activity and SO42– uptake in intact canola. Plant
Physiol 1996, 111:147-157.
57.
••

Lappartient AG, Vidmar JJ, Leustek T, Glass ADM, Touraine B: Inter
organ signaling in plants: regulation of ATP sulfurylase and
sulfate transporter genes expression in roots mediated by
phloem-translocated compound. Plant J 1999, 18:89-95.
An impressive report that suggests a role for GSH as a phloem-translocated signal molecule that represses the expression of genes encoding ATP
sulfurylase and a sulfate transporter. These conclusions are based on the
results of ‘split root’ experiments using A. thaliana and B. napus.

58. Yoo B-C, Harmon AC: Regulation of recombinant soybean serine
acetyltransferase by CDPK. Plant Physiol Suppl 1997, 114:267.
59. Davies JP, Yildiz F, Grossman A: Sac1, a putative regulator that is
critical for survival of Chlamydomonas reinhardtii during sulfur
deprivation. EMBO J 1996, 15:2150-2159.
60. Davies JP, Yildiz FH, Grossman AR: Sac3, an Snf1-like
•
serine/threonine kinase that positively and negatively regulates
the responses of Chlamydomonas to sulfur limitation. Plant Cell
1999, 11:1179-1190.
This paper and [59] demonstrate the benefits of using the unicellular
green algae Chlamydomonas as a model organism in which to study the
effects of nutritional stress on signal transduction. Sac3 cDNA was identified by its complementation of a Chlamydomonas Sac3 mutant, which
lacked the proper responses to sulfate deprivation. The Sac3 product is
a Snf1-related serine/threonine kinase. The involvement of the same
product in responses to sulfur deprivation in higher plants is an intriguing possibility.
61. Azevedo RA, Arruda P, Turner WL, Lea PJ: The biosynthesis and
metabolism of the aspartate derived amino acids in higher plants.
Phytochemistry 1997, 46:395-419.
62. Matthews BF: Lysine, threonine, and methionine biosynthesis. In
•
Plant Amino Acids Biochemistry and Biotechnology. Edited by
Singh BJ. New York: Marcel Dekker, Inc.; 1999:205-225.
An excellent review describing the up-to-date information on biosynthesis of
amino acids in the aspartate family.
63. Ravanel S, Droux M, Douce R: Methionine biosynthesis in higher
plants. I. Purification and characterization of cystathione
γ-synthase from spinach chloroplasts. Arch Biochem Biophys
1995, 316:572-584.
64. Ravanel S, Gakiere B, Job D, Douce R: Cystathione γ-synthase
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from Arabidopsis thaliana: purification and biochemical
chracterization of the recombinant enzyme overexpressed in
Escherichia coli. Biochem J 1998, 331:639-648.
The authors describe the first biochemical characterization of recombinant
cystathione γ-synthase from plants.
65. Kim J, Leustek T: Cloning and analysis of the gene for cystathione
γ-synthase from Arabidopsis thaliana. Plant Mol Biol 1996,
32:1117-1124.
66. Riedel K, Mangelsdorf C, Streber W, Willmitzer L, Höfgen R, Hesse H:
•
Cloning and characterization of cystathione γ-synthase from
Solanum tuberosum L. Plant Biol 1999, 1:638-644.
The cloning of cystathione γ-synthase from potato is reported. The expression pattern of the gene encoding this enzyme exhibits a diurnal rhythm and
is induced by light.
67.

Droux M, Ravanel S, Douce R: Methionine biosynthesis in higher
plants. II. Purification and characterization of cystathione β-lyase
from spinach chloroplasts. Arch Biochem Biophys 1995,
316:585-595.

68. Ravanel S, Gakiere B, Job D, Douce R: The specific features of
•• methionine biosynthesis and metabolism in plants. Proc Natl
Acad Sci USA 1998, 95:7805-7812.
An excellent article describing the current status of our knowledge of Met
biosynthesis in plants.
69. Ravanel S, Ruffet M-L, Douce R: Cloning of an Arabidopsis thaliana
cDNA encoding cystathione β-lyase by functional complementation
in Escherichia coli. Plant Mol Biol 1995, 29:875-882.
70. Turner WL, Pallett KE, Lea PJ: Cystathione β-lyase from Echinochloa
colonum tissue culture. Phytochemistry 1998, 47:189-196.
71. Eichel J, Gonzalez JC, Hotze M, Matthews RG, Schröder J:
Vitamin-B12-independent methionine synthase from higher plant
(Catharanthus roseus). Molecular characterization, regulation,
heterologous expression, and enzyme properties. Eur J Biochem
1995, 230:1053-1058.
72. Petersen M, Van Der Straeten D, Bauw G: Full-length cDNA clone
from Coleus blumei (GenBank Z49150) with high similarity to
cobalamine-independent methionine synthase. Plant Physiol
1995, 109:338.
73. Chiba Y, Ishikawa M, Kijima F, Tyson RH, Kim J, Yamamoto A,
•• Nambara E, Leustek T, Wallsgrove RM, Naito S: Evidence for
authoregulation of cystathione γ-synthase mRNA stability in
Arabidospsis. Science 1999, 286:1371-1374.
The authors present a molecular analysis of Arabidopsis mutants that overproduce Met. They propose a unique mechanism through which stable levels of the mRNA encoding cystathione γ-synthase are maintained by the

Regulation of sulfate transport and synthesis of sulfur-containing amino acids Saito

repression of this enzyme by its own translational product (i.e. cystathione γsynthase) in the presence of Met (or its metabolites).
74. Kim J, Leustek T: Repression of cystathione γ-synthase in
Arabidopsis thaliana produces partial methonine auxotrophy and
developmental abnormalities. Plant Sci 2000, 151:9-18.
75. Bourgis F, Roje S, Nuccio ML, Fisher DB, Tarczynski MC, Li C,
•
Herschbach C, Rennenberg H, Pimenta MJ, Shen T-L et al.:
S-Methylmethionine plays a major role in phloem sulfur transport

195

and is synthesized by a novel type of methyltransferase. Plant
Cell 1999, 11:1485-1497.
Analysis of phloem sap moving towards wheat ears showed that
S-methylmethionine is the major sulfur-containing compound in phloem
sap. S-methylmethionine concentration in the phloem sap was 1.5 times
that of GSH, suggesting that S-methylmethionine is the major form of
sulfur transported in phloem. A unique methyltransferase that is responsible for the production of S-methylmethionine was cloned from several
plant species.