The Plant Journal (2010) 62, 1–11
doi: 10.1111/j.1365-313X.2009.04118.x
Genes of primary sulfate assimilation are part of the
glucosinolate biosynthetic network in Arabidopsis thaliana
Ruslan Yatusevich1, Sarah G. Mugford2, Colette Matthewman2, Tamara Gigolashvili1, Henning Frerigmann1, Sean Delaney2,
Anna Koprivova2, Ulf-Ingo Flügge1 and Stanislav Kopriva2,*
1
Botanisches Institut der Universität zu Köln, Otto-Fischer-Str. 6, D-50674 Köln, Germany, and
2
Department of Metabolic Biology, John Innes Centre, Norwich NR4 7UH, UK
Received 2 December 2009; accepted 10 December 2009; published online 1 February 2010.
*
For correspondence (fax +44 1603 450014; e-mail stanislav.kopriva@bbsrc.ac.uk).
SUMMARY
Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their
synthesis is the transfer of a sulfo group from 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) onto a desulfo
precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation. The sulfate donor for this reaction is
synthesized from sulfate in two steps catalyzed by ATP sulfurylase (ATPS) and adenosine 5¢-phosphosulfate
kinase (APK). Here we demonstrate that R2R3-MYB transcription factors, which are known to regulate both
aliphatic and indolic glucosinolate biosynthesis in Arabidopsis thaliana, also control genes of primary sulfate
metabolism. Using trans-activation assays we found that two isoforms of APK, APK1, and APK2, are regulated
by both classes of glucosinolate MYB transcription factors; whereas two ATPS genes, ATPS1 and ATPS3,
are differentially regulated by these two groups of MYB factors. In addition, we show that the adenosine
5¢-phosphosulfate reductases APR1, APR2, and APR3, which participate in primary sulfate reduction, are also
activated by the MYB factors. These observations were confirmed by analysis of transgenic lines with
modulated expression levels of the glucosinolate MYB factors. The changes in transcript levels also affected
enzyme activities, the thiol content and the sulfate reduction rate in some of the transgenic plants. Altogether
the data revealed that the MYB transcription factors regulate genes of primary sulfate metabolism and that the
genes involved in the synthesis of activated sulfate are part of the glucosinolate biosynthesis network.
Keywords: glucosinolates, MYB transcription factors, phosphoadenosine phosphosulfate, adenosine
5¢-phosphosulfate kinase, ATP sulfurylase, APS reductase.
INTRODUCTION
Plants produce a great variety of natural compounds with
various functions. One of the best-studied groups of secondary metabolites is the glucosinolates (GSs). Glucosinolates are a large group of sulfur-rich amino acid-derived
metabolites, found in the Brassicaceae (Fahey et al., 2001;
Halkier and Gershenzon, 2006). Glucosinolates play an
important role in plant defense against herbivores and
insects (Halkier and Gershenzon, 2006). Upon tissue
damage, the vacuole-stored GSs come into contact with the
enzyme myrosinase, which catalyzes their degradation into
the active deterrents, volatile isothiocyanates or nitriles
(Matile, 1980; Rask et al., 2000). The reaction with myrosinase, and thus the biological activity of GSs, depends on the
sulfate group (Ratzka et al., 2002). Glucosinolates are also
important nutritionally; they are responsible for the smell
and taste of cruciferous vegetables (Fenwick et al., 1983). In
addition, their breakdown products have been shown to
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd
have anticarcinogenic activity (Verhoeven et al., 1997;
Mithen et al., 2003). They can, however, have a negative
impact when present in large amounts in animal feed
derived from rapeseed (Schöne et al., 1997).
The two major classes of GSs, aliphatic and indolic GSs,
are derived from the amino acids methionine and tryptophan, respectively. The pathway of their synthesis has been
elucidated and the corresponding genes have been identified (for review see Yan and Chen, 2007; Halkier and
Gershenzon, 2006; Grubb and Abel, 2006). Glucosinolate
biosynthesis is regulated by a complex network of transcription factors in response to biotic and abiotic stress
(Celenza et al., 2005; Levy et al., 2005; Maruyama-Nakashita
et al., 2006; Skirycz et al., 2006; Gigolashvili et al., 2007a,b,
2008; Hirai et al., 2007; Sønderby et al., 2007; Malitsky et al.,
2008). The best characterized of these factors belong to the
R2R3-MYB family, and can be divided into two groups
1
2 Ruslan Yatusevich et al.
according to their sequence (Gigolashvili et al., 2007a,b,
2008; Hirai et al., 2007; Sønderby et al., 2007). The first clade,
formed by MYB28, MYB76, and MYB29, alternatively called
high aliphatic glucosinolate (HAG) 1–3, respectively, is
specifically involved in the control of synthesis of aliphatic
GSs (Gigolashvili et al., 2007b, 2008; Hirai et al., 2007;
Sønderby et al., 2007), while the other consists of MYB51,
MYB122, and MYB34, alternatively high indolic glucosinolate (HIG)-1, -2, and -3 (also named ATR1), and affects the
synthesis of indolic GSs (Celenza et al., 2005; Gigolashvili
et al., 2007a; Malitsky et al., 2008). Manipulation of the
expression of these transcription factors by overexpression,
RNAi, or T-DNA insertions results in both distinct and
overlapping alterations of mRNA levels of GS biosynthetic
genes as well as GS accumulation (Celenza et al., 2005;
Gigolashvili et al., 2007a,b, 2008; Hirai et al., 2007; Sønderby
et al., 2007; Malitsky et al., 2008). Apart from the MYB
factors, GS synthesis is regulated by the DNA-binding-withone-finger (DOF) transcription factor OBP2, which is inducible by herbivory and methyl jasmonate (Skirycz et al., 2006).
In addition, modulation of expression of the calmodulinbinding IQD protein results in significant but moderate
alterations in GS levels (Levy et al., 2005). Responses of GSs
to sulfur availability, i.e. a strong reduction during sulfur
starvation (Hirai et al., 2005; Falk et al., 2007), are controlled
by sulfur limitation 1 (SLIM1), an ethylene-insensitive3-like
transcription factor, which represents a central regulator of
plant response to sulfur starvation (Maruyama-Nakashita
et al., 2006).
The biosynthesis of GS is closely connected to primary
sulfur metabolism. The final step of GS core synthesis is the
sulfation of the desulfo-GS precursors (Figure 1; Underhill
et al., 1973). The sulfation is catalyzed by sulfotransferases
(SOTs) which transfer activated sulfate from 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) to a hydroxylated
Figure 1. Glucosinolate (GS) biosynthesis.
The last step in glucosinolate synthesis, sulfation of desulfo-GSs by
sulfotransferases (SOT) is connected to the synthesis of the activated sulfate
3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) and linked to primary sulfate
assimilation. Sulfate is activated by adenylation to adenosine 5¢-phosphosulfate (APS) by ATP sulfurylase (ATPS). The APS is either phosphorylated by
APS kinase (APK) to PAPS for synthesis of GSs and other sulfated compounds
or is reduced by APS reductase (APR) to sulfite. Sulfite is reduced to sulfide by
sulfite reductase (SiR). Sulfide is incorporated into O-acetylserine (OAS) by
OAS thiollyase (OAS-TL) to form cysteine. The enzyme with names printed in
bold were investigated in this study.
substrate. The PAPS is synthesized from sulfate in two
ATP-dependent steps (Figure 1). Sulfate is first adenylated
by ATP sulfurylase (ATPS) to adenosine 5¢-phosphosulfate
(APS), which is then further phosphorylated by APS kinase
(APK) to PAPS. The importance of PAPS for GS synthesis
was recently demonstrated in Arabidopsis by the finding of
very low levels of GS in plants disrupted in two of the four
APK genes (Mugford et al., 2009). However, APS is also an
intermediate in primary sulfate assimilation, where it is
reduced by APS reductase (APR) to sulfite, and after further
reduction to sulfide it is incorporated into O-acetylserine
(OAS) to form cysteine (Figure 1; for recent review see
Kopriva, 2006). Genes involved in PAPS synthesis as well as
in APS reduction were reported to be affected by misexpression of the GS MYB factors based on microarray
analyses (Sønderby et al., 2007; Malitsky et al., 2008).
We here demonstrate that at least some of the ATPS, APK,
and APR genes are directly regulated by the R2R3-MYB
transcription factors involved in the regulation of core GS
biosynthesis. Using trans-activation assays and analysis of
plants disrupted in or overexpressing these MYB factors,
evidence is presented that several genes involved in primary
sulfur metabolism are members of the glucosinolate biosynthesis network.
RESULTS
Regulation of ATPS and APK by MYB transcription factors
To test the hypothesis that genes involved in the provision of
activated sulfate for GS synthesis are under the same control
as those involved in core GS biosynthesis, we used the
co-transformation assay developed to identify regulatory
genes in the GS synthesis network (Berger et al., 2007).
Since disruption of APK1 and APK2 isoforms of APS kinase
reduced foliar GS levels by 90% (Mugford et al., 2009), the
APK genes were the first to be tested. Reporter constructs
consisting of promoters of the four APK genes fused to the
uidA (GUS) reporter gene were simultaneously expressed
with constructs overexpressing the aliphatic GS controlling
MYB28, MYB76, and MYB29 (Gigolashvili et al., 2007a) and
MYB51, MYB122, and MYB34, regulating indolic GSs, in
cultured Arabidopsis thaliana cells. It was revealed by GUS
activity staining that all six effectors strongly activated
expression from APK1 and APK2 promoters (Figure 2a). On
the other hand, APK3-derived expression seemed to be only
weakly activated, while the APK4 promoter did not trigger
any GUS activity. No obvious differences between the transactivation potentials of the two MYB factor groups on APK
were observed.
To test whether ATPS, the other enzyme necessary for
PAPS synthesis, is also directly linked to the regulation of
GSs, we isolated promoters of the four A. thaliana ATPS
genes and used them in the same trans-activation assays as
APK. The GUS-dependent blue staining was observed only
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
Glucosinolate biosynthesis and sulfate assimilation 3
APK3
GSs, MYB28, MYB76, and MYB29, appeared to induce a
stronger reaction with the ATPS1 promoter than with
ATPS3, while the opposite was true for all three indolic GS
transcription factors (Figure 2b). Thus, ATPS1 seems to be
strongly associated with the control of synthesis of aliphatic
GSs, whereas ATPS3 may be linked to regulation of indolic
GSs.
APK4
Overexpression of MYB factors affects mRNA levels of APK
and ATPS
(a)
No TF
MYB28 MYB76 MYB29 MYB51 MYB122 MYB34
APK1
APK2
(b)
No TF
The trans-activation assays revealed that the six MYB transcription factors are capable of interacting with the promoters of APK1, APK2, ATPS1, and ATPS3, and partly with
APK3. To test whether this is also the case in vivo, we
compared by quantitative RT-PCR (qPCR) the steady-state
mRNA levels of the APK and ATPS genes in leaves of plants
constitutively overexpressing the MYB factors and plants in
which single MYB factor genes were disrupted (Gigolashvili
et al., 2007a,b, 2008). The 35S promoter-driven expression
of the MYB factors leads to 17-, 110-, and 7-fold increases in
mRNA encoding MYB28, MYB76, and MYB29, respectively,
compared with the wild type (Col-0), and to 40-, 8-, and
26-fold increases in transcripts for MYB51, MYB122, and
MYB34. The transcript levels of APK1 and -2 isoforms were
greatly increased in the leaves of all overexpressing plants
compared with the wild type, except APK1 in MYB76_ox
plants (Figure 3a,b). The mRNA levels of APK3 were significantly higher only in the transgenic plants expressing
MYB28 and MYB51, the major regulators of aliphatic and
indolic GS biosynthesis, respectively, but the increase was
3.5- and 2.5-fold, respectively, not as dramatic as with APK1induced 16- and 9-fold increases in these plants, for example. In contrast, APK4 expression was not significantly
MYB28 MYB76 MYB29 MYB51 MYB122 MYB34
ATPS1
ATPS2
ATPS3
ATPS4
Figure 2. Trans-activation assay of APK and ATPS promoters.
Trans-activation assay of (a) APK and (b) ATPS promoters with MYB
transcription factors. The promoters of all four APK and four ATPS isoforms
were fused to the uidA (GUS) reporter gene. Cultured A. thaliana Col-0 cells
were inoculated with the supervirulent Agrobacterium tumefaciens strain
LBA4404.pBBR1MCS.virGN54D containing either only the reporter construct
or the reporter construct and, in addition, Pro35S:MYB effector constructs.
The GUS staining indicates trans-activation of a promoter by an effector.
in cells expressing the reporter from ATPS1 and ATPS3
promoters (Figure 2b). All six MYB factors were capable of
activating the promoters; however, the intensity of the
staining was not equal. The factors associated with aliphatic
(a)
(b)
(c)
(d)
Figure 3. Transcript levels of adenosine 5¢-phosphosulfate kinase (APK) isoforms.
Transcript levels of APK isoforms in plants (a) overexpressing MYB factors involved in the regulation of aliphatic glucosinolates (GSs), (b) expressing MYB factors
regulating indolic GSs, (c) disrupted in genes of MYB factors involved in the control of aliphatic GSs, and (d) disrupted in genes of MYB factors affecting indolic GSs.
RNA was isolated from mature leaves and subjected to quantitative RT-PCR with primers specific to the four APK isoforms. The mRNA levels were compared to
actin. The levels in wild-type plants are set to 1. Results are presented as means SD from three independent RNA preparations. Values marked with asterisks are
significantly (Student’s t-test; P £ 0.05) different from wild-type plants.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
4 Ruslan Yatusevich et al.
(a)
(b)
(c)
(d)
Figure 4. Transcript levels of ATP sulfurylase (ATPS) isoforms.
Transcript levels of ATPS isoforms in plants (a) overexpressing MYB factors involved in the regulation of aliphatic glucosinolates (GSs), (b) expressing MYB factors
regulating indolic GSs, (c) disrupted in genes of MYB factors involved in the control of aliphatic GSs, and (d) disrupted in genes of MYB factors affecting indolic GSs.
RNA was isolated from mature leaves and subjected to quantitative RT-PCR with primers specific to the four ATPS isoforms. The mRNA levels were compared to
actin. The levels in wild-type plants are set to 1. Results are presented as means SD from three independent RNA preparations. Values marked with asterisks are
significantly (Student’s t-test; P £ 0.05) different from wild-type plants.
increased (Figure 3a,b). To test whether any of the MYB
factors is essential for the expression of APK genes we also
analyzed steady-state mRNA levels in mutants of the individual MYB genes. No disruption in any of the MYB genes
resulted in a reduction in APK transcript levels; on contrary,
APK3 was induced in myb34 mutants (Figure 3c,d).
The results of qPCR for members of the ATPS gene family
also agreed well with the results of the trans-activation
assays (Figure 4). Overexpression of all six MYB factors
resulted in the accumulation of ATPS1 and ATPS3 transcripts compared with wild-type plants, while the mRNA
levels for ATPS2 and ATPS4 were not affected (Figure 4a,b).
Corresponding to the results of the trans-activation assay,
ATPS1 transcript was significantly more induced in
MYB28_ox, MYB76_ox, and MYB29_ox plants than in those
overexpressing the indolic GS-associated factors MYB51,
MYB122, and MYB34. On the contrary, ATPS3 mRNA was
increased 8–12-fold by overexpression of the latter group
but only 5–7-fold in the plants overexpressing aliphatic
GS-associated factors. Analysis of the MYB factor mutants
again did not reveal any large effects on ATPS transcript
levels, except induction of ATPS4, which was apparent in all
six mutants but significant only in myb122 and myb34
(Figure 4c,d). These results thus reveal that the genes
involved in the provision of PAPS are part of the GS
biosynthesis network controlled by the MYB factors.
Regulation of primary sulfate assimilation by MYB factors
Because previous microarray analyses predicted that genes
involved in primary sulfate reduction may also be regulated
by the MYB factors (Sønderby et al., 2007; Malitsky et al.,
2008) we extended the analysis to the APR gene family,
encoding the key enzyme of the pathway (Vauclare et al.,
No TF
MYB28 MYB76 MYB29 MYB51 MYB122 MYB34
APR1
APR2
APR3
Figure 5. Trans-activation assay of APR promoters with MYB transcription
factors.
The promoters of all three adenosine 5¢-phosphosulfate reductase (APR)
isoforms were fused to the uidA (GUS) reporter gene. Cultured A. thaliana
Col-0 cells were inoculated with the supervirulent Agrobacterium tumefaciens
strain LBA4404.pBBR1MCS.virGN54D containing either only the reporter
construct or the reporter construct and, in addition, Pro35S:MYB effector
constructs. The GUS staining indicates trans-activation of a promoter by an
effector.
2002; Loudet et al., 2007). The trans-activation assays
revealed that all six MYB factors activated expression of
GUS directed from promoters of all three APR genes
(Figure 5). APR1 was strongly activated by all MYB factors,
while APR2 seemed to be activated by MYB122 and MYB76
more strongly than by the others. The qPCR analysis of
MYB-overexpressing plants confirmed that APR1 and APR3
transcripts accumulated to a greater extent in all six transgenic lines analyzed (Figure 6a,b). Also in agreement with
the results of the trans-activation assay, APR2 was elevated
in MYB122_ox, MYB28_ox, and MYB76_ox plants. In the
sulfate assimilation pathway, the sulfite produced by APR is
further reduced to sulfide by sulfite reductase (Figure 1). The
mRNA for the single-copy sulfite reductase (SiR) gene was
significantly increased in plants overexpressing the indolic
GS regulators MYB51, MYB122, and MYB34, as well as the
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
Glucosinolate biosynthesis and sulfate assimilation 5
(a)
(b)
(c)
(d)
Figure 6. Transcript levels of adenosine 5¢-phosphosulfate reductase (APR) isoforms and sulfite reductase (SiR).
Transcript levels of APR isoforms and SiR in plants (a) overexpressing MYB factors involved in the regulation of aliphatic glucosinolates (GSs), (b) expressing MYB
factors regulating indolic GSs, (c) disrupted in genes of MYB factors involved in the control of aliphatic GSs, and (d) disrupted in genes of MYB factors affecting
indolic GSs.
RNA was isolated from mature leaves and subjected to quantitative RT-PCR with primers specific to the three APR isoforms and SiR. The mRNA levels were
compared to actin. The levels in wild-type plants are set to 1. Results are presented as means SD from three independent RNA preparations. Values marked with
asterisks are significantly (Student’s t-test; P £ 0.05) different from wild-type plants.
aliphatic GS regulator MYB28 (Figure 6a,b). Steady-state
levels of APR and SiR mRNA were not different in lines with
disrupted genes encoding the MYB factors regulating aliphatic GSs, myb28_RNAi, myb76, and myb29, and wild-type
plants. On the other hand, disruption of the expression of the
three MYB factors of the indolic GS clade resulted in a strong
elevation of APR2 mRNA, while transcripts of APR1 and
APR3 were increased in myb51 and myb122, respectively
(Figure 6c,d).
Adenosine 5¢-phosphosulfate reductase (APR) and other
components of the sulfate assimilation pathway are also
regulated at the post-transcriptional level (Koprivova et al.,
2008; Kawashima et al., 2009). To test whether the observed
changes in transcript levels are really reflected at the activity
level, we measured the activities of the enzymes ATPS and
APR. Both enzyme activities were significantly elevated in
MYB51_ox and myb51 plants compared with the wild type
(Figure 7a,b). In addition, APR activity was reduced in plants
with reduced expression of MYB28 (Figure 7b). The foliar
concentration of glutathione, the major product of sulfate
assimilation, was reduced in MYB28_ox plants and
increased in myb51 compared with wild-type plants
(Figure 7d). Figure 7(e) shows the changes in GS levels
caused by the expression of, or mutation in, the two MYB
factors. In addition, we have determined whether overexpression and disruption of MYB51 affects the flux through
the sulfate assimilation pathway, to assess the biological
significance of increased transcript levels and enzyme
activities in these plants. Indeed, in both MYB51_ox and
myb51 plants the incorporation of 35S from [35S]sulfate to
thiols and proteins is significantly increased (Figure 8).
Thus, modulation of expression of GS-controlling MYB
factors does indeed affect primary sulfur metabolism.
DISCUSSION
Although the significance of the sulfate group for GS function has been recognized (Ratzka et al., 2002), apart from
simple plant nutrition studies (Falk et al., 2007; Schonhof
et al., 2007) little is known about the interconnection of GS
synthesis and sulfate assimilation. Glucosinolate synthesis
was shown to be coordinately repressed by sulfur starvation, dependent on the SLIM1 factor, which on the other
hand activates sulfate uptake and assimilation in these
conditions (Hirai et al., 2005; Maruyama-Nakashita et al.,
2006). Microarray analyses indicated that at least some
genes involved in sulfate assimilation and PAPS synthesis
might be regulated by MYB factors controlling core GS
synthesis (Sønderby et al., 2007; Malitsky et al., 2008);
however, no direct evidence has been presented so far.
Very recently, a reduced capacity to synthesize PAPS was
shown to significantly affect the accumulation of GS and its
biosynthetic pathway (Mugford et al., 2009). Simultaneous
disruption of two of the four APK isoforms in A. thaliana,
APK1 and APK2, resulted in an approximately 90% decrease
in GS levels in the leaves and seeds. Both aliphatic and
indolic GSs were affected (Mugford et al., 2009). In the
leaves of apk1 apk2 plants, the desulfo-GS precursors
accumulated to very high levels and transcripts of most
genes involved in GS synthesis were induced compared
with wild-type plants. Based on these data it was hypothesized that the different APK isoforms provide PAPS for
different sets of sulfotransferases in synthesis of different
sulfated metabolites. This would imply that the different
genes would be under different regulatory circuits, with
APK1 and APK2 most probably co-expressed and co-regulated with the GS synthesis genes. Indeed, two independent
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
6 Ruslan Yatusevich et al.
(a)
(b)
Figure 8. Flux through sulfate assimilation.
Two-week-old seedlings of Col-0, MYB51_ox, and myb51 were incubated for
4 h with 0.2 mM [35S]sulfate and incorporation of 35S into thiols (glutathione
and cysteine) and proteins was quantified. Data are presented as means SD
from three biological replicates. Values marked with different indices are
significantly different (P < 0.05).
(c)
(d)
(e)
Figure 7. Regulation of ATP sulfurylase (ATPS) and adenosine 5¢-phosphosulfate reductase (APR) enzyme activities, and cysteine and glutathione
accumulation in Arabidopsis thaliana.
Regulation of (a) ATPS and (b) APR enzyme activities, and (c) cysteine and (d)
glutathione accumulation in A. thaliana plants overexpressing MYB28 and
MYB51 or in plants with reduced expression of these effectors. Results are
presented as means SD from three independent biological replicates.
Values marked with asterisks are significantly (Student’s t-test; P £ 0.05)
different from wild-type plants.
(e) For comparison, the accumulation of glucosinolates in these plants is
shown as reported in Gigolashvili et al. (2007a,b).
web-based tools, ‘Expression angler’ from the Bio-Array
Resource for Arabidopsis Functional Genomics (BAR)
(Toufighi et al., 2005) and the ‘Drawing gene networks and
searching co-expressed genes’ tool at the ATTED-II database
(http://atted.jp/top_tool.shtml) (Obayashi et al., 2009) indicated that expression of APK1 and APK2 is co-regulated with
the GS biosynthetic genes, while expression of APK3 and
APK4 is not (data not shown). However, these were only
in silico data, and the hypothesis that APK1 and APK2 are
regulated by transcription factors controlling GS synthesis
had to be tested by a direct experimental approach. Therefore, we turned to the trans-activation assay (Berger et al.,
2007), which was employed previously to identify the targets
of R2R3-MYB factors (Gigolashvili et al., 2007a,b, 2008). The
analysis indicated that APK1 and APK2 are regulated by all
six MYB factors, while the trans-activation of APK3 was
much weaker and APK4 did not seem to be activated by the
MYB effectors (Figure 2). These observations were confirmed by measuring the levels of APK transcripts in leaves
of plants overexpressing the MYB factors (Figure 3). Also the
microarray analysis of MYB factors overexpressing plants
revealed increased mRNA levels of APK1 and APK2 but not
the other APK isoforms (Malitsky et al., 2008). This is
especially important, since in these transgenic plants the
MYB factors were not controlled by the constitutive 35S
promoter as in this study but by a late 650 promoter
(Malitsky et al., 2008). Thus, pleiotropic effects due to high
transcript and protein accumulation of the trans factors
expressed from the 35S promoter can probably be excluded.
We therefore conclude that APK1 and APK2 are part of the
GS synthesis network controlled by the MYB factors
(Figure 9). APK3 and APK4 are capable of providing at least
some PAPS for GS synthesis, as apk1 apk2 plants still
contained about 10% of normal GS levels (Mugford et al.,
2009), but seem to be only loosely, if at all, associated with
this network.
Since two APK isoforms were indeed co-regulated with
GS biosynthetic genes, we subjected the other gene family
involved in PAPS synthesis, ATPS, to the same analysis.
ATPS catalyses the entry step of sulfate into both primary
and secondary metabolism. It is regulated in a demanddriven manner; when the demand for reduced sulfur is low,
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
Glucosinolate biosynthesis and sulfate assimilation 7
Figure 9. Glucosinolate biosynthetic network.
The aliphatic and indolic glucosinolate biosynthesis from methionine and chorismate is indicated and the traditional view is supplemented
by the synthesis of 3¢-phosphoadenosine
5¢-phosphosulfate (PAPS). Confirmed regulation
by the MYB factors is indicated by arrows, the
different extent of regulation of ATPS1 and
ATPS3 is indicated by full and interrupted lines.
Grey lines: known regulatory functions.
for example due to nitrogen deficiency or in the presence of
reduced sulfur compounds, the enzyme activity is decreased
(Reuveny et al., 1980; Lappartient and Touraine, 1996;
Lappartient et al., 1999). When the demand for reduced
sulfur is high, such as during sulfur deficiency or abiotic
stress (Reuveny et al., 1980; Farago and Brunold, 1990; Heiss
et al., 1999; Harada et al., 2000), the activity is increased. The
links of ATPS with abiotic and biotic stress (Heiss et al., 1999;
Rausch and Wachter, 2005) and the importance of PAPS for
GS synthesis (Mugford et al., 2009) indicated that at least
some ATPS isoforms might also be co-regulated with genes
controlling GS biosynthesis. This has indeed been the case
for ATPS1 and ATPS3 in the in silico co-transcriptional
analyses, similar to APK1 and APK2 (data not shown).
Similar to APK, both direct approaches – the trans-activation
assays and analysis of MYB factor-overexpressing plants –
revealed that two ATPS isoforms, ATPS1 and ATPS3, are
strongly regulated by the MYB effectors while the other two
are not. However, while the trans-activation assays suggested that ATPS1 is much more strongly activated by the
aliphatic GS-controlling factors than by the indolic GS ones,
with the opposite being true for ATPS3, the differences in the
transgenic plants were less marked (Figures 2 and 4).
Possibly, other unknown factors modulate the binding of
the MYB factors to ATPS promoters in the plant so that they
are not as discriminating as in the trans-activation assay.
Again, the data correspond well with the microarray analysis
by Malitsky et al. (2008).
Mugford et al. (2009) showed that limitation of PAPS
synthesis and GS content result in an increased synthesis of
thiols through the reductive part of the sulfate assimilation
pathway. In addition, genes involved in primary sulfate
reduction were found by microarray analyses to be induced
in plants overexpressing several of the studied MYB factors
(Sønderby et al., 2007; Malitsky et al., 2008). Since APR has a
very high control over the pathway (Vauclare et al., 2002;
Loudet et al., 2007), we tested the possibility that the APR
genes are also under the control of the MYB factors
(Figures 5, 6). Indeed, the trans-activation assays clearly
indicate the potential of the MYB effectors to regulate APR.
The results of the trans-activation assays again agreed well
with qPCR data for the MYB-overexpressing plants, showing
that APR1 and APR3 are under the control of all the MYB
factors while APR2 is regulated only by some. Sulfite
reductase was also induced in MYB-overexpressing plants;
the indolic GS controlling factors had a higher effect than the
aliphatic ones (Figure 6). Thus, the expression of genes
involved in sulfate assimilation is affected by the GSs
controlling MYB factors.
To complement knowledge about the regulation of sulfate
assimilation by the GS-associated MYB factors we also
compared the expression of these genes in plants in which
the expression of single MYB factors was disrupted by
T-DNA insertions or RNAi. Such disruption had very little
effect on mRNA levels of APK, ATPS, and SiR genes,
especially on the isoforms shown to be highly activated by
the overexpressed factors. There are several explanations
for this phenomenon. Firstly, the genes are regulated by
multiple MYB factors (Figures 2–4), therefore disruption of a
single MYB gene does not have a large effect on the
accumulation of mRNA of the target gene. This, however,
does not seem to be the case since, for example, the genes
involved in synthesis of aliphatic GSs are activated by all
three MYB factors – MYB28, MYB76, and MYB29 – but their
transcripts are still significantly reduced in MYB28_RNAi
plants (Gigolashvili et al., 2007b, 2008). Secondly, since
ATPS and APK have other functions apart from GS biosynthesis, their expression is likely to be controlled by multiple
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
8 Ruslan Yatusevich et al.
trans factors. The GS-associated MYB effectors might thus
be important to rapidly induce the transcription of these
genes when demand for GSs increases, but not to keep their
basal mRNA levels. Interestingly, APR was much more
strongly affected by the MYB disruptions. Whereas the APR
transcript levels were not affected by disruption of the
aliphatic GS MYBs, mutants in the indolic group showed
highly increased levels of APR2 and in some cases also of
APR1 and APR3.
How can disruption and overexpression of the same
transcription factor result in the same response of the target
gene? The key enzyme in the control of sulfate assimilation
is APR (Vauclare et al., 2002), and the APR2 isoform in
Arabidopsis contributes most to the total APR activity
(Loudet et al., 2007). Overexpression of the MYB factors
leads to an increased demand for reduced sulfur to incorporate in the thioglucoside bond and for the Met-derived
side chains of aliphatic GS, which consequently induces
APR. In the MYB_ox plants, increased GS production of
approximately 10 nmol (mg dry weight))1 compared with
the wild type (Figure 7) requires 17 nmol (mg dry weight))1
of reduced S (10 nmol for the thioglycoside bond and
7 nmol for the approximately 70% Met-derived GSs). This
corresponds to a S demand of approximately 1.7 lmol S
(g fresh weight))1 that is about sixfold higher than the
amount of sulfur in the GSH pool. To sustain such an
increased rate of sulfate reduction the capacity of the
assimilatory pathway has to be increased, which seems to
be achieved by co-regulation of the key genes, such as APR
and SiR, with GS synthesis via the MYB factors. Disruption
of the MYB factors on the other hand results in a reduction of
GS accumulation (Gigolashvili et al., 2007a,b, 2008; Hirai
et al., 2007; Sønderby et al., 2007). The indolic GSs are very
important for the protection of plants against insects, but
they also have a broad antifungal and antibacterial activity in
the pen2 pathway (Kim and Jander, 2007; Schlaeppi et al.,
2008; Bednarek et al., 2009; Clay et al., 2009). Therefore,
when synthesis of the indolic GSs is compromised, e.g. in
myb51 plants, reduced sulfur compounds may substitute for
GSs in defense against pests (Rausch and Wachter, 2005).
Indeed, reduced synthesis of GSs in apk1 apk2 plants
resulted in increased thiol synthesis (Mugford et al., 2009).
Therefore, the increase in APR2 mRNA may reflect the
increased requirement for sulfate reduction to synthesize
the alternative defense compounds, which are especially
needed in the absence of indolic GSs. Indeed, GSH accumulates in the myb51 plants, similar to apk1 apk2 plants. The
regulatory circuit to increase levels of APR transcript in these
conditions, however, can be completely different from the
two groups of R2R3-MYB factors and thus unaffected by the
disruption of MYB51, MYB34, or MY122 genes. APR is often
regulated differently from other genes in the pathway, e.g. in
the sulfur deficiency response it is not part of the SLIM1
regulatory network (Maruyama-Nakashita et al., 2006).
Indeed, APR is upregulated by many hormones involved in
stress signaling, including jasmonate, salicylate, ethylene,
and nitric oxide (Koprivova et al., 2008). Signaling by these
compounds may be triggered by the low levels of indolic GS
in myb51 plants, in a similar way that the synthesis of GSs
was induced in apk1 apk2 plants due to low levels of GSs.
Importantly, the changes in transcript levels of the sulfate
assimilation genes were at least also partly translated to
changes in enzyme activities and thiol levels (Figure 7). Both
overexpression and disruption of MYB51 had the same
effect on ATPS and APR activity. For both enzymes, mRNA of
at least one isoform was increased in the corresponding
plant material. Correspondingly, the increased activities
indeed resulted in higher rate of sulfate reduction in both
genotypes. However, in MYB51_ox plants the increased flux
through sulfate assimilation did not result in higher GSH
levels. The extra cysteine and GSH synthesized (Figure 8)
were most probably immediately used as a sulfur source for
the increased GS synthesis in these plants and not for
increasing the size of the GSH pool. On the other hand, in
myb51 plants the increase in flux also resulted in increased
glutathione levels, as the extra reduced sulfur was not used
for GSs. Increase in GSH correlating with decrease in
GS levels was observed previously in apk1 apk2 plants
(Mugford et al., 2009). The effects of mis-expression of
MYB28 on APR and ATPS activity are not so easy to explain.
Although overexpression of this trans factor increased
transcript levels for ATPS1, ATPS3, and the three APR
genes, the enzyme activities were not affected. Thus, it
appears that a post-transcriptional and/or post-translational
regulation prevented the increase in the enzyme activities.
Similar phenomena were observed in the regulation of APR
by salt, where in several signaling mutants APR transcript
levels were increased but the activity was not (Koprivova
et al., 2008). It is also possible that the enzymes were directly
inhibited by intermediates in aliphatic GS synthesis, which
during methionine elongation reactions increases the concentration of reduced sulfur compounds in the plastids.
Indeed, APR is known to be redox regulated in a posttranslational manner, to which these intermediates may
contribute (Bick et al., 2001). Because APR activity was not
induced, the extra S used for increased GS synthesis
depleted the GSH pool and the GSH content decreased in
MYB28_ox plants (Figure 7). Disruption of MYB28 had very
little effect on primary sulfate assimilation despite a dramatic reduction of GS content in the MYB28_RNAi plants. It
is feasible to argue that the aliphatic GSs are less important
for plant defense than indolic GSs, and therefore the low rate
of their synthesis does not induce the synthesis of alternative compounds such as GSH. Thus, there is no need for an
increase in flux through sulfate assimilation and APR and
ATPS transcripts are not affected. The reduction in APR
activity in MYB28_RNAi compared with Col-0 might actually
be caused by a very high activity measured in these
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
Glucosinolate biosynthesis and sulfate assimilation 9
particular control plants compared with other Col-0 material.
Regulation of APR takes place on many levels and is known
to respond strongly to small alterations in growth conditions
(Kopriva, 2006).
Our analysis of the control of sulfate assimilation by the
GS-biosynthesis-regulating MYB factors thus resulted in two
significant findings. We could successfully place four new
enzymes, namely APK, ATPS, APR, and SiR, into the GS
biosynthesis network (Figure 9). At least the synthesis of
activated sulfate, the first step of which is simultaneously a
part of primary sulfate assimilation, has to be considered as
an integral part of the GS network. In addition, we have
significantly improved our knowledge of molecular mechanisms of regulation of primary sulfate reduction. Six novel
transcription factors have been revealed to regulate this
pathway. While gel shift assays or chromatin immunoprecipitation are necessary to unequivocally prove direct binding of the effectors to the corresponding promoters, the
effects of the MYB factors on ATPS and APR enzyme
activities as well as the stimulation of flux through the
pathway corroborate the results of the trans-activation
assays and expression analysis. Despite a sound understanding of the regulation of plant sulfate assimilation at the
physiological level and responses of enzyme activities and
gene expression to various stimuli, only one transcription
factor, SLIM1, has been identified to directly control the
pathway (Maruyama-Nakashita et al., 2006). The identification of MYB factors controlling GS biosynthesis also as
regulators of ATPS and APR thus shows the complexity of
regulatory interactions between primary and secondary
sulfur metabolism.
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
The construction and selection of plants overexpressing the MYB
factor was described previously (Gigolashvili et al., 2007a,b, 2008).
For the expression analysis the plants were grown in soil in shortday conditions (8-h light, 16-h dark) at 22–25C and 40% humidity.
Overexpression mutants used for the qPCR and biochemical analysis had only moderately increased levels of corresponding
transgenes. Gain-of-function mutants of indolic GS regulators used
for the analysis were HIG1-1D, Pro35S:MYB122-11, and Pro35S:ATR1-17 lines (called here MYB51_ox, MYB122_ox, and MYB34_ox), whereas gain-of-function mutants of aliphatic GS
regulators were Pro35S:MYB28-15, Pro35S:MYB76-23, and
Pro35S:MYB29-6 (MYB28_ox, MYB76_ox, and MYB29_ox). Loss-offunction mutants used in this work were MYB28-RNAi-10 (accumulating approximately 20% of WT transcript levels), myb76
(hag2), myb29 (hag3), and myb51 (hig1-1) described previously
(Gigolashvili et al., 2007a,b, 2008), and myb122 and myb34 knockout plants (SALK collection) isolated recently (T. Gigolashvili,
unpublished data).
Arabidopsis Col-0 suspension culture for the trans-activation
assays was grown in 50 ml of A. thaliana (AT) medium [4.3 g L)1
MS basal salt medium (Duchefa, http://www.duchefa.com/), 1 mg
L)1 2,4-dichlorophenoxyacetic acid (2,4-D), 4 ml of a vitamin B5
mixture (Sigma, http://www.sigmaaldrich.com), and 30 g L)1
sucrose pH 5.8]. Suspension cell culture was diluted weekly to 1:4
or 1:5 with fresh AT medium and gently agitated at 150 rpm in the
dark at 22C.
Bioinformatics analysis
In order to find which genes are co-expressed with the different
ATPS and APK isoforms the web-based data-mining tool ‘Expression angler’ from BAR was utilized (Toufighi et al., 2005). Existing
microarray data were compared with a chosen query gene, and
Pearson correlation coefficients were calculated to identify genes
with similar expression and response patterns. Parameters were set
to return the 25 best hits from the AtGenExpress global stress
expression data set (Kilian et al., 2007) for APK and from the complete NASC data set for ATPS.
The co-expression analysis based on the publicly available 1388
microarray data of AtGenExpress using Pearson’s correlation
coefficients between all combinations of 22 263 Arabidopsis genes
was performed using the ‘Drawing gene networks and searching
co-expressed genes’ tool at the ATTED-II database (http://atted.jp/
top_tool.shtml) (Obayashi et al., 2009). The gene set used to build
the network was based on ‘Glucosinolate metabolism’ in the Kazusa
Plant Pathway Viewer (http://kpv.kazusa.or.jp/kappa-view/) (Tokimatsu et al., 2005) and the ATPS and APK gene families.
Cloning of promoters of ATPS, APK, and APR genes and
trans-activation assays
To generate reporter constructs, the promoter regions of the ATPS
[5 kbp (ATPS1 and ATPS4) and 2.5 kbp (ATPS2 and ATPS3) upstream of the ATG], APK [3 kbp (APK1 and APK3) and 3.5 kbp (APK2
and APK4) upstream of the ATG], and APR [3 kbp upstream of the
ATG] genes were amplified from genomic DNA of A. thaliana with
specific primers (Table S1 in Supporting Information) and cloned
into the Gateway entry vector (Invitrogen, http://www.invitrogen.com). The promoter sequences were then subcloned into the
binary plant transformation vector pGWB3i, resulting in translational fusions with the gus reporter gene. As effectors, the
constructs Pro35S:MYB28, Pro35S:MYB76, Pro35S:MYB29,
Pro35S:MYB51, Pro35S:MYB122, and Pro35S:MYB34 were used
as described previously (Gigolashvili et al., 2007a,b, 2008). The
reporter and effector constructs were used to transform the supervirulent Agrobacterium strain LBA4404.pBBR1MCS.virGN54D
(kindly provided by Dr J. Memelink, University of Leiden, Netherlands). For transient expression assays in the cell culture, agrobacteria containing the effector constructs, the anti-silencing 19-K
protein, or one of the reporter constructs were taken from fresh
yeast extract broth (YEB) plates, grown overnight, and resuspended
in 1 ml AT medium. The agrobacteria were mixed in a 1:1:1 ratio,
and 75 ll of this suspension was added to 3 ml cultured A. thaliana
root cells, which were then grown for 3–5 or 7 days in the dark and
subsequently used for GUS activity measurements or staining
(Berger et al., 2007).
RNA extraction and expression analysis
The expression of ATPS, APK, APR, and SiR genes was analysed by
real-time quantitative RT-PCR using the fluorescent intercalating
dye SYBR Green in a GeneAmp 5700 sequence detection system
(Applied Biosystems, http://www3.appliedbiosystems.com/). The
Arabidopsis ACTIN2 gene was used as a standard. First, total RNA
was isolated from 5-week-old rosette leaves using TRIsure (Bioline,
http://www.bioline.com/) and reverse-transcribed into cDNA, using
the FirstStrand cDNA Synthesis SSII kit (Bioline) according to the
manufacturer’s instructions. Subsequently, the cDNA was used as a
template in real-time PCR experiments with gene-specific primers
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
10 Ruslan Yatusevich et al.
(for primer sequences see Table S2). Real-time PCR was performed
using the SYBR Green master mix system (Applied Biosystems)
according to the manufacturer’s instructions. The Ct, defined as the
PCR cycle at which a statistically significant increase of reporter
fluorescence is detected, was used as a measure of the transcript
level of the target gene. Relative quantification of the expression
levels was performed using the comparative Ct method (manufacturer’s instructions, bulletin 2, Applied Biosystems). Three independent RNA preparations from independently grown plants were
analyzed with two technical replicates for the qPCR.
Enzyme assays
The activity of APS reductase was determined as the production of
[35S]sulfite, assayed as acid volatile radioactivity formed in the
presence of [35S]APS and dithioerythritol as reductant (Koprivova
et al., 2008). The ATPS was measured as the APS and pyrophosphate-dependent formation of ATP (Cumming et al., 2007). The
protein concentrations were determined according to Bradford with
bovine serum albumin as a standard.
HPLC analysis of low molecular weight thiols
The analysis of cysteine and GSH was performed as described
(Koprivova et al., 2008). Twenty to thirty milligrams of leaf material
was ground in liquid nitrogen and extracted in a 10-fold volume of
0.1 M HCl. After centrifugation at 15 000 g, 25 ll of supernatant was
neutralized by 25 ll of 0.1 M NaOH and 1 ll of 100 mM dithiothreitol
was added to reduce disulfides. After 15 min at 37C, 35 ll water,
10 ll 1 M 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) pH 8.0,
and 5 ll of 100 mM monobromobimane (Thiolyte MB; Calbiochem, http://www.merck-chemicals.co.uk) were added and derivatization of thiols was allowed to proceed for 15 min at 37C in the
dark. The reaction was stopped and the conjugates were stabilized
by the addition of 100 ll of 9% acetic acid. Bimane conjugates were
separated by HPLC (Spherisorb ODS2; 250 · 4.6 mm, 5 lm;
Waters, http://www.waters.com/) using 10% (v/v) methanol, 0.25%
(v/v) acetic acid (pH 9.3) as solvent A and 90% (v/v) methanol, 0.25%
(v/v) acetic acid (pH 9.3) as solvent B. The elution protocol employed
a linear gradient from 96 to 82% A in B within 20 min, and the flow
rate was kept constant at 1 ml min)1. Bimane derivates were
detected fluorimetrically (474 detector, Waters) with excitation at
390 nm and emission at 480 nm.
Determination of flux through sulfate assimilation
The flux through sulfate assimilation was measured as incorporation of 35S from [35S] sulfate to thiols and proteins essentially as
described in Kopriva et al. (1999) and Vauclare et al. (2002). Col-0,
MYB51_ox, and myb51 plants were grown for 14 days on vertical
MS-phytogel plates. The plants were transferred into 24-well plates
containing 2 ml of MS nutrient solution adjusted to a sulfate concentration of 0.2 mM and supplemented with 5.6 lCi [35S]sulfate
(Hartmann Analytic, http://www.hartmann-analytic.com/) to specific
activity of 1860 kBq (nmol sulfate))1 and incubated in the light for
4 h. After the incubation the seedlings were washed three times
with 2 ml of cold non-radioactive nutrient solution, carefully blotted
with paper tissue, weighed, transferred into 1.5 ml tubes, and frozen
in liquid nitrogen. The plant tissue was extracted 1:10 (w/v) in 0.1 M
HCl. Ten microliters of the extract was added to 1 ml of Optiphase
HiSafe3 scintillation cocktail (Perkin Elmer, http://www.perkinelmer.
com/) and the radioactivity was measured in a scintillation counter
(Beckmann, http://www.beckmancoulter.com/) to determine sulfate
uptake. To measure 35S incorporation into proteins, these were
precipitated from 100 ll of the extract with 25 ll 100% trichloroacetic acid (TCA) as described in Kopriva et al. (1999). After 15 min
on ice the precipitate was collected by centrifugation 15 000 g,
washed once in 100 ll 1% TCA and once in 200 ll ethanol (EtOH)
and dissolved in 100 ll 0.1 M NaOH. The radioactivity was
determined after the addition of 1 ml scintillation cocktail in the
scintillation counter.
To determine the radioactivity in thiols 100 ll of the extract was
mixed with 100 ll 0.1 M NaOH and 2 ll 0.1 M DTT and incubated in
the dark at 37C for 15 min. Afterwards 23 ll of 1 M TRIS pH 8.0 and
10 ll 100 mM monobromobimane were added, mixed, and incubated in the dark at 37C for 15 min. Then 22.5 ll of 50% acetic acid
was added, mixed and centrifuged for 15 min at 15 000 g. Two
hundred microliters of the solution was transferred into HPLC
vials. Standard thiol analysis was performed as described above
with an injection volume of 100 ll. The HPLC was connected to a
fraction collector and fractions of 0.8 ml were collected in 6 ml
scintillation vials. The radioactivity in these fractions was determined in a scintillation counter after the addition of 2 ml scintillation
solution.
ACKNOWLEDGEMENTS
This research was supported by the Deutsche Forschungsgemeinschaft and the UK Biotechnology and Biological Sciences
Research Council (grant BB/D009596/1). We would like to thank
Dr Bok-Rye Lee for critical reading of the manuscript.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Table S1. Sequences of primers used for cloning of APK, ATPS, and
APR promoters.
Table S2. Primers for quantitative real-time RT-PCR analysis.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
REFERENCES
Bednarek, P., Pilewska-Bednarek, M., Svato, A. et al. (2009) A glucosinolate
metabolism pathway in living plant cells mediates broad-spectrum antifungal defense. Science, 323, 101–106.
Berger, B., Stracke, R., Yatusevich, R., Weisshaar, B., Flügge, U.I. and
Gigolashvili, T. (2007) A simplified method for the analysis of transcription
factor-promoter interactions that allows high-throughput data generation.
Plant J. 50, 911–916.
Bick, J.A., Setterdahl, A.T., Knaff, D.B., Chen, Y., Pitcher, L.H., Zilinskas, B.A.
and Leustek, T. (2001) Regulation of the plant-type 5¢-adenylyl sulfate
reductase by oxidative stress. Biochemistry, 40, 9040–9048.
Celenza, J.L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A.R., Normanly,
J. and Bender, J. (2005) The Arabidopsis ATR1 Myb transcription factor
controls indolic glucosinolate homeostasis. Plant Physiol. 137, 253–262.
Clay, N.K., Adio, A.M., Denoux, C., Jander, G. and Ausubel, F.M. (2009)
Glucosinolate metabolites required for an Arabidopsis innate immune
response. Science, 323, 95–101.
Cumming, M., Leung, S., McCallum, J. and McManus, M.T. (2007) Complex
formation between recombinant ATP sulfurylase and APS reductase of
Allium cepa (L.). FEBS Lett. 581, 4139–4147.
Fahey, J.W., Zalcmann, A.T. and Talalay, P. (2001) The chemical diversity and
distribution of glucosinolates and isothiocyanates among plants. Phytochemistry, 56, 5–51.
Falk, K. L., Tokuhisa, J. G. and Gershenzon, J. (2007) The effect of sulfur
nutrition on plant glucosinolate content: physiology and molecular
mechanisms. Plant Biol. 5, 573–581.
Farago, S. and Brunold, C. (1990) Regulation of assimilatory sulfate reduction
by herbicide safeners in Zea mays L. Plant Physiol. 94, 1808–1812.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11
Glucosinolate biosynthesis and sulfate assimilation 11
Fenwick, G.R., Heaney, R.K. and Mullin, W.J. (1983) Glucosinolates and their
breakdown products in food and food plants. Crit. Rev. Food Sci. Nutr. 18,
123–201.
Gigolashvili, T., Berger, B., Mock, H.P., Müller, C., Weisshaar, B. and Flügge,
U.I. (2007a) The transcription factor HIG1/MYB51 regulates indolic glucosinolate biosynthesis in Arabidopsis thaliana. Plant J. 50, 886–901.
Gigolashvili, T., Yatusevich, R., Berger, B., Müller, C. and Flügge, U. I. (2007b)
The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. Plant J.
51, 247–261.
Gigolashvili, T., Engqvist, M., Yatusevich, R., Müller, C. and Flügge, U.I. (2008)
HAG2/MYB76 and HAG3/MYB29 exert a specific and coordinated control
on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis
thaliana. New Phytol. 177, 627–642.
Grubb, C.D. and Abel, S. (2006) Glucosinolate metabolism and its control.
Trends Plant Sci. 11, 89–100.
Halkier, B.A. and Gershenzon, J. (2006) Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 57, 303–333.
Harada, E., Kusano, T. and Sano, H. (2000) Differential expression of genes
encoding enzymes involved in sulfur assimilation pathways in response to
wounding and jasmonate in Arabidopsis thaliana. J. Plant Physiol. 156,
272–276.
Heiss, S., Schäfer, H.J., Haag-Kerwer, A. and Rausch, T. (1999) 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. 39, 847–
857.
Hirai, M.Y., Klein, M., Fujikawa, Y. et al. (2005) Elucidation of gene-to-gene
and metabolite-to-gene networks in Arabidopsis by integration of metabolomics and transcriptomics. J. Biol. Chem. 280, 25590–25595.
Hirai, M.Y., Sugiyama, K., Sawada, Y. et al. (2007) Omics-based identification
of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate
biosynthesis. Proc. Natl Acad. Sci. USA, 104, 6478–6483.
Kawashima, C.G., Yoshimoto, N., Maruyama-Nakashita, A., Tsuchiya, Y.N.,
Saito, K., Takahashi, H. and Dalmay, T. (2009) Sulfur starvation induces the
expression of microRNA-395 and one of its target genes but in different cell
types. Plant J. 57, 313–321.
Kilian, J., Whitehead, D., Horak, J., Wanke, D., Weinl, S., Batistic, O.,
D’Angelo, C., Bornberg-Bauer, E., Kudla, J. and Harter, K. (2007) The
AtGenExpress global stress expression data set: protocols, evaluation and
model data analysis of UV-B light, drought and cold stress responses. Plant
J. 50, 347–363.
Kim, J.H. and Jander, G. (2007) Myzus persicae (green peach aphid) feeding
on Arabidopsis induces the formation of a deterrent indole glucosinolate.
Plant J. 49, 1008–1019.
Kopriva, S. (2006) Regulation of sulfate assimilation in Arabidopsis and
beyond. Ann. Bot. 97, 479–495.
Kopriva, S., Muheim, R., Koprivova, A., Trachsel, N., Catalano, C., Suter, M.
and Brunold, C. (1999) Light regulation of assimilatory sulfate reduction in
Arabidopsis thaliana. Plant J. 20, 37–44.
Koprivova, A., North, K.A. and Kopriva, S. (2008) Complex signaling network
in regulation of adenosine 5¢-phosphosulfate reductase by salt stress in
Arabidopsis roots. Plant Physiol. 146, 1408–1420.
Lappartient, A.G. and Touraine, B. (1996) Demand-driven control of root ATP
sulfurylase activity and SO42) uptake in intact canola. The role of phloemtranslocated glutathione. Plant Physiol. 111, 147–157.
Lappartient, A. G., Vidmar, J. J., Leustek, T., Glass, A. D. M. and Touraine, B.
(1999) Inter-organ signaling in plants: regulation of ATP sulfurylase and
sulfate transporter genes expression in roots mediated by phloemtranslocated compound. Plant J. 18, 89–95.
Levy, M., Wang, Q., Kaspi, R., Parrella, M.P. and Abel, S. (2005) Arabidopsis
IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate
accumulation and plant defense. Plant J. 43, 79–96.
Loudet, O., Saliba-Colombani, V., Camilleri, C., Calenge, F., Gaudon, V.,
Koprivova, A., North, K.A., Kopriva, S. and Daniel-Vedele, F. (2007) Natural
variation for sulfate content in Arabidopsis thaliana is highly controlled by
APR2. Nat. Genet. 39, 896–900.
Malitsky, S., Blum, E., Less, H., Venger, I., Elbaz, M., Morin, S., Eshed, Y. and
Aharoni, A. (2008) The transcript and metabolite networks affected by the
two clades of Arabidopsis glucosinolate biosynthesis regulators. Plant
Physiol. 148, 2021–2049.
Maruyama-Nakashita, A., Nakamura, Y., Tohge, T., Saito, K. and Takahashi,
H. (2006) Arabidopsis SLIM1 is a central transcriptional regulator of plant
sulfur response and metabolism. Plant Cell, 18, 3235–3251.
Matile, P.H. (1980) The mustard oil bomb – compartmentation of the myrosinase system. Biochem. Physiol. Pflanz. 175, 722–731.
Mithen, R., Faulkner, K., Magrath, R., Rose, P., Williamson, G. and Marquez, J.
(2003) Development of isothiocyanate-enriched broccoli, and its enhanced
ability to induce phase 2 detoxification enzymes in mammalian cells.
Theor. Appl. Genet. 106, 727–734.
Mugford, S. G., Yoshimoto, N., Reichelt, M. et al. (2009) Disruption of adenosine-5¢-phosphosulfate kinase in Arabidopsis reduces levels of sulfated
secondary metabolites. Plant Cell, 21, 910–927.
Obayashi, T., Hayashi, S., Saeki, M., Ohta, H. and Kinoshita, K. (2009) ATTED-II
provides coexpressed gene networks for Arabidopsis. Nucleic Acids Res.
37, D987–D991.
Rask, L., Andréasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B. and Meijer,
J. (2000) Myrosinase: gene family evolution and herbivore defense in
Brassicaceae. Plant Mol. Biol. 42, 93–113.
Ratzka,A., Vogel, H., Kliebenstein,D.J.,Mitchell-Olds,T. and Kroymann, J.(2002)
Disarming the mustard oil bomb. Proc. Natl Acad. Sci. USA, 99, 11223–11228.
Rausch, T. and Wachter, A. (2005) Sulfur metabolism: a versatile platform for
launching defence operations. Trends Plant Sci. 10, 503–509.
Reintanz, B., Lehnen, M., Reichelt, M., Gershenzon, J., Kowalczyk, M.,
Sandberg, G., Godde, M., Uhl, R. and Palme, K. (2001) Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain
aliphatic glucosinolates. Plant Cell, 13, 351–367.
Reuveny, Z., Dougall, D.K. and Trinity, P.M. (1980) Regulatory coupling of
nitrate and sulfate assimilation pathways in cultured tobacco cells. Proc.
Natl Acad. Sci. USA, 77, 6670–6672.
Schlaeppi, K., Bodenhausen, D., Buchala, A., Mausch, F. and Reymond, P.
(2008) The glutathione-deficient mutant pad2-1 accumulates lower
amounts of glucosinolates and is more susceptible to the insect herbivore
Spodoptera littoralis. Plant J. 55, 774–786.
Schöne, F., Rudolph, B., Kirchheim, U. and Knapp, G. (1997) Counteracting the
negative effects of rapeseed and rapeseed press cake in pig diets. Br. J.
Nutr. 78, 947–962.
Schonhof, I., Blankenburg, D., Muller, S. and Krumbein, A. (2007) Sulfur and
nitrogen supply influence growth, product appearance, and glucosinolate
concentration of broccoli. J. Plant Nutr. Soil Sci. 170, 65–72.
Skirycz, A., Reichelt, M., Burow, M. et al. (2006) DOF transcription factor
AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate
biosynthesis in Arabidopsis. Plant J. 47, 10–24.
Sønderby, I.E., Hansen, B.G., Bjarnholt, N., Ticconi, C., Halkier, B.A. and
Kliebenstein, D.J. (2007) A systems biology approach identifies a R2R3
MYB gene subfamily with distinct and overlapping functions in regulation
of aliphatic glucosinolates. PLoS ONE, 2, e1322.
Suter, M., Lavanchy, P., von Arb, C. and Brunold, C. (1986) Regulation of sulfate
assimilation by amino acids in Lemna minor L. Plant Sci. 44, 125–132.
Tokimatsu, T., Sakurai, N., Suzuki, H., Ohta, H., Nishitani, K., Koyama, T.,
Umezawa, T., Misawa, N., Saito, K. and Shibata, D. (2005) KaPPA-view: a
web-based analysis tool for integration of transcript and metabolite data on
plant metabolic pathway maps. Plant Physiol. 138, 1289–1300.
Toufighi, K., Brady, S.M., Austin, R., Ly, E. and Provart, N.J. (2005) The Botany
Array Resource: e-northerns, expression angling, and promoter analysis.
Plant J. 43, 153–163.
Underhill, E.W., Wetter, L.R. and Chisholm, M.D. (1973) Biosynthesis of glucosinolates. Biochem. Soc. Symp. 38, 303–326.
Vauclare, P., Kopriva, S., Fell, D., Suter, M., Sticher, L., von Ballmoos, O.,
Krähenbuhl, U., Op den Camp, R. and Brunold, C. (2002) Flux control of
sulfate assimilation in Arabidopsis thaliana: adenosine 5¢-phosphosulfate
reductase is more susceptible to negative control by thiols than ATP sulfurylase. Plant J. 31, 729–740.
Verhoeven, D.T., Verhagen, H., Goldbohm, R.A., van den Brandt, P.A. and van
Poppel, G. (1997) A review of mechanisms underlying anticarcinogenicity
by brassica vegetables. Chem. Biol. Interact. 103, 79–129.
Yan, X. and Chen, S. (2007) Regulation of plant glucosinolate metabolism.
Planta, 226, 1343–1352.
ª 2010 The Authors
Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 62, 1–11