Photosynthesis Research (2005) 86: 491508

DOI: 10.1007/s11120-005-7386-8

! Springer 2005

Regular paper

Expression profiling of metabolic genes in response to methyl jasmonate

reveals regulation of genes of primary and secondary sulfur-related
pathways in Arabidopsis thaliana
Ricarda Jost1,8, Lothar Altschmied1, Elke Bloem2, Jochen Bogs3, Jonathan Gershenzon4,
Urs Hahnel1, Robert Hansch5, Tanja Hartmann6, Stanislav Kopriva6, Cordula Kruse3,
Ralf R. Mendel5, Jutta Papenbrock7, Michael Reichelt4, Heinz Rennenberg6, Ewald
Schnug7, Ahlert Schmidt7, Susanne Textor4, Jim Tokuhisa4, Andreas Wachter3, Markus
Wirtz3, Thomas Rausch3 & Rudiger Hell3,*
1

Department of Molecular Cell Biology, Institut fur Pflanzengenetik und Kulturpflanzenforschung (IPK),
06466 Gatersleben, Germany; 2Institut fur Pflanzenernahrung und Bodenkunde (FAL), 38116 Braunschweig,
Germany; 3Heidelberger Institut fur Pflanzenwissenschaften (HIP), Universitat Heidelberg, 69120 Heidelberg, Germany; 4Max-Planck-Institute of Chemical Ecology, 07745 Jena, Germany; 5Institut fur Pflanzenbiologie, Universitat Braunschweig, 38023 Braunschweig, Germany; 6Institut fur Forstbotanik und
Baumphysiologie, Universitat Freiburg, 79085 Freiburg, Germany; 7Institut fur Botanik, Universitat
Hannover, 30419 Hannover, Germany; 8Present address: Research School of Biological Sciences, The Australian National University, 0200 Canberra, ACT, Australia; *Author for correspondence (e-mail: rhell@hip.
uni-heidelberg.de; fax: +49-6221-545859)

Received 27 April 2005; accepted in revised form 11 May 2005

Key words: glucosinolates, pathogen resistance, sulfur metabolism, sulfur-rich peptides, thionin

Abstract
The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467
selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster
analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time
course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than
2fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfurrelated genes were by far strongest aected. Gene expression and metabolite patterns of sulfur metabolism
were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes
encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were
rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate
metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only
an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed
rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response.
These results document for the first time how comprehensively the regulation of sulfur-related genes and
plant defense are connected. This interaction is discussed as a new approach to dierentiate between
supply- and demand-driven regulation of the sulfate assimilation pathway.
Abbreviations: AOP 2-oxoglutarate-dependent dioxygenase; AOS allene oxide synthase; APR adenosinephosphosulfate reductase; APS ATP sulfurylase; ESP epi-thiospecifier protein; FW fresh

492
weight; GSH glutathione; GST glutathione-S-transferase; MeJA methyl jasmonate; OAS O-acetylserine; SIR sulfite reductase; VSP vegetative storage protein

Introduction
In recent years the significance of sulfate supply
for the resistance of crop plants to pathogens has
been recognized as atmospheric sulfur depositions
in central Europe dropped from about 50 kg/ha
in 1980 to about 10 kg/ha in 1990 due to the
eective desulfurisation of anthropogenic emissions (Dammgen et al. 1998). Increased susceptibility to fungal infections and loss of yield were
concomitantly observed for oilseed rape, a crop
that is known for its high sulfur requirement. The
symptoms could only be relieved by application of
sulfate fertilizers or fungicides (Schnug et al. 1995;
Ceccotti et al. 1998). Indeed, rape plants grown
under controlled conditions at limiting or optimal
sulfate supply displayed dierent susceptibility to
several fungal pathogens. The eect correlated
with enhanced antimicrobial activity of extracted
fractions obtained from high sulfate grown plants
(Dubuis et al. 2005). In contrast, nitrogen fertilization above the recommended rate for a crop is
known to increase susceptibility to pathogens. For
instance rice blast, Pyricularia grisea, progressed
more eciently in field experiments with increased
nitrogen treatment (Long et al. 2000; Konishi et al.
2001). Hence, plant sulfur assimilation appears to
be important for plant pathogen defense.
Plants have developed numerous inducible
defense mechanisms against bacterial and fungal
pathogens. These are often characterized by a
hypersensitive response in incompatible interactions and are mediated by the plant hormones
jasmonate, salicylate and ethylene. Many of the
defense compounds that are formed in response to
pathogen contact contain reduced sulfur moieties
as functional groups. Examples are the sulfur-rich
peptide classes of thionins and defensins with 3 to
4 disulfide bridges resulting in cysteine contents of
at least 15% (Garcia-Olmedo et al. 2001). While
thionins and defensins are most abundant in seeds,
both are expressed in vegetative and generative
tissues in either constitutive or pathogen- and
methyl jasmonate (MeJA)-inducible isoforms
(Pieterse and van Loon 1999). An Arabidopsis
thaliana mutant with constitutive expression of the

thionin and defensin genes THI2.1 and PDF2.1

showed enhanced resistance to powdery mildew
infection (Ellis and Turner 2001).
Low molecular weight sulfur-containing
defense metabolites in Arabidopsis include glucosinolates and the phytoalexin camalexin (Glazebrook 2001; Wittstock and Gershenzon 2002).
Glucosinolates are thioglucosides that contain up
to 3 atoms of reduced and oxidized sulfur. Their
toxicity results from hydrolysis products that are
released during their degradation by myrosinase
upon wounding or pathogen attack (Tierens et al.
2001; Wittstock and Gershenzon 2002). The deficiency of major glucosinolates and their isothiocyanates in the Arabidopsis mutant gsm1-1 resulted
in enhanced susceptibility to Fusarium oxysporum
(Tierens et al. 2001). Camalexin is the principal
phytoalexin that accumulates in Arabidopsis upon
infection by fungi or bacteria (Rogers et al. 1996).
The Arabidopsis mutant pad3 is camalexin deficient due to a mutation in a biosynthetic gene and
significantly more susceptible to the fungal pathogen Alternaria brassicicola (Thomma et al. 1999).
Even depositions of elemental sulfur have been
correlated with enhanced resistance of defined
cultivars of several plant species against fungal
pathogens and are linked to transient increases in
cysteine, glutathione and sulfate pools of adjacent
tissues (Williams and Cooper 2004).
The regulation of primary sulfur metabolism
has so far mostly been investigated with respect to
supply-dependent responses. Sulfate limitation
results in the rapid transcriptional activation of a
plethora of genes. Most aected are members of
the sulfate transporter family followed by genes of
sulfate assimilation (Buchner et al. 2004; Saito
2004), but macroarray analyses covering 7000 to
9000 Arabidopsis genes revealed the induction of
downstream genes of sulfur-related and so far
considered unrelated pathways (Hirai et al. 2003;
Maruyama-Nakashita et al. 2003; Nikiforova et al.
2003). Because their internal concentrations
respond rapidly to external sulfate depletion,
sulfate, O-acetylserine (OAS), the intermediate
metabolite of cysteine synthesis, cysteine itself and
glutathione are discussed as regulatory triggers of

493
the transcriptional response. Although local concentrations are not known, cytosolic sulfate and
OAS levels are the most likely candidates in this
respect (see Saito 2000; Leustek et al. 2000;
Buchner et al. 2004; Saito 2004 for review). Metabolic regulation seems to be exerted by OAS and
sulfide via the reversible interaction of the subunits
of the cysteine synthase complex (Hell et al. 2002)
and by cysteine-feedback inhibition of serine acetyltransferase (SAT) that catalyses the first step of
cysteine synthesis (Saito 2000).
Interestingly, macroarray analyses of plants
transferred to sulfate-deprived medium revealed
that genes of jasmonate biosynthesis are concomitantly induced within 24 to 48 h (Hirai et al.
2003; Maruyama-Nakashita et al. 2003; Nikiforova et al. 2003). Although jasmonate concentrations have not been determined in these
experiments, the finding that several jasmonateregulated genes are also activated after feeding of
OAS suggests an interaction between both pathways (Hirai et al. 2003; Saito 2004). Whether
this is part of an intricate network of various
nutritional stresses and hormonal responses
remains to be confirmed but this assumption is
supported by the recent finding of transcriptional
regulation of jasmonic acid biosynthetic genes by
potassium starvation and re-suppy (Armengaud
et al. 2004).
Therefore, the signalling pathway mediated by
jasmonate might also aect the expression of genes
of primary sulfur metabolism to coordinate the
timing and localization of the corresponding
enzymes and metabolites with defense responses at
infection sites. Such specific expression is well
documented for genes involved in the formation of
some of the end products, such as the sulfur-rich
thionins and defensins (Bohlmann et al. 1998;
Thomma et al. 1998). Also the inducing eect of
intense MeJA treatment on glucosinolate content
of leaves of rape and Arabidopsis has been reported (Doughty et al. 1995; Harada et al. 2000). In
Arabidopsis seedlings this treatment induced the
expression of four out of nine genes of primary
sulfur metabolism tested (Harada et al. 2000).
Furthermore, the genes for synthesis and reduction of glutathione, a sulfur-containing tripeptide
involved in numerous biotic and abiotic stress
reactions, were up-regulated by MeJA application
(Xiang and Oliver 1998). Array analyses covering
limited numbers of Arabidopsis genes suggested

up-regulation of genes of the defense-related signalling pathways and single sulfur-related genes
after MeJA or 12-oxo-phytodienoic acid treatment
or infection with the fungal pathogen Alternaria
brassicicola. These included genes encoding
methionine synthase, thaumatin, PDF, glutathione-S-transferase (GST), c-glutamylcysteine synthetase (ECS; the first enzyme of glutathione
synthesis), and myrosinase-binding protein
(Schenk et al. 2000; Sasaki et al. 2001; SasakiSekimoto et al. 2003; Schenk et al. 2003; van Wees
et al. 2003). A more comprehensive array experiment with 13.000 ESTs was designed for analysis
of primary metabolic pathways after infection of
Arabidopsis with Pseudomonas syringae and
revealed induction of numerous housekeeping
genes such as reductive carbon and amino acid
metabolism, but no expression of sulfur-related
genes was reported (Scheideler et al. 2002). However, in none of these experiments sulfur metabolism was investigated more closely and
consequently knowledge on gene expression and
metabolic reactions of this pathway in response to
MeJA or pathogens is still rather fragmentary.
It was the aim of this study to explore the
possibility of a regulatory interaction between
defense signalling and sulfur metabolism more
throughly using methyl jasmonate as one potent
inducer of defense reactions in Arabidopsis. While
salicylate is a signalling molecule in the response
of Arabidopsis to many biotrophic pathogens,
MeJA functions in wound responses and has been
recognized as a transducer of a dierent set of
defense eector genes, e.g. against necrotrophic
pathogens like the fungus Alternaria brassicicola
(Thomma et al. 1998; Pieterse and Van Loon
1999; Glazebrook 2001; Ramonell and Somerville
2002; Glazebrook et al. 2003). Here the expression
profiles of 2467 non-redundant genes on a macroarray as well as concentrations of sulfur
metabolites, amino acids and sugars were monitored in a time course following the initial 24 h of
response after MeJA application. We found that,
to a much higher extent than other pathways, key
genes throughout the entire primary and secondary metabolism of sulfur were rapidly and coordinately up-regulated in response to low
concentrations of MeJA and that the signaling
pathway involved is probably dierent from the
one mediating the response to external sulfate
supply.

494
Materials and methods

Selection of clones for multiparallel expression

analysis and array hybridization

Plant growth and MeJA treatment

Arabidopsis thaliana, ecotype Columbia C24, was
grown axenically on half-strength MS (Murashige
and Skoog 1962) solid medium in culture vessels
(v = 900 ml, Duchefa, The Netherlands) under
short day conditions (10 h light / 150 lE, 21 "C
(day), 19 "C (night)) in a Percival Arabidopsis
growth chamber (CLF GmbH, Emersacker, Germany). 4-week-old plants were sprayed with a final
concentration of 0.2 lM MeJA in the gas phase of
each culture vessel or mock treated with 0.9 ml
0.1% (v/v) Tween 20 solution. The treatments
were applied 2 h after the beginning of the light
period. Rosettes of MeJA treated and control
plants were harvested after 0.5, 3, 6 and 24 h. The
control and MeJA treatments were carried out in 3
replicates, if not indicated otherwise.
As a positive control for MeJA induction a
transgenic reporter line of Arabidopsis thaliana
ecotype Col-2 expressing a gene for b-glucuronidase (GUS) from E. coli under the control of the
thionin 2.1 promoter (THI2.1-GUS; Vignutelli
et al. 1998), generously provided by K. Apel and
H. Bohlmann, ETH Zurich, Switzerland, was
treated in the same way as described above.
Metabolite analyses
Cysteine, glutathione, sulfite and amino acids
including O-acetylserine (OAS) as well as sulfate
were determined from 100 mg fresh weight (FW)
plant tissue after extraction either in 0.1 N HCl or
80% (v/v) ethanol (sulfate). Analysis by HPLC
followed established protocols (Hell and Bergmann 1990; Wirtz et al. 2004). These are described
in detail at the website http://pgrc.ipk-gatersleben.de/gpsg/. For glucosinolate analysis of frozen
samples (!100 mg FW) an internal standard
(4-hydroxybenzyl glucosinolate, 1 mM) and desulfation was applied. HPLC separation and identification of at least 17 desulfoglucosinolates was
carried out as described (Brown et al. 2003). The
list of detected compounds and more detailed
procedure is given at http://pgrc.ipk-gatersleben.
de/gpsg/. Concentrations of hexoses (glucose and
fructose) and sucrose in leaf tissue were determined enzymatically according to Stitt et al.
(1989).

All clones used for array analysis are listed at

http://pgrc.ipk-gatersleben.de/gpsg/. 138 cDNA
clones or RT-PCR fragments were collected from
the German Plant Sulfur Group or generously
donated by the following cooperation partners:
K. Saito, Chiba University, Japan (Sultr3;1,
Sultr2;1 and Sultr2;2), J.C. Davidian, University
of Montpellier, France (Sultr1;1), Y.-F. Tsay,
University of Taipei, Taiwan (NTR1;1, NTR1;2),
F. Mauch, University of Fribourg, Switzerland
(11 GST clones), M. Bucher, ETH Zurich, Switzerland (AtPT1; AtPT2, APA, Ara(c)tip, elF4A),
I. Feuner, University of Gottingen, Germany
(AtHPL, AtAOS, AtLOX1-6, 7 Lipases),
K. Palme, Max-Delbruck-Laboratory, Cologne,
Germany (CYP79F1, CYP79F2), D. Kliebenstein,
MPI Jena, Germany (AOP2, DOG, ESP) and
B. Halkier, Royal Veterinary and Agricultural
University, Copenhagen, Denmark (CYP79B2).
Clones listed without a cDNA or EST accession
number were in the vector pGEM-T (Promega,
Madison, USA). They were obtained from
mRNA of root or leaf material from control or
MeJA-treated plants using oligo(dT) primer and
AMV reverse transcriptase according to the supplier s protocol (Promega, Madison, USA) followed by PCR amplification of cDNAs using
ORF-specific primers. All other clones were ESTs
that had been selected according to their proposed
function from a collection provided by Michigan
State University (Newman et al. 1994) and distributed by the ABRC (Arabidopsis Biological
Resource Center at Ohio State University, USA).
About 10% of the EST clones were re-sequenced.
From these clones 24% showed variation from
the expected sequences. The spotted ESTs
encoding unknown or hypothetical proteins
were selected due to predicted signal peptides in
their amino acid sequence for chloroplast or
mitochondrial import.
The amplification of EST clones, macroarray
preparation and hybridization with [a-33P]dCTP
as well as scanning followed established procedures. A detailed description has been deposited at
http://pgrc.ipk-gatersleben.de/gpsg/. cDNA probes
from three independent replicates for each sampling
point were subsequently hybridized to the same
filter. One individual filter was used for only one

495
time point of either three replicas of control or
MeJA treatment, i.e. altogether 8 filters were
employed. To verify the filter-to-filter reproducibility the cDNA probes from the 3 replicas of 3 h
MeJA treatment were also hybridized to the filter
for the 0.5 h MeJA samples and found to yield
essentially the same results as with the first filter
(data not shown).
Processing of signals and data evaluation
The signal intensities for all spots on an array were
determined using the image processing software
ArrayVision 5.1 (Imaging Research Inc., St.
Catharines, Ont., Canada). Sorting of signals into
DNA spots and blanks, calculation of ratios and
average intensities for duplicated spots as well as
normalization and data filtering was performed
using a custom made script for the publicly available statistical package R (http://www.r-project.org/).
This script, designated R1.4.1, is available from L.
Altschmied, IPK Gatersleben. After subtraction of
the local background, all signal intensities smaller
than 0.2x the local background, were set to 0.2x
the local background. For normalization, the
median of logarithmically scaled signal intensities
for each array was set to zero (median centering of
arrays; Eisen et al. 1998) and logarithms were
converted back to normalized signal intensities.
This procedure is equal to a division of all signal
intensities of an array; by a certain factor. For the
experiments described here this factor ranges from
0.8 to 7.69. The average intensity for a spot after
normalization is 20 arbitrary units, the average
maximum spot intensity is 5315 arbitrary units,
whereas the average background intensity is 4
arbitrary units.
For clustering of expression profiles, the mean
of the logarithmically scaled (base 2), normalized
signal intensities for each gene was set to zero
(mean centering of genes, Eisen et al. 1998). This
type of transformed data will yield clusters of
genes with similar expression behaviour regardless
of absolute signal intensities when Euclidean distance measures are used. Data of the 590 selected
DNA fragments were loaded in J-Express V2.1
(Dysvik and Jonassen 2001) and hierarchical
cluster analysis using average linkage and the
!Find most similar" option were used to extract the
most promising MeJA-regulated candidate genes.

Northern analysis
Leaf material from the same experiments analyzed by
macroarray hybridization were pulverized with
mortar and pestle in liquid nitrogen and total RNA
was isolated with the RNAeasy Plant Kit (Qiagen,
Hilden, Germany) according to the manufacturers
instruction. Electrophoresis of RNA was performed
on formaldehyde containing agarose gels at
120 V. RNA was transferred onto Hybond-XP nylon
membranes (Amersham Pharmacia Biotech, Freiburg, Germany) and hybridized with random-primed
32
P-labelled cDNA probes for ATP sulfurylase
(APS), adenosinephosphosulfate reductase (APR),
sulfite reductase (SIR), O-acetylserine (thiol) lyase
and D-ribulose-5-phosphate 3-epimerase from
A. thaliana. The membranes were washed three times
at dierent concentrations of SSC in 0.1% SDS for
20 min, the final washing step being 0.5 SSC, 0.1%
SDS at 65 "C, and exposed to a X-ray film (Kodak
Biomax-MS) at ) 80 "C for 2 to 3 days.
Enzyme activity assays
-Glucuronidase (GUS) activity was determined
from 100 mg fresh weight plant tissue according to
Jeerson et al. (1987). Enzymes related to sulfur
metabolism were assayed from crude desalted
protein extracts of 100 mg plant material that had
been kept at )80 "C after harvest. Extraction and
determination of maximal in vitro activity of APR
was carried out according to Brunold and Suter
(1990) and SAT was measured as described (Wirtz
and Hell 2003). For myrosinase assays a new
method was established based on benzyl glucosinolates cleavage and phenyl cyanide as internal
standard. After extraction with dichloromethane
the benzyl isothiocyanate was analysed using an
Agilent 6890 Series gas chromatograph and flame
ionisation. The detailed protocol can be found
under supplemental material at http://pgrc.
ipk-gatersleben.de/gpsg/.

Results
General quality assessment of transcription
profile experiments
The eect of MeJA treatment on sulfur-containing
metabolites and genes of sulfur metabolism was

496
between 0.89 to 0.93 and comparable slopes of
1.27 and 1.26 (data not shown). As expected from
the threefold repetition of the experiments, the
deviations resulting from individual signals were
suppressed and signals with 2-fold and 0.5-fold
dierences were thus dierentially expressed with
high probability.

investigated in Arabidopsis thaliana by application

of a final concentration of 0.2 lM MeJA in the
culture vessel. Low-dosage and short-term exposure (30 min to 24 h) were chosen to monitor early
responses of gene expression and primary and
secondary metabolites without interference of
senescence-related reactions.
The transcriptional response was determined
using multiparallel expression analysis. The clones
on the filter array were pre-selected from an EST
collection (Newman et al. 1994) and from donations by members of the German Sulfur Research
Group
(http://www.bot.uni-heidelberg.de/hip/
gpsg/GPSG-homepage.htm) and other colleagues
(see Materials and methods). The 18 functional
categories of clones represent key steps in primary
and secondary metabolism, transport and regulation as well as more than 400 unknown proteins
(Table 1). Three independent experiments were
carried out comparing control and MeJA treatment at each of the 4 sampling points, except for
the 24 h values (which were not repeated). Filterto-filter reproducibility was high according to
scatter plots of control versus control and MeJA
versus MeJA signals from independent experiments that yielded linear regression coecients

Quantitative evaluation of transcription profiles

2891 clones representing 2467 non-redundant
genes were double-spotted on nylon membranes,
i.e. each gene under investigation was covered
2.3-fold on average. Signals from 1244 clones were
detectable showing a normalized signal intensity
1.5x above the local background, of which 413
clones turned out to be dierentially expressed
after application of the following evaluation
scheme:
The pattern analysis of signals during the
experimental time course of 24 h had to consider
fluctuations of diurnally regulated genes, due to
the goal of analyzing short-term responses to
MeJA. Many of these may be unaected by MeJA
but may give rise to false-positive signals unless
comparisons for MeJA/control signal ratios are

Table 1. Functional classification of genes on the macroarray. 18 functional categories were defined according to the predicted
properties of the annotated 2467 EST and cDNA clones. The number of genes with at least two-fold induction (>2x MeJA) or repression
(<0.5x MeJA) 6 h after MeJA treatment is indicated together with the percentage of genes (% of changes) aected in each category
Functional category

No. of genes

>2x MeJA (%)

<0.5x MeJA (%)

% of changes

Sulfur
Development
Stress
Redox regulation
Nitrogen
Cellwall / cytoskeleton
Carbon
Membrane trac
Hormones
Secondary metabolites
Protein degradation
Nucleotides
Phosphate
Signal transduction
Unknown proteins
Lipids
Transcription factors
Ribosomal proteins
Total

100
18
150
127
147
146
209
185
78
156
94
17
18
202
435
126
148
111
2467

13
2
11
9
8
5
8
7
3
6
3
0
1
5
10
3
5
1
97

3
0
4
2
4
6
4
5
2
4
3
1
0
6
12
3
2
0
64

16
11
10
10
8
7
7
7
7
7
6
6
6
5
5
4
4
1
7

(13)
(11)
(7)
(7)
(5)
(3)
(4)
(4)
(4)
(4)
(3)
(0)
(6)
(2)
(2)
(2)
(3)
(1)
(4)

(3)
(0)
(3)
(3)
(3)
(4)
(3)
(3)
(3)
(3)
(3)
(6)
(0)
(3)
(3)
(2)
(1)
(0)

497
followed-up along the time course for each individual clone. Hierarchical cluster analysis proved
to be a suitable tool to detect fluctuations during
the time course and to distinguish between diurnally regulated, MeJA regulated and diurnally plus
MeJA regulated expression patterns (Figure 1). Of
the 413 dierentially expressed clones 312 were
diurnally regulated, 221 MeJA regulated and 120
diurnally and MeJA regulated according to unbiased similarity searches. 192 clones showed only
diurnal changes and 101 were only aected by
MeJA. Among all MeJA regulated clones, 141
were up-regulated and 80 were repressed
(Figure 2). Most clones exhibited 2- to 5-fold differences and 16 candidates showed more than
10-fold up-regulation 6 h after MeJA application,
but no gene was found to be repressed by MeJA
more than 10-fold. A complete list of primary and
processed array data can be found at http://
pgrc.ipk-gatersleben.de/gpsg .
Clones with at least 10-fold up- or down-regulation are given in Figure 1. As expected, typical
MeJA response genes like vegetative storage protein 2 (VSP2) and allene oxide synthase (AOS), that
initiates the synthesis of jasmonic acid from
hydroperoxylinolenic acid (Wasternack and Hause
2002, for review), were found on this list. The AOS
signal pattern showed all control signals below and
all signals from MeJA treated samples above the
gene-and-array-centered mean value for this particular gene. Maximum log2 intensity of about 5
was already reached after 3 h and declined to less
than 1 within 24 h, indicating the transient nature of
MeJA eects on the AOS gene (Wasternack and
Hause 2002). GSH-S-transferases of the Tau- and
Phi-classes, the blue copper binding protein and a
significant number of genes related to sulfur
metabolism (i.e. 2-oxoglutarate-dependent dioxygenase AOP2, involved in biosynthesis of aliphatic
glucosinolates, myrosinase, thionin THI1.1 and
GSH-dependent dehydroascorbate reductase)
exhibited induction patterns comparable to AOS.
Genes of sulfur metabolism are up-regulated
by MeJA treatment
In 11 out of the 18 categories of genes on the filter
more genes were up-regulated than down-regulated 6 h after the beginning of MeJA application.
An induction of genes of several of the categories
on the array by MeJA was indeed expected such as

for stress- or redox-related genes (Table 1). However, a ranking of the functional categories
according to the percentage of genes with more
than 2-fold expression changes placed the class of
sulfur-related genes with 16% at the top position,
followed by genes related to stress (10%), redox
regulation (10%) and nitrogen metabolism (8%).
In contrast to the primary pathways of carbon and
nitrogen, the response of the complete set of 100
currently known sulfur-related genes has not been
investigated in that respect before. The prominent
role of sulfur metabolism may be further underscored by the high proportion of up-regulated genes
among the responding genes (13% up- compared
to 3% down-regulated). This rate of up-regulation
is 3-fold higher than the average of all categories,
suggesting a comprehensive activation rather than
repression in response to MeJA.
The average MeJA to control signal ratios of
all genes in the three independent experiments
were calculated for each time point. The results for
the MeJA-induced genes of sulfur metabolism with
gene annotations are given in Table 2. Clones
representing nearly all committed or regulating
steps in sulfur assimilation, sulfur amino acid
biosynthesis and sulfur-related functions with
locations in plastids, mitochondria and the cytosol
showed up-regulation. Strongest dierences were
observed between 3 and 6 h after MeJA application when compared to mock-treated controls.
This includes genes for assimilatory sulfate
reduction, APS2 and APR3, encoding plastidlocalized isoforms of ATP sulfurylase and adenosinephosphosulfate reductase, and the single copy
gene SIR encoding the plastidic sulfite reductase.
SAT3 encodes the mitochondrial serine acetyltransferase that carries out the first step in cysteine
synthesis, the formation of the precursor O-acetylserine. Also up-regulated was the ECS gene
encoding plastid c-glutamylcysteine synthetase
that catalyses the initial step of glutathione (GSH)
synthesis. Accordingly, GSH-S-transferase genes
were induced upon MeJA treatment and also a
GSH-dependent dehydroascorbate reductase-like
gene. Also transcripts for four genes related to
glucosinolates were elevated, including the presumably cytosolic epi-thiospecifier protein (ESP).
THI1 and THI2.1 are members of the sulfur-rich
thionin gene family, while the one member of the
defensin family present on the array (DEF1.4) was
only weakly up-regulated.

498

Figure 1. Hierarchical cluster analysis of multiparallel expression data during the 24 h time course and in response to MeJA application. For this graphical presentation clones were chosen that are at least 10-fold up- or down-regulated in at least one of the samples.
Signal intensities shown as coloured boxes represent array-and-gene-centered logarithmic (base 2) values. Clone identities labeled with
red dots represent genes with reported MeJA induction in Arabidopsis thaliana, those with blue dots represent an Arabidopsis homolog
of a MeJA inducible gene from another plant species.

The array-derived expression data of several

genes of sulfur assimilation were verified by
Northern hybridization using the same plant material but independent RNA extractions (Figure 3a).

APS2, APR isoforms 1 to 3 and SIR showed

strongest expression 0.5 to 3 h after MeJA application, whereas OASB (encoding plastid O-acetylserine (thiol) lyase) mRNA levels were not changed.

499
ities increased by 76% and APR activity increased
by 57% after 24 h, while changes of myrosinase
activity 6 h and 24 h after treatment were not significant (Figure 4). It should be noted that in all
three cases increased activities of the individual
isoenzymes had to be assayed against the strong
background delivered by the other forms of the
enzyme. The changes in total enzyme activities that
could be expected were therefore relatively small.

Targeted metabolite profiling in response

to MeJA treatment

Figure 2. Identification of MeJA and diurnally regulated

cDNA clones as obtained from hierarchical cluster analysis.
Dierences of 2-, 5- and 10-fold refer to average values of three
experiments 6 h after MeJA application based on gene-andarray-centered log2 values.

Thus, the patterns obtained from the arrays were

fully confirmed by RNA-DNA hybridization.
In addition, the reporter gene activity of a
transgenic THI2.1 promoter fusion with the
b-glucuronidase gene was used to verify the
THI2.1 expression pattern observed in the array
analysis (Figure 3b). The transgenic line was
treated with MeJA in the same way as the wild
type for the array experiments. A similar time
course of reporter gene induction was observed as
for the THI2.1 gene expression pattern on the
array. It is concluded that the expression profiles
at least of the genes tested here were fully corroborated, thus confirming the accuracy of our
filter hybridization and evaluation procedures.
The activities of three enzymes related to sulfur
metabolism were determined in order to test whether up-regulation of mRNAs after MeJA treatment indeed translated into metabolic activities.
Almost all enzymes in this pathway are encoded by
gene families in the Arabidopsis genome with differential expression and induction patterns. SAT (5
genes), APR (3 genes) and myrosinase (3 genes)
were selected due to their crucial positions for
metabolic flux in the corresponding sections of the
pathway and the induction of at least one member
of the underlying gene family according to our
array data. When compared to extractable in vitro
activities of non-treated control plants (thus taking
into account possible diurnal changes), SAT activ-

Numerous defense compounds are known to

contain sulfur moieties. Since sulfur assimilation
provides the precursors for these compounds, the
concentrations of 9 primary sulfur compounds
were analyzed, but no dierences were observed
between MeJA-treated and control plants. The
plants contained on average 1.0 0.2 lmol/g FW
sulfate and (in nmol/g FW): 2.3 0.6 sulfite,
45 5 L-cysteine, 10 3 c-glutamylcysteine,
10 3 cysteinylglycine, 37352 glutathione and
153 methionine. Cysteine and especially glutathione showed diurnal variation (1.5-fold increase
8 h after the onset of the light period). The direct
precursor of L-cysteine, OAS, was present at
247 nmol/g FW. In addition, the concentrations
of 6 non-sulfur amino acids known to respond to
biotic stress were determined. Mean contents were
(in nmol/g FW): 3105215 arginine, 1515300
asparagine, 42131092 glutamine, 6816 phenylalanine, 18339 proline and 143 tyrosine. In
none of these cases significant dierences were
observed between controls and MeJA treated
plants during the 24 h time course.
Among the sulfur-containing defense compounds in the Brassicaceae, glucosinolates are
often assumed to have antimicrobial activities. The
total glucosinolate content and concentrations of
17 individual glucosinolates were determined after
MeJA treatment. Total glucosinolate content
increased from 3.4 to 4.4 lmol/g FW during the
time course of the experiment, but showed no
significant dierence between controls and MeJA
treated samples (Figure 5a). An approximately
2-fold increase was observed for the indol-3-ylmethylglucosinolate, glucobrassicin (Figure 5b).
MeJA treated plants also contained slightly more
4-methylsulfinylbutyl- and 1-methoxy-indol-3-yl-

500
Table 2. Inducing eect of MeJA treatment on sulfur-related genes in Arabidopsis thaliana. AOS and VSP2 are included as positive
controls for MeJA induction
Gene

Annotation

MIPS code

0.5 h Ratio

3 h Ratio

6 h Ratio

24 h Ratio

SUL
APS1
APS2
APK
APR1
APR2
APR3
SIR
SAT3
MST1
MST2
ECS
PCS
DHA
GST U5
GST U13
GST F6
GST F9
CBL
SAM
AOP2
MYR
MBP
ESP
THI1.1
THI2.1
DEF1.4
VSP2
AOS

Sulfate transporter-like
ATP sulfurylase
ATP sulfurylase
Adenosinephosphosulfate kinase
Adenosinephosphosulfate reductase
Adenosinephosphosulfate reductase
Adenosinephosphosulfate reductase
Sulfite reductase
Serine acetyltransferase
Mercaptopyruvate sulfurtransferase
Mercaptopyruvate sulfurtransferase
c-Glutamylcysteine synthetase
Phytochelatin synthase
GSH-dependent dehydroascorbate reductase-like
GSH-S-transferase
GSH-S-transferase
GSH-S-transferase
GSH-S-transferase
Cystathionine-b-lyase-like
SAM synthetase
2-Oxoglutarate-dependent dioxygenase
Myrosinase
Myrosinase binding protein
Epithiospecifier protein
Thionin
Thionin
Defensin
Vegetative storage protein
Allene oxide synthase

At1g80310
At3g22890
At4g14680
At4g39940
At4g04610
At1g62180
At4g21990
At5g04590
At3g13110
At1g79230
At1g16460
At4g23100
At5g44070
At1g19570
At2g29450
At1g27130
At1g02930
At2g30860
At1g64660
At4g01850
At4g03060
At3g09260
At2g33070
At1g54040
At1g66100
At1g72260
At1g19610
At5g24770
At5g42680

1.9
1.8
3.2
2.8
0.9
0.6
0.9
1.3
3.1
0.9
0.7
2.2
1.1
6.4
8.0
2.5
5.3
1.6
2.1
1.4
4.2
1.1
3.0
1.6
3.2
1.2
1.3
38
7.6

1.0
3.3
2.4
3.4
2.7
1.8
1.9
2.2
2.4
1.4
2.0
3.8
2.6
18
5.3
2.5
3.5
2.0
7.3
1.2
3.7
2.6
3.8
2.6
3.6
4.8
1.2
53
7.1

0.9
1.6
2.1
0.7
1.9
0.9
1.1
0.8
3.6
1.0
1.3
1.6
0.8
8.8
2.1
1.2
1.3
1.2
10.4
1.2
2.5
4.8
5.5
1.2
4.8
6.6
2.0
69
4.7

1.9
1.9
1.7
1.8
1.9
1.3
1.4
1.2
2.5
1.5
1.4
1.0
1.0
2.6
1.6
1.2
1.4
1.6
3.0
1.3
1.9
1.3
0.7
1.9
3.8
2.5
1.1
11
4.9

methylglucosinolate while all other glucosinolate

levels remained unchanged (data not shown). With
respect to sulfur and nitrogen bound in these
compounds it is notable that the increases
observed for individual glucosinolates are in the
same order as the pool sizes of the amino acids and
sulfur compounds measured. This suggests that a
possible up-regulation of flux might not be
detectable for the steady-state concentrations of
the primary compounds during the early responses
to the artificial inducer MeJA.
The total contents of glucose, fructose and
sucrose were investigated as indicators of primary carbon metabolism (Figure 5c). In control
plants, the steady state pools of sucrose and
fructose showed opposite diurnal changes,
whereas glucose content remained almost

unchanged. MeJA-treated plants showed only

minor transient decreases for glucose, whereas
sucrose content decreased transiently by 50%
after 3 h and fructose content started to decrease
by 40% of control levels from 6 h MeJA treatment onwards. MeJA is known to stimulate the
expression of cell-wall invertase in leaves (Zhang
et al. 1996), and in the experiments reported
here, MeJA also stimulated the expression of a
cytosolic invertase (Figure 1). In general,
increased invertase activities often correlate with
higher respiration rates and an activated oxidative pentose phosphate cycle. Thus, MeJA may
cause the observed reduction of sucrose and
fructose contents via stimulating the respiration
rate, while simultaneously stimulating the
expression of cytosolic, cellwall and/or vacuolar

501

Figure 4. Alterations of sulfur-related enzyme activities and

metabolite contents. A. Specific activities of SAT (serine acetyltransferase), APR (adenosinephosphosulfate reductase) and
MYR (myrosinase) of control (black bars) and MeJA treated
(open bars) from four independent experiments, indicated with
standard errors, at the indicated times after MeJA application.
Activities of controls were defined as 100% and were
10.22.1 nmol mg)1 protein min)1 (SAT), 0.860.10 nmol
mg)1 protein min)1 (APR) and 8.81.7 nmol mg)1 protein
min)1 (myrosinase).

Figure 3. Expression patterns of genes related to sulfur

metabolism in response to MeJA treatment. A. Detection of
genes of sulfate assimilation by Northern hybridization. Genespecific probes were applied for plastid adenosinephosphosulfate reductase (APR1, 2 and 3), ATP sulfurylase (APS2) and
OAS-TL (OASB). A probe for ribulose-5-phosphate 3-epimerase (RPE, At5g61410) served as loading control. A representative experiment of control (C) and MeJA (MJ) treated
samples from the time points indicated is shown. B. Expression
pattern of the THI2.1 gene as revealed by b-glucuronidase
(GUS) activity of a transgenic Arabidopsis thaliana line harbouring a THI2.1 promoter-GUS construct (Vignutelli et al.
1998). Plants were cultivated and treated under the same conditions as wild-type plants. Standard error bars refer to 2
independent experiments with 3 protein extractions each.

invertases, resulting in an overall increased flux

through these metabolites.
Discussion
Many constitutive and inducible components of
plant defense against pathogens contain one or

more reduced sulfur moieties. Hence, pathogen

attack may create an increased requirement for
sulfur in order to synthesize defense-related sulfur
compounds such as thionins and glucosinolates.
An up-regulation of primary sulfur metabolism
could help to provide intermediates for these
defensive end-products. Jasmonic acid has been
described as a potent inducer for most of these
compounds (Vignutelli et al. 1998; Mikkelsen et al.
2003). In this study, the established signaling
molecule, MeJA, was applied to Arabidopsis plants
at low concentration (0.2 lM) to avoid any
induction of senescence processes known to result
from harsh MeJA treatments (Wasternack and
Hause 2002; He et al. 2002). The early responses of
more than 2400 unique genes on a macroarray
were monitored together with profiling of key
intermediates of sulfur metabolism during shortterm sampling (up to 24 h) to reduce interference
with pleiotropic downstream eects. The arrays
were subjected to rigorous quality assessment
regarding signal-to-background intensities, double-spotting ratio and filter-to-filter reproducibility. Statistical reliability was achieved by three
completely independent repetitions of control and
MeJA experiments and a time course with four
sampling points. According to these analyses,
33% of all detected clones were dierentially
expressed.

502

Figure 5. Glucosinolate and sugar concentrations in leaves of

Arabidopsis thaliana by MeJA treatment. A: total glucosinolate
content. B: glucobrassicin (indolyl)3-methyl-glucosinolate). C:
Total contents of glucose (G), fructose (F) and sucrose (S).
Black bars represent control plants sprayed with 1 ml 0.1%
Tween 20 solution, white bars represent plants treated with
200 lM MeJA / 0.1% Tween 20. Presented are the averages of
three individual extractions of three independent induction
experiments with error bars indicating standard errors.

Hierarchical cluster analysis was used to distinguish MeJA and diurnally regulated genes. Out
of the 413 dierentially expressed genes, more than
50% were MeJA regulated or diurnally plus MeJA
regulated. This ratio was in the same range as that
obtained for MeJA regulation when results from

pathogen, wounding, MeJA and salicylic acid

treatments of Arabidopsis were combined (Schenk
et al. 2000) and somewhat higher than for MeJa
treatments reported by Sasaki et al. (2001) who
used a higher cut-o for their macroarray signals.
Similarly, the number of diurnally regulated clones
in our study was of the same order of magnitude as
detected in macroarray analysis of diurnally and
circadian-regulated genes (Schaer et al. 2001).
The strong induction of two characteristic markers
for MeJA responsiveness, AOS and VSP2
(Wasternack and Hause 2002), confirmed the
eectiveness of our MeJA treatment. Also the
well-characterized induction of the defense gene
THI2.1 by MeJA (Vignutelli et al. 1998) was
confirmed and the one member of the MeJAinducible defensin family on the array, DEF1.4,
was induced (Thomma et al. 2002).
It is a common observation that alterations of
mRNA steady-state levels determined by array
analyses are not necessarily reflected by fluctuations of the encoded gene products. Using
Northern hybridization of 5 genes and in one case
a promoter/GUS transgenic line, we demonstrate
the reliability of our array experiments. Furthermore, two out of three up-regulated genes of sulfur
metabolism correlated with increased activities of
the encoded enzymes in whole plant extracts.
Remarkably, these increases were observed for
APR and SAT, which catalyse two rate-limiting
steps early in the sulfate assimilation pathway, and
against a background of several other members of
the APR and SAT gene families without up-regulation on the macroarray after MeJA treatment.
Myrosinase, the third tested enzyme, is located at
the very end of the pathway and may form an
exception because of its association with myrosinase binding proteins and the special compartmentalization of the glucosinolate system in
idioblast cells (Wittstock and Gershenzon 2002).
Substantial and rapid shifts in the expression of
metabolic genes in response to infection of Arabidopsis with Pseudomonas have been observed
(Scheideler et al. 2002), but reports on reactions of
sulfur-related genes to wounding, pathogen attack
or MeJA treatment are scarce. MeJA has been
shown to induce the expression of GSH biosynthetic genes (Xiang and Oliver 1998) as well as of
myrosinase and several GSH-S-transferase
encoding genes (Schenk et al. 2000; Sasaki et al.
2001). Harada et al. (2000) reported a transient

503
up-regulation of four genes of sulfate reduction in
10 day old seedlings. In these experiments a
50-fold higher final MeJA concentration (10 lM
compared to 0.2 lM used here) was applied and
transcript contents were not corrected for diurnal
variations as known e.g. for APR (Saito 2004 for
review). An up-regulation of a cluster of sulfurrelated genes, but, very remarkably, no change in
expression levels of known sulfur-deficiency
induced sulfate transporters, after a MeJA treatment has been cited as unpublished results by
Saito (2004). In contrast, we could show that

MeJA application at a low dose induces genes

encoding enzymes of all branches of sulfate
assimilation, sulfur amino acid biosynthesis and
closely related metabolites such as glutathione
within 30 min to 6 h (Figure 6 and Table 2). Again
we found no induction of known S induced sulfate transportes. Furthermore, THI and DEF
genes and several genes involved in glucosinolate
biosynthesis and breakdown responded. Interestingly, a proteomics approach of rice plants infected with blast fungus (Magnaporthe grisea) at
supposedly dierent nitrogen supplies revealed

Figure 6. Pathway chart of MeJA induction of primary and secondary sulfur metabolism and related reactions in Arabidopsis thaliana.
Dierential expression of genes is indicated by squares with increasing intensity. Non-regulated genes are omitted, but genes encoding
proteins for almost all reactions of the pathway were present on the array and can be viewed at http://pgrc.ipk-gatersleben.de/gpsg.
Gene abbreviations are explained in Table 2.

504
changes in the abundance of 12 proteins, one of
which was a sulfur-rich thaumatin-like protein. It
is important to note that this protein was detected
only after infection and at top application of
nitrogen, which in this case was applied as
ammonium sulfate, i.e. nitrogen and sulfur supply
were enhanced simultaneously (Konishi et al.
2001). Thus, genes across the entire sulfur network
responded to MeJA as a signal of tissue wounding
and pathogen attack, providing not only a new
aspect of demand-driven regulation of sulfur
metabolism but also of pathogen defense.
Vice versa, activation of genes of the jasmonate
biosynthetic pathway has been noted in microarray experiments with Arabidopsis plants under
various mineral nutrient deficiencies. Sulfate
deprivation and treatment with the putative trigger
metabolite OAS, but also potassium and nitrogen
starvation, are able to induce several genes of the
octadecanoid acid pathway, including AOS, as
well as of other plant hormones (Hirai et al. 2003;
Nikiforova et al. 2003; Maruyama-Nakashita et al.
2003; Armengaud et al. 2004; Hirai et al. 2004).
These findings are in accordance with our data and
together support the idea of a signalling loop
between sulfur/mineral deficiency or increased
internal demand and hormonal signalling by
MeJA and possibly other hormones (Saito 2004).
Targeted expression studies have already
revealed that distinct members of the sulfate
transporter, APS and APR gene families are
up-regulated by sulfate deficiency, while the genes
downstream in the pathway seemed less responsive
(Takahashi et al. 1997; Koprivova et al. 2000; Hell
et al. 2002). The metabolites sulfate, O-acetylserine, cysteine and glutathione are discussed as
candidates for triggering the transcriptional
response of these genes, particularly those encoding sulfate transporters and APRs (Smith et al.
1997; Lappartient et al. 1999; Vidmar et al. 2000).
Remarkably, instead of the 4 well-known sulfate
transporter genes of Arabidopsis on the array
(Sultr1;2, Sultr2;1, Sultr2;2 and Sultr3;1), a largely
uncharacterized EST clone spotted on the array
encoding a sulfate transporter-like protein (SUL,
equivalent to Sultr5.1; Buchner et al. 2004)
responded to MeJA, suggesting a possible dierence in sulfate allocation patterns in response to
external sulfate starvation versus additional internal demand generated by the MeJA signal. The
MeJA induction of Sultr4;2, a sulfate transporter

supposed to be involved in vacuolar eux of sulfate (H. Takahashi, personal communication) is

also consistent with this suggestion.
While increased mRNA levels throughout the
pathway as well as elevated enzyme activities of
SAT and APR were observed, the primary sulfur
metabolites showed no significant changes in their
steady-state concentrations after our conditions of
MeJA application. This discrepancy may have
dierent causes. For one, the applied MeJA concentrations may have been sucient to prime
rapid responses at the mRNA level throughout the
sulfur and other pathways and a moderate increase
in APR and SAT activities, but too low to cause
measurable changes of the steady-state concentrations of metabolites. Indeed, the 50% increase
in total glucosinolates content observed by Harada
et al. (2000) after 3 h fell back to starting levels
after 24 h and were achieved using a much higher
final MeJA dosis. Primary sulfur metabolites had
not been determined by these authors. Second,
MeJA is only one part of the signalling response
during pathogen attack, salicylate and ethylene
addressing partly independent, partly interconnected pathways (Pieterse and van Loon 1999;
Glazebrook 2003; Devoto and Turner 2005).
Accordingly, full induction of the PDF1.2 gene in
Arabidospis grown under sterile conditions
required the combined treatment of MeJA plus
ethylene (Pennincks et al. 1998). Third, MeJA
treatment only mimics wounding or pathogen
attack and therefore may not result in actual
consumption of sulfur metabolites, such as GSTcatalyzed GSH conjugation or the hydrolysis of
glucosinolates resulting from cell damage, at least
in this short-time experiment.
It is important to note, however, that a change
of flux rate in sulfate assimilation in fact may have
occurred. The intracellular concentration of cysteine in plants is remarkably constant, although
this first organic thiol-containing compound is at
the intersection of sulfur supply and demand (Hell
et al. 1997; Leustek et al. 2000). Flux analysis and
transgenic approaches using Arabidopsis indicate
important control points at sulfate uptake and the
first reduction step encoded by APR without significant accumulation of intermediates (Tsakraklides et al. 2002; Vauclare et al. 2002). The step of
cysteine synthesis seems to be regulated at the
supply side by sensing of sulfide via the cysteine
synthase complex (Hell et al. 2002) and at the

505
demand side by allosteric feedback inhibition of
specific SAT isoforms by cysteine (Saito 2000
2004), together providing stable steady-state concentrations of free cysteine. To clarify unequivocally whether MeJA induced a flux increase in the
primary sulfur pathway would therefore require
determination of synthesis rates with labelling
studies. However, this would be further complicated because GSH, but probably also cysteine,
concentrations dier between the subcellular
compartments (Hell 1997; Leustek et al. 2000 and
references therein). Organellar thiol pools may
react dierently than cytosolic ones, resulting in no
measurable variations on a whole leaf basis.
In contrast, the levels of sucrose and fructose did
change upon MeJA application, suggesting an
enhanced demand for energy and biosynthetic
capacity to support defense reactions. An up-regulation of genes of the pentose phosphate pathway and
other housekeeping functions has been observed after
infection of Arabidopsis with an avirulent Pseudomonas syringae strain (Scheideler et al. 2002), a fact
that may also be reflected in this study by the dierential induction of genes in the carbon and nitrogen
metabolism categories (Table 1).
Also an increase in one class of secondary sulfur compounds, the indole glucosinolates, was
observed, while the amount of aliphatic glucosinolates did not change. In previous studies, MeJA
treatment has often been observed to lead to
increases in indole glucosinolates (Bodnaryk 1994;
Kliebenstein et al. 2001; Mikkelsen et al. 2003),
while increases in aliphatic glucosinolates have
been less frequently reported (Harada et al. 2000;
Mikkelsen et al. 2003). It is unclear whether the
dierences among these studies are due to the use
of dierent growth conditions, ecotypes, or MeJA
concentrations. In the current work, MeJA treatment not only led to an increase in indole glucosinolate content, but also to an increase in four
transcripts associated with glucosinolate biosynthesis, hydrolysis and breakdown: AOP2, myrosinase, a myrosinase-binding protein and ESP
(Table 2). The ESP protein promotes the formation of nitriles instead of isothiocyanates during
glucosinolate hydrolysis (Lambrix et al. 2001).
Therefore, MeJA treatment alters not only the
quality of glucosinolates present, but also the
profile of products formed on glucosinolate
hydrolysis. Since each class of hydrolysis products
has a distinctive pattern of biological activity,

MeJA may alter the overall character of the

defense response as well as its magnitude. Interestingly, the amount of glucosinolates, including
those of the indole class, strongly depends on
sulfate supply and correlates with the susceptibility
of Brassica napus to several fungal pathogens
(Dubuis et al. 2005).
Significant changes of cellular sulfur metabolite
concentrations, including the assumed candidates
for signalling of the sulfur deficiency response, as
well as changes in transcript levels of the primary
targets of this well-studied response, the high- and
low-anity sulfate transporters, were not observed
in this study. This finding indicates, together with
the comprehensive changes in steady-state mRNA
patterns of down-stream sulfur-related genes in
response to MeJA treatment, that responses of
sulfur metabolism to external sulfur deprivation
on one hand side and to enhanced demand for
defense reactions on the other hand may be regulated by dierent signal transduction pathways.
Further work will focus on the reaction of sulfur
metabolism to the attack of fungal pathogens and
the underlying metabolic and hormonal signal
components.
Acknowledgements
The authors of the German Sulfur Research
Group wish to thank the Deutsche Forschungsgemeinschaft for funding of the Research Group
383. We are indebted to M. Bucher, ETH Zurich,
I. Feussner, Universitat Gottingen and F. Mauch,
University of Fribourg, who donated cDNA
clones for the array analysis, and to H. Bohlmann
and K. Apel, ETH Zurich, for generous provision
of the THI2.1 reporter line. We thank Christa
Kallas for excellent technical assistance, M.
Hajirezaei, Molecular Plant Physiology Group,
IPK Gatersleben, for support with anion analysis
and U. Scholz, Bioinformatics Group, IPK Gatersleben, for the web page development.

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