Analysis of Arabidopsis glutathione-transferases in yeast

Article abstract of DOI:10.1016/j.phytochem.2012.04.016

The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large

Full text of DOI:10.1016/j.phytochem.2012.04.016

Phytochemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Analysis of Arabidopsis glutathione-transferases in yeast
Matthias P. Krajewski a, Basem Kanawati b, Agnes Fekete b, Natalie Kowalski a, Philippe Schmitt-Kopplin b,
Erwin Grill a,
⇑
a Lehrstuhl für Botanik, Technische Universität München, Emil-Ramann Straße 4, D-85354 Freising, Germany
b Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, Analytical Biogeochemistry, D-85764 Neuherberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in
seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as her-
bicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In
this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows
screening for GST substrates and identifying substrate preferences within the plant GST family. To this
end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The result-
ing yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-
dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple
mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression
of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays
and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was
unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass
spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members dem-
onstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently,
the established yeast system was explored for its potential to screen the Arabidopsis GST family for con-
jugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased lev-
els of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi,
tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of
which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast
extracts allowed the simultaneous detection and semiquantification of anilazine conjugates as well as
catabolites.
Dedicated to the memory of Meinhart H.
Zenk
Keywords:
Fungicide
Detoxification
Xenobiotics
ICR-FTMS
Isotope labeling
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
mers. GSTs have a conserved N-terminal domain involved in
GSH-binding, the so-called G-site. The GSTs decrease the pKa of
GSTs (EC 2.5.1.18) are a superfamily of multifunctional proteins
that play a prominent role in glutathione (GSH) metabolism of
many living organisms (Hayes et al., 2005; Dixon et al., 2010). GSTs
catalyze a number of reactions including redox reactions, acting as
peroxidases or reductases, and particularly as transferases in the
conjugation of lipophilic compounds to GSH (Edwards and Dixon,
2005; Cummins et al., 2011). GSTs can form homo- and heterodi-
the substrate GSH resulting in its deprotonation and the formation
of a more reactive thiolate anion. The anion of GSH is stabilized in
the active site of the GSTs through an interaction with either cys-
teine or serine. The C-terminal domain referred to as the H-site ac-
cepts a variety of hydrophobic co-substrates and is structurally
more variable.
The formation of GSH-S-conjugates (GS-conjugates) with xeno-
biotics, such as pesticides, is part of a detoxification mechanism in
plants and animals. Xenobiotics can either be directly conjugated
to GSH or functional groups are introduced into the chemical com-
pound prior to conjugation (Hayes et al., 2005; Dixon et al., 2010).
In plants, the generated GS-conjugates are further catabolized and
frequently transported to the vacuole. Plants recruit different path-
ways for the catabolism of GS-conjugates. One pathway is initiated
by the removal of the glycine residue and involves the cytosolic en-
zyme phytochelatin synthase (Beck et al., 2003; Grzam et al., 2006;
Abbreviations: AZ, anilazine; CDNB, 1-chloro-2,4-dinitrobenzene; DHAR, dehy-
droreductase; ICR-FTMS, ion cyclotron resonance Fourier transform mass spec-
trometry; GFP, green fluorescent protein; GGT,
c-glutamyltranspeptidase; GSH,
reduced glutathione; GST, glutathione transferase; GSTF, phi-class glutathione
transferase; GSTL, lambda-class glutathione transferase; GSTT, theta-class gluta-
thione transferase; GSTU, tau-class glutathione transferase; m/z, mass-to-charge
ratio; NBD-Cl, 4-chloro-7-nitro-2,1,3-benzoxadiazole.
⇑
Corresponding author. Tel.: +49 8161 5433.
E-mail address: erwin.grill@wzw.tum.de (E. Grill).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.04.016
Please cite this article in press as: Krajewski, M.P., et al. Analysis of Arabidopsis glutathione-transferases in yeast. Phytochemistry (2012), http://dx.doi.org/
10.1016/j.phytochem.2012.04.016

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M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
Blum et al., 2007, 2010). A second route is initiated by the cleavage
of the glutamate residue via the activity of glutamyltransferase, 5-
oxo-prolinase, or glutamylpeptidase (Grzam et al., 2007; Ohkama-
Ohtsu et al., 2007a,b, 2008; Geu-Flores et al., 2011). Different path-
ways lead to the common catabolite cysteinyl-conjugate, which
may be subjected to further degradation (Brazier-Hicks et al.,
2008).
By analogy to the classification of mammalian GSTs, plant GSTs
have been grouped into seven clades referred to as tau (U), phi (F),
lambda (L), theta (T), zeta (Z), dehydroascorbate reductase (DHAR)
and tetrachlorohydroquinone dehalogenase-like (TCHQD), which
has a single member in Arabidopsis (Wagner et al., 2002). In plants,
a plethora of genes encoding GSTs exist with more than 50 mem-
bers in Arabidopsis or rice (Banerjee and Goswami, 2010), which
considerably exceeds the number of counterparts encoded by
mammalian genomes. In Arabidopsis, the phi and tau class repre-
sent by far the largest clades with 13 and 28 members,
respectively.
Transcriptional profiling of Arabidopsis GSTs, as well as in vitro
analyses, have implicated tau class members in the detoxification
of xenobiotics (Dixon et al., 2009; Skipsey et al., 2011). The most
abundant member of this subgroup is AtGSTU19, which is together
with AtGSTF2 induced by the herbicide safeners benoxacor and
fenclorim (DeRidder et al., 2002; Smith et al., 2004; DeRidder
and Goldsbrough, 2006). AtGSTU7, AtGSTU19, and AtGSTU24 con-
jugate fenclorim to GSH (Brazier-Hicks et al., 2008), and the subse-
quent catabolism of the GS-conjugate generates products that act
as inducers of GST activity (Liu et al., 2009). The functional role
of the phi class GSTs is largely unknown. AtGSTF8 is the only mem-
ber of this group for which significant conjugating activity to a
xenobiotic compound has been assigned (Dixon et al., 2009). Mem-
bers of the phi class are induced by salicylic acid and may be impli-
cated in pathogen defense (Sappl et al., 2004). AtGSTF9, AtGSTF10,
and AtGSTF11 have been proposed to be involved in the biosynthe-
sis of glucosinolates (Sonderby et al., 2010) and AtGSTF6 in cama-
lexin production (Su et al., 2011). However, the simultaneous
knockdown of AtGSTF6, AtGSTF7, AtGSTF9, and AtGSTF10 expres-
sion revealed no altered GST activity (Sappl et al., 2009). The pres-
ence of multiple GSTs with overlapping functions and substrate
specificities might preclude the observation of phenotypic alter-
ation in knockout mutants (Sappl et al., 2009). Hence, the cellular
function of most GST members in detoxification and plant metab-
olism remain elusive in spite of considerable research efforts. Re-
cently, heterologous expression of plant GSTs in bacteria (Dixon
and Edwards, 2009) and in vitro substrate-fishing experiments
(Dixon and Edwards, 2010) offer additional avenues for elucidating
the role of plant GSTs. In this study, GST-deficient yeast is proposed
as a model system for assaying the substrate specificity of Arabid-
opsis GSTs.
jugating activity to ethacrynic acid (Garcera et al., 2006). Two
translation elongation factors eEF1B (Tef3p, Tef4p) indicate an
c
N-terminal domain similar to the GSH-binding site of GSTs and
for Tef4p a GST activity has been postulated (Jeppesen et al., 2003).
A variety of yeast mutants were generated with multiple dis-
ruptions of GST and GST-related genes and their residual GST activ-
ity were analyzed (Fig. 1). Combined deletion of GTT1, GTT2, GRX1,
and GRX2, comparable to the previously analyzed quadruple mu-
tant (Collinson and Grant, 2003), showed a marginal change of to-
tal GST activity against CDNB (25%) in cell-free protein extracts
(Fig. 1A). The findings indicated the presence of other major GST
activities and, therefore, the contribution of several other genes
was analyzed including the GTO cluster. The triple knockout
GTO1–3 displayed also only a marginal change in total GST activity
towards CDNB (Fig. 1A). In contrast, inactivation of TEF4 resulted in
a 50% decline of total GST activity and in the quintuple knockout,
D
tef4Dgtt1Dgtt2Dgrx1Dgrx2, a residual level of activity was ob-
served equivalent to approximately 35% of the wild type, respec-
tively (Fig. 1A). This observation is in accordance with the
claimed GST activity of Tef4p (Jeppesen et al., 2003) and with the
reported GST-activity of a plant Tef4p homolog (Kobayashi et al.,
2001), while Tef3p lacks the serine residue critical for GSH coordi-
nation. Tef3p and Tef4p are both
c subunits of the elongation factor
1B in yeast, which act together with the catalytic subunit as a
nucleotide exchange factor for the elongation factor 1A.
The various yeast lines were also analyzed with NBD-Cl as an
alternative GST substrate for which GTT2 encodes a major GSH-
conjugating activity (Ma et al., 2009). The in vitro analysis of
NBD-Cl conjugation by cell-free protein extracts showed a clearly
reduced
gtt1 gtt2
hanced decrease
tef4 gtt1 gtt2 grx1
GST
activity
grx2 by approximately 70%, and an even en-
(85%) in the quintuple mutant
grx2 compared to the wild type (Fig. 1B).
in
the
quadruple
mutant
D
D
D
grx1
D
D
D
D
D
D
The enzymatic analyses with two different GST substrates consis-
tently established the lowest GST activity in the quintuple mutant,
which was favored in subsequent analysis.
In a next step, the CDNB-sensitivity of GST-deficient yeast was
examined. The quadruple mutant showed no increased sensitivity
of the mutant cells towards CDNB in a growth assay (Fig. 2A). In
contrast, the single TEF4 disruption (Dtef4) caused an enhanced
sensitivity to CDNB, which was further pronounced in combination
with deletion of GTT1, GTT2, GRX1 and GRX2 in the quintuple mu-
tant. The single
Dtef4 mutant and the quintuple mutant grew com-
parably to wild type in nutrient-rich YPD medium, while in
supplemented minimal medium the mutants grew slightly slower
(data not shown).
The reduced CDNB-conjugating activity of
Dtef4 and the qua-
druple mutant is a likely cause for the observed CDNB hypersensi-
tivity. In order to examine whether plant GSTs can rescue the
phenotype, three plant GSTs, AtGSTF2, AtGSTF8 and AtGSTU19 with
known CDNB activity were characterized (Dixon et al., 2009).
Expression of the Arabidopsis genes was controlled by a galact-
ose-inducible promotor of a low-copy expression vector (Guelden-
er et al., 2002). In both GST-deficient strains, galactose-dependent
AtGSTF8 and -U19 expression fully complemented the CDNB
hypersensitivity (Fig. 2B), while AtGSTF2 expression only partially
rescued CDNB tolerance.
In order to rule out that the different degrees of complementa-
tion were a result of different GST expression levels, the three plant
enzymes were labeled with a green fluorescence protein (GFP) tag
to monitor GST abundance. Immunological analysis of cell-free
protein extracts from the yeast lines documented strictly galact-
ose-dependent and comparable protein levels for all three plant
GSTs (Fig. S2). Assessment of total GST activity in the yeast lines
showed a strongly increased CDNB-conjugating activity in extracts
from AtGSTF8 and AtGSTU19 expressing cells. GSH–dinitrobenzene
2. Results and discussion
2.1. GST-deficient yeast mutants and Arabidopsis GST expression
A GST-deficient yeast strain was designed for use as a heterolo-
gous system to analyze the GSH-conjugating activity of single
members of the Arabidopsis GST cluster. To reduce the endogenous
GST activity in yeast, GST genes were inactivated by marker-as-
sisted homologous recombination. Yeast has nine genes encoding
GSTs and GST-similar proteins (McGoldrick et al., 2005; Choi
et al., 1998), which are quite diverse as depicted in the phyloge-
netic tree (Fig. S1). Strong reduction of total GST activity has been
reported for a quadruple mutant disrupted in the GST genes GTT1,
GTT2, and the glutaredoxin genes GRX1 and GRX2 (Collinson and
Grant, 2003). In addition, Gto1p, Gto2p, and Gto3p have GSH-con-
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M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
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Fig. 1. Enzymatic characterization of GST-deficient yeast strains. Specific GST activities were determined in cell-free protein extracts of GST wild type yeast (WT) and GST-
deficient deletion mutants towards the GST-standard substrates (A) CDNB and (B) NBD-Cl. The chemical structure of both substrates is presented. The (+) sign indicates the
presence of the wild type allele in respective pairings of deleted genes (D). Error bars represent standard deviations from triplicates. NA: not analyzed.
(GS–DNB) levels remained low in yeasts expressing AtGSTF2
(Fig. 2C), which might be attributed to a poor substrate preference
of this specific GST for CDNB. A second GST substrate was, there-
fore, examined. In the presence of NBD-Cl, extracts of the AtGSTF2
line showed only background levels of the GSH conjugate (GS–
NBD), while AtGSTF8 and AtGSTU19 expression enhanced forma-
tion of GS–NBD by a factor of approximately 4 and 50, respectively.
AtGSTF2 might strongly discriminate against both GST substrates
or, alternatively, was expressed in an inactive form or efficiently
inactivated by posttranslational mechanisms. The analyses support
the notion that the different degrees of complementation observed
in the yeast growth assay are caused by different CDNB conjugat-
ing activities of the plant enzymes.
resolution of masses permits the robust identification of metabo-
lites and the frequent deduction of elemental composition of un-
known compounds. The question is whether the mass
spectrometric analysis provides a quantitative and reliable readout
to compare conjugate levels among different samples. Differential
stable isotope labeling allows discriminating and analyzing of two
different samples in a single probe (Hsu et al., 2003; Synowsky
et al., 2009). This method was used herein to separately label an
extract of CDNB-treated yeast cells either with [1H2]- or [2H2]-
formaldehyde, which dimethylates the free amino group of GS-
conjugates. Subsequently, both samples were combined and ana-
lyzed by ICR-FTMS (Fig. 3A). The mass signals for the dimethylated
isotopologs of GS–DNB showed a perfect match in intensity, which
indicated comparable efficiencies in labeling and detection of the
GS-conjugates. Dimethylated and deuterated GS–DNB was used
as an internal standard to quantify GS–DNB levels in extracts of
yeast expressing either AtGSTF2, AtGSTF8, or AtGSTU19 (Fig. 3B).
The ICR-FTMS analysis established a pattern of GS–DNB levels in
the different lines that was comparable to the results generated
by a parallel HPLC quantification (Fig. 3C). Both methods differed
by up to 30% in absolute values. Based on these results, either
the ICR-FTMS or HPLC analysis were used to (semi)-quantify GS-
metabolite levels in subsequent experiments.
2.2. Analysis of dinitrobenzene-conjugates in yeast extracts
CDNB and NBD-Cl are chromophoric GST substrates that allow
for detection and quantification of the derived GS-conjugates by
chromatography, e.g. high-performance liquid chromatography
(HPLC). Non-chromophoric substrates are much more difficult to
analyze by HPLC. To have a more widely applicable tool for the
analysis of conjugation and turnover of xenobiotic compounds,
flow infusion electrospray ultrahigh-resolution mass-spectrometry
was tested to detect and quantify GS-conjugates in methanolic
yeast extracts. Ion cyclotron resonance Fourier-transformed mass
spectrometry (ICR-FTMS) offers the advantage of providing a reso-
lution beyond 10,000 mass peaks and mass accuracy below
0.5 ppm (Hertkorn et al., 2008; Tziotis et al., 2011). The ultrahigh
2.3. Conjugation of CDNB and NBD-Cl by Arabidopsis GST family
The Arabidopsis GST family comprises 54 members (Fig. S3), for
a number of which no data as to GSH-conjugating activity are
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associated with expression of AtGSTF4, F8, F10, U5, U8, U17, U19,
U24, U25, and U26, yielding conjugate levels more than 6 times
higher than in the CDNB-exposed quintuple mutant and even high-
er amounts than in the yeast wild type strain. The maximum GS–
DNB level was observed with AtGSTU25, providing approximately
24-fold higher amounts than the GST-deficient mutant. Our results
differ from those of a previous study of Arabidopsis GST members
in Escherichia coli, in which heterologously expressed proteins
were purified prior to in vitro analysis of CDNB conjugating activity
(Dixon et al., 2009). In their study, only GSTF8 and -F9 gave prom-
inent activities among the phi class and the specific activities were
more than 7 and 40 times less than the activity of AtGSTU25,
respectively.
NBD-Cl was used as a second substrate to examine the Arabid-
opsis GSTs. Contrary to the CDNB experiment, expression of the
minor clade members of Arabidopsis resulted in elevated GS-con-
jugate levels. An approximately twofold increase in GS–NBD levels
occurred by expression of AtGSTL3, AtDHAR3, AtGSTT1, and
AtGSTT2 (Fig. 4B). AtGSTZ1 expression significantly increased GST
activity. AtGSTZ1 catalyzes isomerization reactions (Wagner
et al., 2002) and its GSH transferase activity has not been known.
Maximal GS–NBD levels were approximately 5-fold increased
compared to the control line carrying an empty expression cas-
sette. Among the GSTs in this analysis, tau class members, such
as AtGSTU5 and AtGSTU15, were the most active. Among the phi
class, only AtGSTF4 and AtGSTF8 expression yielded a clear, more
than twofold increase of conjugate levels. Taken together, the func-
tional analysis of the Arabidopsis GST family established a conju-
gating activity for most of the plant enzymes at least for one of
the two substrates examined.
2.4. Glutathionylation of the fungicide anilizine by Arabidopsis GSTs
Anilazine was used to combat glume blotch disease of wheat
(Liebich et al., 1999). The halogenated compound has a known
affinity to sulfhydryl groups but the biotransformation and catab-
olism of the fungicide in vivo is only understood rudimentarily. To
test a possible conjugation of anilazine to GSH, yeast lines express-
ing plant GSTs were analyzed for GS-conjugates. Anilazine has a
chromophore suitable for HPLC analysis and methanolic extracts
from yeast grown in the presence of anilazine were examined
(Fig. 5A). The expression of the plant GSTs AtGSTF4 and AtGSTU5
clearly increased two UV-absorbing fractions in comparison to
the quintuple mutant harboring an empty expression cassette.
Both fractions were collected, subjected to mass spectrometric
analysis by ICR-FTMS, and identified as the mono- and diglutath-
ionylated conjugates (Fig. 5B). The mono- and diglutathionylated
anilazine conjugates were quantified in extracts of yeast lines
Fig. 2. Complementation of GST-deficient strains. (A) The reference strain (WT) and
the quadruple mutant
D
gtt1
D
gtt2D
grx1D
grx2 (D
4), the single GST mutant
Dtef4,
and the quintuple mutant
D
tef4
D
gtt1
D
gtt2 grx1D
D
grx2 (M) were analyzed for
growth after 5 days at 30 °C. Dilution of overnight-grown cultures were transferred
onto solidified minimal medium containing glucose as a carbon source (Glc) and
either 60
the spotted yeast suspension is indicated. (B) Galactose-inducible expression of
Arabidopsis GSTs in tef4 and the quintuple mutant labeled and analyzed as above.
lM CDNB or 0.1% EtOH solvent as a control. The optical density (OD600) of
D
expressing the Arabidopsis GST family in the presence of 1 lM fun-
Expression of AtGSTF2, -F8 and -U19 was either repressed by the presence of
sucrose (upper panel, ꢀGal.) or induced by galactose as a carbon source (lower
panel, +Gal.). (C) GST activity of cell-free protein extracts from yeasts as shown in
(B) towards CDNB and NBD-Cl as a second substrate are indicated by black and gray
bars, respectively.
gicide (Fig. 5C). In most cases, the plant GSTs elevated the GS-anil-
azine content by more than a factor of 6 above the level present in
the anilazine-exposed quintuple mutant. All 10 analyzed GSTs of
the phi class enhanced GS-conjugate formation with varying effi-
ciency. Highest GS-conjugate levels were observed for AtGSTF2,
AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF9, and AtGSTF10. In the previ-
ous analysis with CDNB and NBD-Cl (Fig. 2C and 4), AtGSTF2 had
not been significantly active. The presence of AtGSTF2, however,
stimulated the GS-anilazine formation more than 20-fold. The
selective conjugating activity indicates a strong substrate specific-
ity of this phi class GST known to bind a number of endogenous
compounds such as the phytoalexin camalexin (Dixon et al., 2011).
The highest GS-anilazine levels were generated by the tau class
members AtGSTU5 and AtGSTU25 that yielded a 35-and 40-fold in-
crease in GS-conjugates compared to the control, respectively.
Three tau class GSTs had no detectable anilazine-conjugating activ-
ity in our assay, namely AtGSTU4, AtGSTU16, and AtGSTU27. The
available to date. To analyze the GST family in yeast, a total of 42
Arabidopsis GSTs was cloned (Fig. S3, and Supplementary Table 1)
into a galactose-inducible expression vector (Gueldener et al.,
2002). The cloned GSTs encompass six of the seven clades includ-
ing 24 of a total of 28 tau members, 10 out of 13 phi, 2 of 3 lambda,
2 of 3 theta, 1 of 2 zeta, and 3 of 4 the dehydroascorbate reductase
class. The GSTs were expressed in the yeast quintuple mutant chal-
lenged with CDNB to test the functionality of the Arabidopsis en-
zymes (Fig. 4A). The analysis showed
a widespread GSH-
conjugating activity for CDNB throughout the major Arabidopsis
GST subfamilies phi and tau, while the other class members were
either marginally active or inactive. Prominent activities were
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M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
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Fig. 3. Quantification of glutathione conjugates of CDNB. (A) Stable isotope labeling of the cell-free extracts from CDNB-exposed yeast cells by [1H2]- and [2H2]-formaldehyde-
generated dimethyl-modified GS-conjugates (dimethyl-GS–DNB). The extract from AtGSTU19 expressing cells was split in half and differentially labeled, combined, and
analyzed by high resolution mass spectrometry. The perfect match in signal intensity for both dimethyl-GS–DNB isotopologues indicates a robust detection and
quantification by this method. The masses and the difference of the isotopologs are indicated. (B) Quantification of GS–DNB conjugate levels in extracts of various lines (see
Fig. 2C) by ICR-FTMS and using [2H4]-dimethyl-GS–DNB as internal standard. (C) Parallel analysis of the samples of (B) by LC/UV for quantification of GS–DNB levels. The
values for the AtGSTU19 expressing line were set as 100%. In (B) the absolute amount of GS–DNB was 93 16 nmol/109 cells, and in (C) 122 4 nmol/109 cells.
Fig. 4. Conjugation of CDNB and NBD-Cl by members of the Arabidopsis GST family. The GST-deficient yeasts expressing Arabidopsis GSTs were analyzed for
glutathionylation of (A) CDNB (50 lM, 40 min exposure) and (B) NBD-Cl (1 lM, 30 min). The subclasses of GSTs phi, tau, lambda, zeta, DHAR, and theta are marked along with
the numbering of the GST in that clade. The reference strain (WT) and the quintuple mutant (M) served as controls. Error bars represent standard deviations from triplicates.
N.A., not analyzed.
clear GST activity observed for AtGSTF11, AtGSTF14, AtGSTU14 and
AtGSTT2 is interesting. AtGSTF11, AtGSTF14 and AtGSTU14 lack the
conserved serine residue in the active G-site considered critical for
GSH binding. A weak CDNB-conjugating activity had been detected
for AtGSTU14 attributed to two presumed compensatory acting
serine residues (Dixon et al., 2009; Dixon and Edwards, 2010).
AtGSTF14 has also a serine residue (Ser17) near the conserved site
that might account for the observed conjugating activity, but not
AtGSTF11. The assigned activity of AtGSTF11 needs further analy-
sis. In our previous experiment, no significant GST activity of AtG-
STU14 with CDNB was observed (Fig. 4A). In contrast, AtGSTU14
expression increased the anilazine-conjugate level by approxi-
mately 20-fold (Fig. 5C). This finding corroborated the GST activity
of this member and uncovered a clear substrate preference of AtG-
STU14 for the fungicide. AtGSTT2 had been characterized as a GSH-
dependent peroxidase implicated in lipid metabolism (Dixon et al.,
2009). Our analysis thus demonstrated efficient anilazine glutath-
ionylation in the presence of AtGSTT2 indicating a new functional
capacity of this enzyme in the conjugation of
compound.
a xenobiotic
In general, the analysis of the Arabidopsis GST family showed
that individual plant GSTs can rescue the impaired conversion of
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M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
Fig. 5. Anilazine conjugation by Arabidopsis GSTs. (A) Chromatograms of cell-free extracts from anilazine-exposed cells of controls (WT, quintuple mutant M) and AtGSTF4
and -U5 expressing lines analyzed by HPLC. The induction of two UV-absorbing fractions upon 1 M fungicide treatment are marked with 1 and 2. (B) The ICR-FTMS analysis
l
of fractions 1 and 2 collected from the AtGSTU5-expressing line shown in (A) yielded the (di)-GS-conjugate and (mono)-GS-conjugate of anilazine, respectively. The
isotopologic pattern of the GS-conjugates reflects the presence of [13C] and [37Cl]-isotopes and the main mass signal is marked by an arrow. (C) GST-deficient yeasts
expressing the Arabidopsis GST family were analyzed for glutathionylation of anilazine (1 lM, 30 min). The analysis was carried out as shown in Fig. 4.
CDNB, NBD-Cl, and anilazine in the GST-deficient yeast. GST
members with low or no detectable activity in yeast for one sub-
strate frequently showed activity with another substrate.
Prominent examples for such cases were AtGSTF2 and AtGSTF3,
which had no significant effect on conjugate levels of CDNB
and NDB-Cl, but were active against anilazine. Other examples
were AtGSTU4 and AtGSTU15, which had only a clear effect on
GS-NDB conjugation. These results partly differ from data ob-
tained with an in vitro study of Arabidopsis GSTs (Dixon et al.,
2009). Several GSTs, which had low conjugation activity in that
study, showed good activity in our yeast analysis. Yeast as a
eukaryotic expression system seems to provide a suitable envi-
ronment to analyze plant GSTs in intact cells. The yeast system
has the advantage of avoiding potential loss of enzyme activity
that may be caused by disruption of cellular integrity and pro-
tein purification.
2.5. Catabolism of anilazine conjugates
Previous analysis of chlorobimane-conjugate turnover in yeast
(Wünschmann et al., 2010) established that initial steps in the pro-
cessing of conjugates were de-glycination and removal of the glu-
tamic acid residue from the GSH moiety yielding the cysteinyl-
conjugate (Fig. 6A). In order to assess whether the various pre-
dicted intermediates are also formed for the GS-anilazine conju-
gates and whether there are alterations in the catabolite profiles
among yeasts expressing different Arabidopsis GSTs, the conjugate
catabolism via mass spectrometry was examined. Anilazine is chlo-
rogenated at 3 positions that might serve as target sites for the
GST-catalyzed nucleophilic attack of the thiolate anion of GSH.
Hence, conjugates glutathionylated at 1 to 3 positions (see graph-
ical abstract) could be expected. However, only the mono- and
diglutathionylated derivatives and their catabolites were identified
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M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
7
Fig. 6. Catabolism of anilazine conjugates in yeast. (A) Schematic presentation of glutathionylation of xenobiotic compounds (R) by GST and subsequent processing of the
conjugate by the carboxypeptidases CPC and CPY to the glutamylcysteinyl-adduct ( -EC-R) or by the -glutamyltranspeptidase encoded by CIS2 to the cysteinylglycine-
c
c
conjugate (CG-R). Subsequently, the dipeptidyl-conjugates are catabolized to the cysteinyl-adduct (C-R). (B) Possible chemical structures of GS- anilazine metabolites. The
fungicide offers three halogenated sites suitable for GSH-conjugation. Tri-glutathionylated conjugates were not detected. The structures of the mono-glutathionylated
anilazine and its possible catabolites, followed by the structures of di-glutathionylated metabolites are shown. The exact position of glutathionylation has not been resolved
and the depicted structures provide one form of the possible conjugates. Abbreviations: c-glutamyl residue (c-E); cysteinyl (C); glycine (G); anilazine (AZ). (C) Identification
of the different GS-anilazine conjugates and catabolites of yeast exposed for 1 h to 1 lM anilazine and expressing the Arabidopsis GST family. The abundance of the individual
catabolite is semiquantatively presented based on the signal intensity observed by ICR-FTMS ranging from noise level (below 106, white fields), above noise 106 (gray fields),
to strong signals 108 (black) in relative units.
in the cell-free extracts of fungicide-exposed cells (Fig. 6B). The
mass spectrometric analysis allowed for semi-quantitative readout
of the individual catabolites identified by their exact mass/charge
ratios (Fig. 6C). However, it did not provide information as to the
stereochemistry, i.e. at what position of the anilazine molecule
the glutathionylation occurred and which of two GSH adducts is
processed. As expected, the catabolites comprise the mono- and
dicysteinyl conjugates and all combinations of possible intermedi-
ates during GSH processing, in which either the C-terminal glycine
residue has been cleaved off by the carboxypeptidases C and Y
(CPC, CPY) or the glutamyl residue removed by the single yeast
to access the halogen atom of anilazine of the diglutathionylated
anilazine conjugate, or by efficient conversion of the primary con-
jugate to the catabolic pathway. The analysis corroborates the
power of the method to quickly assess the presence of the various
catabolites. The results confirm the postulated pathway for GS-
conjugate turnover (Wünschmann et al., 2010), and show a hith-
erto unknown and widespread capacity of Arabidopsis phi and
tau GSTs to conjugate a fungicide. Among the minor clade GSTs,
AtGSTT2, AtGSTL1, and AtGSTL3 expression resulted in a significant
increase of catabolites of the diglutathionylated anilazine pathway
compared to the quintuple mutant. Mono-glutathionylated conju-
gate levels were marginal in AtGSTL1 and AtGSTL3 lines and did
not differ from the content in the GST-deficient yeast. The results
are compatible with a rapid turnover of the GS-conjugate once
formed, thereby preventing the accumulation of GS-anilazine.
One option for increasing these low levels of GS-conjugates in fu-
ture experiments would be the use of yeast mutants deficient in
CPC, CPY, and CIS2. The triple mutant lacks all enzymes of the initial
degradation pathway (Fig. 6A) and as a consequence GS-conjugates
strongly accumulate (Wünschmann et al., 2010).
c-glutamyltransferase Cis2p (Fig. 6B andC). Expression of 31 phi
and tau GSTs resulted in high levels of monoglutathionylated anil-
azine in 23 cases consistent with the results shown in Fig. 5C. High
levels of the diglutathionylated form was associated with 7 plant
GSTs. Interestingly, the diglutathionylated anilazine adduct was
not found at high levels in the GST wild type yeast but several
catabolites thereof such as the cysteinyl–GS–anilazine and cystei-
nylglycine-glutamyl cysteine-anilazine (Fig. 6C). Both catabolic
intermediates were highly abundant in 11 plant GST-expressing
strains. Other catabolites detected at high levels associated with
plant GST expression were cysteinylglycine–GS–anilazine and
catabolites of monoglutathionylated anilazine such as cysteinyl-
glycine–anilazine. Cysteinyl–anilazine was a dominant catabolite
in the wild type yeast strain and in 19 yeast lines expressing differ-
ent phi and tau GSTs. While the quintuple mutant still had low
catabolite levels of GS-anilazine turnover, no compound of the
diglutathionylated anilazine pathway was observed.
3. Conclusion
In yeast and plants, GSH conjugating activities are provided by a
number of different GSTs. In this study, combined inactivation of
five prominent GST genes of yeast GTT1, GTT2, GRX1, GRX2, and
TEF4 lead to CDNB hypersensitivity. Both in vivo and in vitro, the
quintuple mutant displayed a strongly reduced total GST activity
against three GST substrates tested. Thus, the strain is a suitable
The failure to detect the triple glutathionylated derivative
throughout the analysis might be accounted for by steric hindrance
Please cite this article in press as: Krajewski, M.P., et al. Analysis of Arabidopsis glutathione-transferases in yeast. Phytochemistry (2012), http://dx.doi.org/
10.1016/j.phytochem.2012.04.016

8
M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
eukaryotic host to examine the substrate activities of heterolo-
gously expressed GSTs.
pounds were isolated and identified by high-resolution mass spec-
trometry ICR-FTMS (Apex Qe & Solarix) coupled to 12 Tesla magnet
and Apollo II ionization source (Bruker, Bremen, Germany) in the
negative mode as described (Janz et al., 2010). ICR-FTMS analysis
of GS–DNB yielded signal intensities of 8.1 ꢁ 107 37%,
The analysis of the Arabidopsis GST family in this yeast strain
illustrates the potential of the system for screening GST substrates
and for identifying the most active GSTs for specific substrates. The
study documents the widespread capacity of Arabidopsis phi and
tau class GSTs to conjugate CDNB and the fungicide anilazine.
Members of the minor clades lambda, theta, and zeta and most
of the tau class were active in NDB-Cl conjugation. Of all the differ-
ent Arabidopsis GSTs expressed in this yeast system and examined
for the 3 substrates CDNB, NDB-Cl, and anilazine, not a single one
failed to show a significant conjugation activity for at least one
substrate. This observation highlights both the suitability of the
yeast system to functionally express the plant GSTs and the impor-
tance of testing a broad range of substrates for functional GST
analysis.
Xenobiotic compounds, such as pesticides, are subjected to bio-
transformation after uptake into cells. In a first phase, functional
groups might be incorporated into the foreign compounds by, for
example, hydroxylation recruiting P450-dependent monooxygena-
ses (Hayes et al., 2005; Yuan et al., 2007). The possible biotransfor-
mation of the xenobiotic compound prior to conjugation generates
a major challenge in identifying such modified GS-conjugates by
the help of analytical methods. The analysis of GS-conjugates and
catabolites with high-resolution mass spectrometry combined
with differential isotopic labeling offers a powerful tool in order
to analyze such pathways as exemplified for anilazine turnover.
The system established in this study paves the way to screen for
GST-mediated conjugates of xenobiotic and endogenous com-
pounds. The analysis can be performed on a GST family-wide scale.
The tool allows also dissecting the catabolic pathway of GS-conju-
gates and we will explore the yeast system to reconstruct the GS-
conjugate metabolism of plants.
3.0 ꢁ 108 27%, and 2.1 ꢁ 109 14% for 0.25
lM, 1 lM and 5 lM
GS-conjugate solutions, respectively, which were provided at a
flow rate of 1,2 ll per minute.
4.3. Yeast strains and growth conditions
Yeast strains of the wild type BY4741 (MATa; his3
D
1; leu2
D
0;
tef4
gtt2 (Acc. No. Y01548) of Saccharomyces
cerevisiae were obtained from Euroscarf (Frankfurt, Germany). In
this study, the multiple mutants gtt1 gtt2 grx1 grx2 and
tef4 gtt1 gtt2 grx1 grx2 were generated by stepwise homolo-
met15D0; ura3D0; Acc. No. Y00000) and the isogenic strains D
(Acc. No. Y09430) and
D
D
D
D
D
D
D
D
D
D
gous recombination of each locus (Gueldener et al., 2002). Briefly,
the genes GTT1 (YIR038c) and GRX2 (YDR513w) were deleted in the
genetic background of
(His5+ marker) and pUG73 (Leu2 marker). Subsequently, the multi-
ple mutant gtt1 gtt2 grx2 was transformed with pSH47 (Guel-
Dgtt2 using the disruption cassettes pUG27
D
D
D
dener et al., 2002) for expression of CRE recombinase to remove
the introduced selection markers. In a subsequent step, this GST
triple mutant served as a material to inactivate the GRX1 (YCL035c)
and TEF4 (YKL081w, EFC1), again via the use of pUG73 and pUG72
(URA3 marker) for homologous recombination, respectively. The
selection markers present in the mutants
Dgtt1Dgtt2Dgrx1Dgrx2
and tef4 gtt1 gtt2 grx1 grx2 were removed using pSH62.
D
D
D
D
D
Yeast cells were grown on minimal medium either under inducible
(2% galactose) and non-inducible (2% sucrose) conditions, or in YPD
medium as previously described (Wünschmann et al., 2007, 2010).
Liquid medium was solidified by the addition of 1.5% agar (w/v).
4. Experimental
4.1. Chemicals
4.4. Cloning and expression of Arabidopsis GSTs
1-Chloro-2,4-dinitrobenzene,
adiazole, anilazine, and all other chemicals were purchased from
Fluka of highest available purity.
4-chloro-7-nitro-2,1,3-benzox-
GSTs were amplified from cDNA clones obtained by cDNA syn-
thesis after RNA isolation from Arabidopsis cell cultures (Col-0)
according to the supplier (Qiagen). A few cDNA clones were pro-
vided by RIKEN (Japan) and ABRC Stockcenter (Norwich, United
Kingdom). The 42 different GST genes were cloned via the unique
BamHI/SalI, BamHI/XhoI or EcoRI/SalI restriction sites into the low
copy pSH62 expression vector by replacing the CRE-recombinase
gene (Gueldener et al., 2002). The nucleotide sequences of the
cloned GSTs were determined to verify correctness according to
the TAIR database (list of genes and primers, Table S1, Supplemen-
tary data). The empty vector control lacks the CRE-recombinase.
4.2. Synthesis and analysis of conjugates
Standards of glutathionylated 1-chloro-2,4-dinitrobenzene, 4-
chloro-7-nitrobenzo-2-oxa-1,3-diazole, and anilazine were gener-
ated with equine liver GST (Sigma, Steinheim, Germany). Aqueous
and degassed solutions (20 mM Tris–HCl, pH 7.8) of 0.1 mM xeno-
biotic compound were incubated with 1 mM GSH and 40 mU/ml of
equine GST for 0.5–3 h at 30 °C. Samples were analyzed by high
performance liquid chromatography (Prontosil 120–3 C18;
100 mm ꢁ 2 mm, 3
lm, Bischoff Chromatography, Leonberg, Ger-
4.5. GST assays and Western blot analysis
many). The conjugates were detected by UV absorption as indi-
cated. For analysis of CDNB, NBD-Cl, and anilazine, a gradient by
mixing solvent A (10% MeOH/0.1% HCO2H, v/v) with solvent B
(MeOH) was employed to elute the GS-conjugates. CDNB–GS-con-
jugate (k = 340 nm): 00–10: 25% B, 10–80: gradient from 25% to 60% B,
80–100: 60% B, 100–110: gradient 60% to 80% B, 110–120: 80% B, 120–
140: gradient 80% to 25% B, 140–150; 25% B. NBD–GS-conjugate
(k = 419 nm): 00–50: 0% B, 50–80: gradient 0%–30% B, 80–110: 30% B,
110–130: gradient 30%0–40% B, 130–160: gradient from 40% to 75%
B, 160–18.50: gradient from 75% to 0% B, 18.50–200: 0% B. Anil-
azine–GS-conjugates (k = 254/285 nm):00–20: 25% B, 20–50: gradient
from 25% to 50% B, 50–100: gradient 50%–75% B, 100–11.50: 75% B,
11.50–130: gradient from 75% to 25% B, 130–150: 25% B. The com-
The method for isolation of yeast cell-free extracts was per-
formed as described (Wünschmann et al., 2010). Conditions for
the in vitro analysis are according to Collinson and Grant (2003),
however, a minimum of 300 lg protein of crude yeast extracts
was required per sample (0.2 ml) to yield a signal ratio above noise.
Non-enzymatic conjugation rates of GSH with CDNB, NBD-Cl and
anilazine were 0.44 0.2, 0.87 0.2 and 0.15 0.1 pkat/ml, respec-
tively. The values of in vitro GST activities presented were cor-
rected for the non-enzymatic rates. For Western blot analysis, the
GST was N-terminally fused to GFP and immunodetected by using
a GFP-antibody (Mouse-anti-a-GFP, Santa-Cruz, USA) essentially as
described (Wünschmann et al., 2010).
Please cite this article in press as: Krajewski, M.P., et al. Analysis of Arabidopsis glutathione-transferases in yeast. Phytochemistry (2012), http://dx.doi.org/
10.1016/j.phytochem.2012.04.016

M.P. Krajewski et al. / Phytochemistry xxx (2012) xxx–xxx
9
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of phytochelatin synthase. Plant Physiol. 153, 159–169.
4.6. Exposure of yeast to xenobiotics
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Edwards, R., 2008. Catabolism of glutathione conjugates in Arabidopsis
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roles for plant glutathione transferases in xenobiotic detoxification. Drug
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Deridder, B.P., Dixon, D.P., Beussman, D.J., Edwards, R., Goldsbrough, P.B., 2002.
Induction of glutathione S-transferases in Arabidopsis by herbicide safeners.
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Deridder, B.P., Goldsbrough, P.B., 2006. Organ-specific expression of glutathione S-
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For growth assays, a single colony of the yeast strains was inoc-
ulated in 5 ml pre-cultures in minimal media for 8 h in a thermo-
shaker (200 rpm) at 30 °C. Subsequently, the cultures were used to
inoculate 10 ml at a final OD600 = 0.05 and incubated overnight for
16 h at 30 °C. The cell cultures were subsequently diluted with
minimal medium to an OD600 equaling 2.0, 1.0 and 0.5, respec-
tively. A volume of 20
ll of each dilution was spotted onto plates
containing 0.1% EtOH (v/v) as a solvent control or 60
lM CDNB,
and incubated for 5 days at 30 °C. Heterologous expression of the
Arabidopsis GSTs was performed using the shuttle vector pSH62
providing
expression
a
HIS-selectable marker and
cassette. Transformed
grx1 grx2 mutant strains with AtGSTF2, -F8
a
galactose-inducible
D
tef4 and
D
tef4D
gtt1
D
gtt2D
D
and -U19 clones were spotted and cultivated under inductive or
non-inducing conditions. For GS-conjugate analysis of metabolites,
yeast cultures were treated in midlog phase (OD600 = 0.5–0.7) with
Dixon, D.P., Edwards, R., 2009. Selective binding of glutathione conjugates of fatty
acid derivatives by plant glutathione transferases. J. Biol. Chem. 284, 21249–
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transferases in flavonoid metabolism in plants as identified by ligand fishing. J.
Biol. Chem. 285, 36322–36329.
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subcellular localization of members of the Arabidopsis glutathione transferase
superfamily. J. Exp. Bot. 60, 1207–1218.
Dixon, D.P., Sellars, J.D., Edwards, R., 2011. The Arabidopsis phi class glutathione
transferase AtGSTF2: binding and regulation by biologically active heterocyclic
ligands. Biochem. J. 438, 63–70.
1 lM anilazine for 1 h (ICR-FTMS metabolite screen) or 30 min
(HPLC quantification). Cells were harvested, disrupted with glass
beads (Retsch Mill, Haan, Germany) and extracted twice with
MeOH, 0.1% HCO2H and MeOH-H2O containing 0.1% HCO2H (1:1,
v/v), 0.1% formic acid. Supernatants were combined and a 1:25
dilution was analyzed by ICR-FTMS.
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plant secondary metabolism. Phytochemistry 71, 338–350.
Edwards, R., Dixon, D.P., 2005. Plant glutathione transferases. Methods Enzymol.
401, 169–186.
Garcera, A., Barreto, L., Piedrafita, L., Tamarit, J., Herrero, E., 2006. Saccharomyces
cerevisiae cells have three Omega class glutathione S-transferases acting as 1-
Cys thiol transferases. Biochem. J. 398, 187–196.
Geu-Flores, F., Moldrup, M.E., Bottcher, C., Olsen, C.E., Scheel, D., Halkier, B.A., 2011.
Cytosolic gamma-glutamyl peptidases process glutathione conjugates in the
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Grzam, A., Martin, M.N., Hell, R., Meyer, A.J., 2007. Gamma-glutamyl transpeptidase
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Grzam, A., Tennstedt, P., Clemens, S., Hell, R., Meyer, A.J., 2006. Vacuolar
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4.7. Stable isotope analytics
GS-conjugates were dimethylated via reductive amination
using formaldehyde and sodium cyanoborohydride as reductant.
Cell-free extracts were labeled with [2H1]-formaldehyde or its
[2H2]-isotopomer as described previously (Synowsky et al., 2009).
Briefly, the yeast extract or standard conjugates (1 ml) were incu-
bated in the presence of ammonia (0.15% v/v) for 1 h at 25 °C. Sub-
sequently, HCO2H (0.25% v/v) was added to the reaction and the
mixture was loaded onto a C18 cartridge (100 mg Baker Bond™
C18 SPE, Deventer, Holland), washed with deionized H2O (5 ml),
and bound compounds eluted by MeOH (3 ꢁ 5 ml). After evapora-
tion to dryness, the labeled extract was resuspended in MeOH-H2O
(3:1, v/v) and analyzed by ICR-FTMS.
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We are grateful to Jana Wünschmann for her help and expertise
in yeast genetics, and members of the Botany Department for use-
ful suggestions. Thanks to Farhah Assaad for critically reviewing
the manuscript. We thank the Deutsche Forschungsgemeinschaft,
grant DFG EG938/4 for financial support. The provision of Arabid-
opsis GST cDNAs by ABRC stockcenter, Norwich, and RIKEN, Japan,
is gratefully acknowledged.
Appendix A. Supplementary data
Kobayashi, S., Kidou, S., Ejiri, S., 2001. Detection and characterization of glutathione
S-transferase activity in rice EF-1betabeta’gamma and EF-1gamma expressed in
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Liu, J., Brazier-Hicks, M., Edwards, R., 2009. A kinetic model for the metabolism of
the herbicide safener fenclorim in Arabidopsis thaliana. Biophys. Chem. 143, 85–
94.
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.phytochem.2012.04.016.
Ma, X.X., Jiang, Y.L., He, Y.X., Bao, R., Chen, Y., Zhou, C.Z., 2009. Structures of yeast
glutathione-S-transferase Gtt2 reveal a new catalytic type of GST family. EMBO
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Please cite this article in press as: Krajewski, M.P., et al. Analysis of Arabidopsis glutathione-transferases in yeast. Phytochemistry (2012), http://dx.doi.org/
10.1016/j.phytochem.2012.04.016