Báo cáo khoa học: Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana potx

Hydroperoxide reduction by thioredoxin-specific
glutathione peroxidase isoenzymes of Arabidopsis thaliana
Aqib Iqbal
1
, Yukinori Yabuta
2
, Toru Takeda
2
, Yoshihisa Nakano
1
and Shigeru Shigeoka
2
1 Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Japan
2 Department of Advanced Bioscience, Kinki University, Nara, Japan
The generation of reactive oxygen species (ROS) is
an inevitable process in living organisms and poses a
hazard when present in high concentrations by irre-
versibly damaging different macromolecules such as
protein, lipid and DNA [1]. Plants, because of their
sessile nature, are at greater risk of alterations in redox
homeostasis resulting in oxidative stress [2]. However,
at moderate concentrations ROS play an important
role in signaling processes as the regulatory mediators.
Thus, many ROS-mediated responses actually protect
cells against oxidative stress and re-establish redox
homeostasis [3].
In mammals, glutathione peroxidase (GPX) iso-
enzymes (EC 1.11.1.9) play a key role in protecting
cells against oxidative damage. At least five GPX iso-
enzymes have been identified in mammals and these
differ with respect to structure, substrate specificity

and tissue distribution [4]. cDNA with homology to
mammalian GPX isoenzymes has been cloned from
Nicotiana sylvestris leaves [5] and since then more have
been isolated from different plant species and photo-
synthetic organisms [6–14].
Recently, we reported that GPX proteins (Gpx-1,
Gpx-2) from Synechocystis PCC 6803 are able to util-
ize NADPH, but not glutathione (GSH), as an elec-
tron donor, and unsaturated fatty acid hydroperoxides
or alkyl hydroperoxides as acceptors [15]. In addition,
it has been reported that yeast GPX isoenzymes can
utilize both GSH and thioredoxin (Trx) as a reducing
agent [16]. This profile was also observed in GPX iso-
enzymes from sunflower, tomato and Chinese cabbage
[11,17]. Thus it is evident that in addition to GSH,
GPX proteins can utilize other physiological substrates
for hydroperoxide reduction.
GSH and Trx from eukaryotic cells are two major
reducing compounds that maintain cellular redox
Keywords
Arabidopsis; glutathione peroxidase;
hydroperoxide; thioredoxin
Correspondence
T. Takeda, Department of Advanced
Bioscience, Faculty of Agriculture,
Kinki University, 3327-204 Nakamachi,
Nara 631–8505, Japan
Fax: +81 742 43 8976
Tel: +81 742 43 8179
E-mail:

(Received 22 August 2006, revised 18
October 2006, accepted 20 October 2006)
doi:10.1111/j.1742-4658.2006.05548.x
Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs
(AtGPX1–8). Four mature GPX isoenzymes with different subcellular dis-
tributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli
and characterized. Interestingly, these recombinant proteins were able to
reduce H
2
O
2
, cumene hydroperoxide, phosphatidylcholine and linoleic acid
hydroperoxides using thioredoxin but not glutathione or NADPH as an
electron donor. The reduction activities of the recombinant proteins with
H
2
O
2
were 2–7 times higher than those with cumene hydroperoxide. K
m
values for thioredoxin and H
2
O
2
were 2.2–4.0 and 14.0–25.4 lm, respect-
ively. These finding suggest that GPX isoenzymes may function to detoxify
H
2
O
2

and organic hydroperoxides using thioredoxin in vivo and may also
be involved in regulation of the cellular redox homeostasis by maintaining
the thiol ⁄ disulfide or NADPH ⁄ NADP balance.
Abbreviations
ABA, abscisic acid; AtGPX, Arabidopsis thaliana homolog of glutathione peroxidase; GPX, glutathione peroxidase; GSH, glutathione;
Nbs
2
, 5,5¢-dithiobis(2-nitrobenzoic acid); PhGPX, phospholipid hydroperoxide glutathione peroxidase; ROS, reactive oxygen species;
SeCys, selenocysteine; TPx, Trx-dependent peroxidase; Trx, thioredoxin.
FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS 5589
balance and interact with various transducer and effec-
ter molecules to bring about specific responses [18].
GSH not only acts as a redox sensor of environmental
cues, but also forms part of multiple regulatory path-
ways coordinating the expression of defense genes [19].
Trx is a ubiquitous protein that is present in prokaryotes
and eukaryotes, and shows particularly wide diversity in
photosynthetic organisms [20,21]. In higher plants, Trxs
are distributed in the cytoplasm, plasma membrane,
endoplasmic reticulum, nucleus, apoplast, mitochon-
drion and chloroplast [18,22], and are involved in the
regulation of different metabolic processes.
Eight genes with homology to mammalian GPX iso-
enzymes have been identified in Arabidopsis thaliana.
Seven of these, AtGPX1–7, are differently expressed in
various plant tissues. However, cDNAs encoding five
GPX isoenzymes, AtGPX1, -2, -3, -5 and -6, were
detected in the ESTs database and the expression of
five genes was clearly different in response to different
abiotic stresses and plant hormones [23]. Two proteo-

mic studies have identified AtGPX1 which is located in
the thylakoid membrane of chloroplast [24,25]. In
addition, AtGPX3 green fluorescent fusion protein has
been localized in the cytoplasm [26]. Based on the
deduced amino acid sequences of the other cDNAs, it
seems likely that AtGPX2, -5 and -6 are putatively dis-
tributed in the cytosol, endoplasmic reticulum and
mitochondria ⁄ cytosol, respectively [23]. GPX proteins
localized in the cytosol have been previously character-
ized only from plants and photosynthetic organisms;
thus there is no information available about the enzy-
matic properties of the isoenzymes localized in other
subcellular compartments. In this study, large amounts
of four types of recombinant GPX proteins, AtGPX1,
-2, -5 and -6 with different subcellular distribution
were obtained. It was found that the recombinant pro-
teins can reduce H
2
O
2
, cumene hydroperoxide, phos-
phatidyl choline and linoleic acid hydroperoxides using
Trx as an electron donor, but not GSH or NADPH.
Their physiological roles in plants is also discussed.
Results
Expression of proteins of AtGPX1, -2, -5 and -6
and AtTrx h2 and h3 in E. coli
Chimeric primers were designed to amplify AtGPX1,
-2, -5 and -6 fragments comprising the amino acid
residues encoding the mature proteins without transit

peptides that target them to different subcellular
compartments. Each recombinant protein was induced
in E. coli BL21 (DE3) pLysS cells in an exponentially
growing culture according to the procedure described
in the Experimental procedures. SDS ⁄ PAGE analysis
of the soluble proteins from the host cells showed that
the proteins are successfully expressed using this sys-
tem, resulting in the presence of a prominent band at
 22.5 kDa, corresponding to the expected molecular
mass of each AtGPX isoenzyme tagged with the histi-
dine residues (Fig. 1A). The recombinant proteins pro-
duced were 20–25% of the total soluble proteins.
Recombinant AtGPX1, -2, -5 and -6 proteins of near
electrophoretic homogeneity were obtained using a
HiTrap
TM
chelating HP column. Recombinant thio-
redoxin h2 (17.0 kDa) and h3 (15.5 kDa) were also
purified using the same procedure (Fig. 1B). The pro-
teins of recombinant AtGPX and Trx were used for
the enzymatic analysis.
A B
Fig. 1. SDS ⁄ PAGE analysis of the expression of Arabidopsis recombinant proteins in E. coli using a pColdII vector system. (A) Recombinant
AtGPX1, -2, -5 and -6 (GenBank
TM
accession numbers NP_180080, NP_180715, NP_191867 and AAK63967, respectively); (B) recombinant
Txr h2 and h3 (GenBank
TM
accession numbers NP_198811 and NP_199112, respectively). The soluble crude extract (10 lg) and each puri-
fied recombinant protein (1 lg) were separated by 15% SDS ⁄ PAGE and stained with Coomassie Brilliant Blue. The sizes of the molecular

mass markers are shown on the left of the panel. Lane 1, empty pColdII vector. i, Total soluble proteins; ii, purified recombinant proteins.
A. thaliana glutathione peroxidases A. Iqbal et al.
5590 FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS
Detection of enzyme activities of GPX
isoenzymes
When the recombinant AtGPX isoenzymes were used
with GSH or NADPH as a reducing agent and H
2
O
2
,
cumene hydroperoxide, unsaturated fatty acids or lipid
hydroperoxides as electron acceptor, no hydroperoxide
reduction activities were detected (Table 1). However,
it was found that AtGPX isoenzymes were able to
reduce H
2
O
2
with an E. coli Trx as the reducing sub-
strate. In addition, these isoenzymes were found to
utilize Trx for the reduction of cumene hydroperoxide,
unsaturated fatty acids hydroperoxides or lipid hydro-
peroxides. The V
max
values for the purified recombinant
AtGPX isoenzymes with cumene hydroperoxide and
Trx were between 60 ± 16.2 and 233 ± 16.7 nmolÆ
min
)1

Æmg
)1
protein. However, AtGPX5 was not able
to reduce cumene hydroperoxide with either Trx or
GSH. The V
max
values for AtGPX isoenzymes with
H
2
O
2
and Trx were approximately two- to sevenfold
higher than those of cumene hydroperoxide. Activities
toward unsaturated fatty acid hydroperoxides and lipid
hydroperoxides were very low compared with those
toward cumene hydroperoxide (Table 1). The reduc-
tion of hydroperoxides with two cytosolic Trx’s, h2
and h3 from Arabidopsis were also measured in an
assay coupled with E. coli Trx-reductase. However, the
AtGPX isoenzymes were not able to reduce hydroper-
oxides with either Trx h2 or h3 (Table 1).
Enzymatic properties of recombinant GPX
isoenzymes
Double reciprocal plots of 1 ⁄ [activity] against 1⁄ [Trx]
for Trx and H
2
O
2
were linear and reproducible for
each AtGPX isoenzyme (data not shown). The V

max
values for AtGPX1, -2, -5 and -6 with a fixed
concentration of H
2
O
2
(0.1 mm) were 423 ± 22.5,
Table 1. Reduction of alkyl hydroperoxide (cumene hydroperoxide), H
2
O
2
, polyunsaturated fatty acids hydroperoxide (PUFAOOH, linoleic
acid) or phosphatidylcholine hydroperoxide (PCOOH) with GSH, NADPH or Trx by AtGPX isoenzymes and comparison with other plant GPXs.
ER, endoplasmic reticulum; mit, mitochondria; ND, not determined.
Species Name and location Substrate
Activity (nmolÆmin
)1
Æmg
)1
protein)
Arabidopsis
Trx h2 Trx h3
GSH NADPH Trx (E. coli)
Arabidopsis GPX1, Plastids CumOOH 0 0 233 ± 16.7 0 0
H
2
O
2
0 0 423 ± 22.5 0 0
PUFAOOH 0 0 35 ± 4.1 ND ND

PCOOH 0 0 0 ND ND
GPX2, Cytosol CumOOH 0 0 60 ± 16.7 0 0
H
2
O
2
0 0 346 ± 33.5 0 0
PUFAOOH 0 0 29 ± 0.5 ND ND
PCOOH 0 0 12 ± 2.9 ND ND
GPX5, ER CumOOH 0 0 0 0 0
H
2
O
2
0 0 409 ± 29.4 0 0
PUFAOOH 0 0 16 ± 3.3 ND ND
PCOOH 0 0 8 ± 1.3 ND ND
GPX6, Cytosol ⁄ mit CumOOH 0 0 65 ± 5.8 0 0
H
2
O
2
0 0 433 ± 64.2 0 0
PUFAOOH 0 0 38 ± 2.8 ND ND
PCOOH 0 0 18 ± 2.8 ND ND
Citrus
a
GPX1, Cytosol PCOOH 40 ND ND ND ND
Tomato
b

GPXle1, Cytosol PUFAOOH 28 ± 0.04 ND 147 ± 1.11 ND ND
AlkylOOH 58 ± 1.3 ND 147 ± 1.34 ND ND
PCOOH – ND 108 ± 0.56 ND ND
H
2
O
2
0 ND 153 ± 1.79 ND ND
Sunflower
b
GPXha1, Cytosol PUFAOOH 42 ± 0.11 ND 147 ± 1.11 ND ND
AlkylOOH 39 ± 0.61 ND 161 ± 0.93 ND ND
PCOOH – ND 127 ± 0.05 ND ND
H
2
O
2
0 ND 147 ± 2.12 ND ND
Yeast
c
GPX2, Cytosol AlkylOOH 295 ND 1000 ND ND
H2O2 270 ND 2600 ND ND
Chinese cabbage
d
PHCC-TPx, Cytosol H
2
O
2
0 ND 17 200 ND ND
CumOOH 0 ND 7600 ND ND

a
[6],
b
[17],
c
[16],
d
[11].
A. Iqbal et al. A. thaliana glutathione peroxidases
FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS 5591
346 ± 33.5, 409 ± 29.4 and 433 ± 64.2 nmolÆmin
)1
Æ
mg
)1
protein. The K
m
values for AtGPX1, -2, -5 and
-6 with a fixed concentration of H
2
O
2
(0.1 mm)
for Trx were 4.0, 2.2, 3.1 and 2.3 lm, respectively.
Thus the K
cat
values for AtGPX1, -2, -5 and -6 for
Trx were 13.4 · 10
)2
, 11.0 · 10

)2
, 13.0 · 10
)2
and
13.7 · 10
)2
Æs
)1
and K
cat
⁄ K
m
values were 3.35 · 10
4
,
5.0 · 10
4
, 4.19 · 10
4
and 5.96 · 10
4
m
)1
Æs
)1
, respect-
ively (Table 2).
V
max
and K

m
values for these isoenzymes for H
2
O
2
using a fixed concentration of 4 lm E. coli Trx were
also calculated. The V
max
values for AtGPX1, -2, -5
and -6 were 262 ± 10.2, 218 ± 8.5, 247 ± 9.8 and
269 ± 9.1 nmolÆmin
)1
Æmg
)1
protein and the K
m
values
were 17.1 ± 0.8, 15.3 ± 1.4, 25.4 ± 1.6 and 14.0 ±
1.2 lm, respectively. Accordingly, the K
cat
values for
AtGPX1, -2, -5 and -6 calculated for H
2
O
2
were
8.30 · 10
)2
, 6.90 · 10
)2

, 7.80 · 10
)2
and 8.5 · 10
)2
Æs
)1
and the K
cat
⁄ K
m
values were 4.9 · 10
3
, 4.5 · 10
3
,
3.1 · 10
3
and 6.1 · 10
3
m
)1
Æs
)1
, respectively (Table 3).
Discussion
Based on amino acid sequence homology, GPX isoen-
zymes from photosynthetic organisms, including higher
plants, are closely related to mammalian GPX4 (phos-
pholipid hydroperoxide GPX; PhGPX). However, these
GPX isoenzymes contained a conserved Cys in their

catalytic site, unlike mammalian PhGPX, which has
a selenocysteine (SeCys) [4]. It has been shown that
replacement of SeCys with a Cys via point mutation in
pig heart GPX resulted in a drastic decrease in enzyme
activity [27]. Furthermore, sequence alignments for
plasma GPX (GPX3) and GPX4 from mammals showed
that the amino acid residues necessary for GSH binding
are not conserved. Consequently, GPX isoenzymes from
photosynthetic organisms were not able to reduce sev-
eral hydroperoxides utilizing GSH. However, even when
they could reduce hydroperoxides with GSH as an elec-
tron donor, they had a very low activity [15,28–30].
Similarly, the non-SeCys PhGPXs recently identified in
mammals showed little GSH-dependent GPX activity
in vitro [31]. These findings suggest that GSH is unlikely
to be the sole physiological electron donor for GPX iso-
enzymes under all circumstances and hence the reduc-
tion activity of the AtGPX isoenzymes toward
hydroperoxides with GSH is lost, which is in accordance
with the results for non-SeCys GPX isoenzymes repor-
ted previously [11,16,27,31].
Interestingly, AtGPX isoenzymes were able to utilize
Trx as a sole electron donor for the reduction of H
2
O
2
or hydroperoxides (Table 1). GPX isoenzymes from
Table 2. Comparison of kinetic characteristics of Arabidopsis, tomato and sunflower GPX isoenzymes towards thioredoxin using fixed con-
centration of H
2

O
2
or t-butyl hydroperoxide (100 lM). Kinetic parameters of AtGPX isoenzymes were calculated using H
2
O
2
(100 lM) and that
of GPXle1 and GPXha2 were calculated with t-butyl hydroperoxide (100 l
M) as the substrates. Data for GPXle1 and GPXha2 were taken
from Herbette et al. [17].
V
max
nmolÆmin
)1
Æmg
)1
protein
K
m
lM
K
cat
s
)1
K
cat
⁄ K
m
M
)1

Æs
)1
AtGPX1 423 ± 22.5 4.0 ± 0.4 13.4 · 10
)2
3.35 · 10
4
AtGPX2 346 ± 33.5 2.2 ± 0.1 11.0 · 10
)2
5.00 · 10
4
AtGPX5 409 ± 29.4 3.1 ± 0.2 13.0 · 10
)2
4.19 · 10
4
AtGPX6 433 ± 64.2 2.3 ± 0.1 13.7 · 10
)2
5.96 · 10
4
GPXle1 263 ± 0.4 2.2 ± 0.3 8.4 · 10
)2
3.8 · 10
4
GPXha2 244 ± 0.4 1.5 ± 0.06 7.8 · 10
)2
5.2 · 10
4
Table 3. Comparison of kinetic characteristics of Arabidopsis, tomato, sunflower and yeast GPX isoenzymes toward H
2
O
2

using fixed con-
centration of thioredoxin (4 l
M). Data for GPXle1 and GPXha2 were taken from Herbette et al. [17], data for yeast GPX2 were taken from
Tanaka et al. [16].
V
max
nmolÆmin
)1
Æmg
)1
protein
K
m
lM
K
cat
s
)1
K
cat
⁄ K
m
M
)1
Æs
)1
AtGPX1 262 ± 10.2 17.1 ± 0.8 8.3 · 10
)2
4.9 · 10
4

AtGPX2 218 ± 8.5 15.3 ± 1.4 6.9 · 10
)2
4.5 · 10
4
AtGPX5 247 ± 9.8 25.4 ± 1.6 7.8 · 10
)2
3.1 · 10
4
AtGPX6 269 ± 9.1 14.0 ± 1.2 8.5 · 10
)2
6.1 · 10
4
GPXle1 154 ± 1.79 13.7 ± 0.02 4.9 · 10
)2
2.2 · 10
4
GPXha2 147 ± 1.34 13.9 ± 0.20 4.7 · 10
)2
3.1 · 10
4
Yeast GPX2 2.60 · 10
3
20.0 9.6 · 10
)2
4.8 · 10
3
A. thaliana glutathione peroxidases A. Iqbal et al.
5592 FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS
several other plants, such as tomato, sunflower and
Chinese cabbage, and microorganisms such as Synecho-

cystis PCC 6803, Saccharomyces cerevisiae and Plasmo-
dium falciparum have also been found to utilize other
physiological electron donors, such as NADPH or Trx,
for the reduction of hydroperoxides [11,15–17].
GPX isoenzymes from photosynthetic organisms
contain three conserved Cys; the third Cys is outside
the classical GPX catalytic domains (Fig. 2). The first
and third Cys residues conserved in yeast GPX2 and
Chinese cabbage Trx-dependent peroxidase (TPx) are
needed to form a disulfide bond within the GPX
monomer in vivo, suggesting that the first Cys func-
tions as a peroxidatic site attacked by hydroperoxides
to produce H
2
O and Cys-SOH which then form an in-
tramolecular disulfide bond with the third Cys [an
atypical 2 Cys peroxiredoxin (Prx)-type reaction]
[11,16]. The process for reduction of the disulfide bond
Fig. 2. Alignment of the amino acid sequence of the GPX proteins. The deduced amino acid sequences aligned for comparison are from
A. thaliana (AtGPX1–8), Helianthus annuus (HaPhGPX), Lycopersicon esculentum (LePhGPX), Chinese cabbage (PHCC-TPx), Setaria italica
(SiPhGPX), Raphanus sativus (RsPhGPX), S. cerevisciae (yeast GPX2) and non-SeCys type GPX from human and mouse. The three con-
served Cys of these GPX isoenzymes are shown by inverted triangles. The GPX conserved regions are shown by a horizontal line below the
amino acid residues and represented by I, II and III. GenBank
TM
accession numbers for AtGPX1, -2, -5 and -6 are shown in Fig. 1. The acces-
sion numbers of other isoenzymes are AtGPX3 (NP_181863), AtGPX4 (NP_566128), AtGPX7 (NP_194915), AtGPX8 (NP_176531), HaPhGPX
(CAA75009), LePhGPX (CAA75054), PHCC-TPx (AF411209), RsPhGPX (AF322903), SiPhGPX (AAS47590), yeast GPX2 (NP_009803), human
NPGPx (BC032788) and mouse NPGPx (BC003228).
A. Iqbal et al. A. thaliana glutathione peroxidases
FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS 5593

in target proteins by the reduced Trx has already been
elucidated [32,33]. The Arabidopsis and other plant
GPX isoenzymes also contained the same Cys arrange-
ment and thus may possess a similar mode of action
for the reduction of hydroperoxide utilizing Trx
(Fig. 2).
K
m
values for both Trx and H
2
O
2
of AtGPX isoen-
zymes were nearly similar to those of GPXs from sev-
eral plants and yeast (Tables 2 and 3). K
m
values for
AtGPXs for Trx were in lm, which were compatible
with Trx levels in vivo and thus within physiological
limits. However, the TPx activities with H
2
O
2
or alkyl
hydroperoxides of Arabidopsis, sunflower and tomato
GPXs were significantly lower than those of yeast
GPX2 and Chinese cabbage TPx (Tables 2 and 3).
There are no significant differences between these
enzymes with respect to the arrangement of the well-
conserved secondary structural elements, that is, the

existence of three Cys residues and deletion of the
region for tetramerization (Fig. 2). Accordingly, it is
necessary to undertake a more detailed investigation to
clarify why Arabidopsis GPX isoenzyme has low TPx
activity with H
2
O
2
or alkyl hydroperoxides.
Tomato and sunflower GPX isoenzymes had
approximately similar reduction activity with H
2
O
2
,
alkyl and lipids hydroperoxide in the presence of Trx.
In contrast, those from Arabidopsis and Chinese cab-
bage, and yeast GPX2 had higher activity with H
2
O
2
than with the other hydroperoxides (Table 1), indica-
ting a gradual evolution toward a more specialized
function of H
2
O
2
reduction [11,16,17]. Even though
the GPX isoenzymes from photosynthetic organisms
and Prx are weakly homologous at the amino acid

level, substrate specificity, reaction mechanism, affinity
and catalytic efficiency toward various hydroperoxides
are very similar [36]. As the three conserved Cys resi-
dues appear to be essential for the thioredoxin-depend-
ent reduction activity [11,16], it is suggested that GPX
isoenzymes from photosynthetic organisms may be
classified as a distinct group of 3-Cys peroxiredoxins
(Fig. 2).
Although plant GPX isoenzymes were able to utilize
E. coli Trx as a substrate in vitro and have been identi-
fied as potential Trx targets in different proteomic
approaches [34], AtGPX isoenzymes were not able to
reduce hydroperoxides with the two cytosolic Trxs h2
and h3 from Arabidopsis [21]. The two mitochondrial
Trxs, poplar PtTrxh2 and Arabidopsis AtTrxo1, could
not serve as electron donors to either the mitochond-
rial PtGPX3 or cytosolic PtGPX1 and -5 of poplar,
whereas the cytosolic Trx isoforms, PtTrxh1 and
PtTrxh3, could reduce them [22]. It has been reported
that various types of Trx isoforms show considerable
differences in structure and electrostatic potentials
around the redox active site [35]. These findings sug-
gest that plant Trxs show functional redundancy and a
high degree of specificity toward target proteins [35].
Although we checked hydroperoxide reduction activity
in only two cytosolic Trx isoforms from Arabidopsis,it
is possible that AtGPX isoenzymes could utilize a par-
ticular Trx with the respective subcellular distribution
or some other unidentified reductant, as reported pre-
viously [22]. Accordingly, it is imperative to identify

the in vivo reductant for each GPX isoenzyme because
the various thiol buffers, including Trx, can affect a
number of redox reactions in the cells.
Thiol-dependent redox regulation is more diverse
in plants than in animals, bacteria or fungi [36]. Thiol–
disulfide exchange reactions, which are rapid and read-
ily reversible, are ideally suited to controlling protein
function via the redox state of structural or catalytic
SH groups [37–39]. The occurrence of thiol-dependent
activities in the GPX isoenzymes suggests that they
may be involved in redox modification and signal
transduction in plant cells. Anti-apoptotic activities
have been reported for mitochondrial PhGPX [40] and
tomato GPX1 [41]. Similarly, it has been reported that
tomato plants overexpressing an eukaryotic selenium-
independent GPX (GPX5) maintained a significantly
higher photosynthesis rate and fructose-1,6-bisphos-
phatase activity under chilling stress, because of the
sustenance of the cellular redox homeostasis [42]. Fur-
thermore, plant GPX isoenzymes are induced to
remarkable levels by various stress conditions surmi-
sing involvement in defense [6,23,43]. Overexpression
of Chlamydomonas GPX in tobacco plants either in
the cytosol (TcGPX) or chloroplasts (TpGPX) also
resulted in the maintenance of a higher photosynthetic
capacity and increased tolerance to various abiotic
stresses [44]. Recently, it has been reported that the
atgpx3 mutation in Arabidopsis disrupted abscisic acid
(ABA) activation of calcium channels and the expres-
sion of ABA and stress-responsive genes [26]. GPXs

may thus work in tandem with peroxiredoxins, the
other antioxidant enzymes utilizing Trx, to detoxify
H
2
O
2
and organic hydroperoxides and also be involved
in the regulation of the redox homeostasis by main-
taining the thiol ⁄ disulfide or NADPH ⁄ NADP balance.
Experimental methods
Plant materials and chemicals
NADPH, H
2
O
2
, GSH, E. coli Trx and Trx reductase were
purchased from Sigma Aldrich (St Louis, MO). Hydroper-
oxides of unsaturated fatty acids were prepared by oxygen-
A. thaliana glutathione peroxidases A. Iqbal et al.
5594 FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS
ation with 2,2¢-azobis (2-amidinopropane) dehydrochloride
and purified using RP-HPLC in the mobile phase as repor-
ted previously [45]. The amount of hydroperoxide was
calculated from the UV absorption at 234 nm (e ¼
27400 m
)1
Æcm
)1
). Arabidopsis plants (ecotype Columbia)
were grown for 15 days in MS medium containing 3%

sucrose under a white light of  75–100 lEÆm
)2
Æs
)1
at
25 °C with a 16 h light period.
Cloning of AtGPX isoenzymes
Total RNA was extracted from 15-day-old Arabidopsis seed-
lings as described previously [46]. First-strand cDNA was
synthesized using oligo(dT
20
) primer and RevTra Ace
(reverse transcriptase; Toyobo, Osaka, Japan) with 5 lg total
RNA as a template according to the manufacturer’s proto-
col. Full-length cDNAs encoding mature AtGPX1 (CAT
ATGGCTGCAGAAAAAACCG ⁄ GGATCCTTTAAGCG
GCAAGCAA), AtGPX2 (CATATGGCGGATGAATC
TCCAA ⁄ GGATCCTCCTCCTCCTGGTGAT), AtGPX5
(CATATGGGTGCTTCATCATCAT ⁄ GG ATCCCTGTG
CGTGTTCACAA) and AtGPX6 (CATATGGC AGC
AGAGAAGTCTG ⁄ GGATCCAGTTATCCAGATTGAA)
without transit peptides or Trx h2 (CATATGGGA
GGAGCTTTATC ⁄ GGATCCGCGTTAACAATGCTCA)
and Trx h3 (CATATGGAAGAGAAGCCGCA ⁄ GGATCC
AAATCAAGCAGCAGC) proteins were amplified by RT-
PCR with chimeric primers (in parenthesis) introducing the
NdeI ⁄ BamHI sites into the respective forward and reverse
primers (bold sequences). The amplification conditions were
30 cycles at 94, 58 and 72 °C for 1 min each and final exten-
sion at 72 °C for 10 min. The PCR product was gel purified

using GFX
TM
PCR Gel Band Purification kit (GE Health-
care, Chalfont St Giles, UK), ligated into pT7 vector (Nov-
agen, EMD Bioscience Inc., La Jolla, CA) and sequenced
using the dideoxy chain terminator method with an automa-
tic DNA sequencer (ABI PRISM
TM
310; Applied Biosys-
tems). The NdeI ⁄ BamHI fragment was obtained and
subsequently ligated into pColdII vector (Takara, Kyoto,
Japan) giving an in-frame fusion with His
6
tag. After con-
firming sequence using the same procedure mentioned above,
E. coli BL21 (DE3) pLysS (Promega Corp., Madison, WI)
cells were transformed and used for the protein expression.
Heterologous expression and purification of
A. thaliana GPX1, -2, -5, -6 and AtTrx h2 and h3
Escherichia coli BL21 (DE3) pLysS cells containing the pos-
itive clones for AtGPX1, -2, -5 and -6, AtTrx h2 and h3
were grown in 50 mL LB medium, containing 50 lgÆmL
)1
of ampicillin and 34 lgÆmL
)1
of chloramphenicol at 37 °C
until the D
600
of the culture is between 0.4 and 0.6. Cells
were kept at 15 °C for 30 min without shaking and the

fusion protein expression was induced by the addition of
0.4 mm isopropyl-thio-b-d-galactoside. Cells were further
grown for 24 h at 15 °C with shaking, collected by centrifu-
gation at 3000 g for 10 min with a himac CR21 centrifuge
(Hitachi, Tokyo, Japan; rotor type R20A2), resuspended in
100 mm Tris ⁄ HCl buffer, pH 8.0, and disrupted by sonica-
tion at 10 kHz for a total of 2 min (five intervals of 40 s
each). Soluble proteins were collected by centrifugation at
15 000 g for 15 min at 4 °C (himac CR21 centrifuge, rotor
type R20A2) and purified using a HiTrap
TM
chelating HP
column (Amersham Biosciences, Uppsala, Sweden) accord-
ing to the manufacturer’s protocol. Briefly, the column was
washed with 10 mL of distilled water, charged with 0.5 mL
of 0.1 m NiCl
2
and washed again with 5 mL of distilled
water. After preparation, the column was equilibrated with
10 vol of binding buffer (20 mm Tris ⁄ HCl, pH 8.0, 0.5 m
NaCl, 5 mm imidazole and 1 mm 2-mercaptoethanol). The
sample was applied using a syringe and the column was
washed with 10 vol of binding buffer containing 20 mm im-
idazole. The recombinant protein was eluted with 5–10 vol
of the binding buffer containing 500 mm imidazole, and
collected in a 1-mL fraction. Protein content was deter-
mined according to the method of Bradford [47]. The
reduced forms of recombinant Trxs were confirmed by
determining the reduction of 5,5¢-dithiobis(2-nitrobenzoic
acid) (Nbs

2
) in the presence of E. coli Trx reductase. One
millilitre of assay mixture contained 100 mm Tris ⁄ HCl,
pH 7.5, 5 mm EDTA, 0.2 mm NADPH, 0.6 mm Nbs
2
and the recombinant proteins. The reaction was started
by the addition of 0.5 U E. coli Trx reductase and the
reduction of Nbs
2
was monitored at 412 nm [e
412
(Nbs
2
) ¼
13 600 m
)1
Æcm
)1
] in a spectrophotometer (data not shown)
[48].
Enzymatic assays
GPX activity with GSH and H
2
O
2
or hydroperoxides was
assayed spectrophotometrically at 340 nm by the decrease
in the absorbance due to the conversion of NADPH in
the presence of GSH reductase, which catalyzes the reduc-
tion of oxidized GSH formed by GPX [14]. The reaction

mixture contained 100 mm Tris ⁄ HCl, pH 7.5, 1 mm GSH,
0.2 mm NADPH, 0.1 mm H
2
O
2
or hydroperoxides, 5 mm
EDTA, 1 U GSH reductase and the enzyme in a total vol-
ume of 0.5 mL. Reduction of hydroperoxides was meas-
ured in the same assay mixture, except that H
2
O
2
was
replaced by 0.1 mm cumene, unsaturated fatty acid or
lipid hydroperoxides. Enzymes activities were calculated
using an e-value of 6220 m
)1
Æcm
)1
. Trx- and NADPH-
dependent reduction activities were measured in a manner
similar to that described previously [15,17]. The GSH and
GSH reductase in the reaction mixture mentioned above
were replaced with E. coli Trx and Trx reductase
(0.3 UÆmL
)1
)orArabidopsis Trx h2 and h3 (4 lm).
NADPH-dependent Trx peroxidase activity was measured
in the same way as the GSH-dependent peroxidase activity
mentioned above.

A. Iqbal et al. A. thaliana glutathione peroxidases
FEBS Journal 273 (2006) 5589–5597 ª 2006 The Authors Journal compilation ª 2006 FEBS 5595
Acknowledgements
We thank Miss Aya Muramoto, Mr Takahisa Ogawa
and Mr Noriaki Tanabe for their excellent technical
assistance and expert advice during the course of this
experiment. This work was supported by CREST, JST
(S.S: 2005-10) and by the ‘Academic Frontier’ Project
for Private Universities: matching fund subsidy from
MEXT (S.S: 2004-08).
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