Botanical Studies (2008) 49: 1-7.
*
Corresponding author: E-mail: boyhlin@gate.sinica.edu.tw;
Fax: +886-2-2782-7954; Tel: +886-2-2789-9590 ext. 320.
INTRODUCTION
Ascorbate (AsA) plays an important role in protecting
plant cells against the action of reactive oxygen species
(Dalton et al., 1986; Hou et al., 2000). In plants, peroxide-
scavenging is accomplished through the AsA-glutathione
pathway, a coupled series of redox reactions involving
four enzymes: AsA-specific peroxidase (EC 1.11.1.11),
monodehydroascorbate (MDA) reductase (EC 1.6.5.4),
dehydroascorbate (DHA) reductase (EC 1.8.5.1), and
glutathione reductase (EC 1.6.4.2) (Dalton et al., 1993;
Leonardis et al., 1995). This pathway has been studied
mainly in chloroplasts, in which the possible reactive
oxygen species produced by photosystem I during
photosynthesis might cause serious damage. However, the
AsA-glutathione pathway has also been found in cytosol
(Borraccino et al., 1986; Elia et al., 1992), mitochondria
(Lunde et al., 2006), and peroxisomes (Jimenez et al.,
1997). When AsA functions as an antioxidant in cells,
it is oxidized to MDA free radical, and MDA reductase
catalyzes the reduction of MDA back to AsA with
NAD(P)H (Hossain et al., 1984). MDA was a sensitive
endogenous index of oxidative stress in leaf tissues (Heber
et al., 1996).
Thioredoxins, the ubiquitous small proteins with a
redox active disulfide bridge, are important regulatory
elements in a number of cellular processes (Buchanan,
1991). They all contain a distinct active site, WCGPC,
which is able to reduce the disulfide bridges of target
proteins. Initially described as hydrogen carriers in
ribonucleotide reduction in E. coli, they were found to
serve as electron donors in a variety of cellular redox
reaction (Holmgren, 1985). From genome sequencing
data, a significant diversity of thioredoxin genes
containing five different multigenic families (f, m, x, o
and h) was observed (Mestres-Ortega and Meyer, 1999;
Meyer et al., 2002; Balmer and Buchanan, 2002). The
ferredoxin-thioredoxin system (thioredoxins f and m)
has been proven to regulate several enzymatic activities
associated with photosynthetic CO
2
assimilation in
chloroplasts. Thioredoxin x contains a transit peptide
similar to those required for chloroplast and mitochondria
targeting; however, its function is not clearly defined
(Mestres-Ortega and Meyer, 1999). A new type of plant
mitochondrial thioredoxin o was also shown to regulate
the activities of several mitochondrial proteins by disulfide
bond reduction (Laloi et al., 2001).
Thioredoxin h is generally assumed to be cytosolic,
which was supported by the absence of a transit peptide in
the genes cloned for the isoforms from tobacco (Marty and
Sweet potato storage root thioredoxin h2 with both
dehydroascorbate reductase and monodehydroascorbate
reductase activities
Guan-Jhong HUANG
1
, Hsien-Jung CHEN
2
, Yuan-Shiun CHANG
1
, Te-Ling LU
3
, and Yaw-Huei
LIN
4,
*
1
Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, Taiwan
2
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
3
School of Pharmacy, China Medical University, Taichung 404, Taiwan
4
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan
(Received August 28, 2007; Accepted September 29, 2007)
ABSTRACT.
Recombinant thioredoxin h (Trx h2) overproduced in E. coli (M15) was purified by Ni
2+
-
chelate affinity chromatography as previously reported (Huang et al., 2004a). The molecular mass of Trx h2
was ca. 14 kDa determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis).
It had antioxidant activity (Huang et al., 2004b) and it reduced dehydroascorbate (DHA) in the presence of
glutathione to regenerate ascorbate (AsA). However, without glutathione, Trx h2 had very low DHA reductase
activity. AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radicals. MDA was
also reduced by Trx h2 to AsA in the presence of NADH mimicking the MDA reductase catalyzed reaction.
These data suggest that Trx h2 has both DHA reductase and MDA reductase activities.
Keywords: Dehydroascorbate reductase; Monodehydroascorbate reductase; Sweet potato storage roots;
Thioredoxin h.
BIOChemISTRy
pg_0002
2
Botanical Studies, Vol. 49, 2008
Meyer, 1991; Brugidou et al., 1993), Arabidopsis (Rivera-
Madrid et al., 1993; 1995), Triticum aestivum (Gautier et
al., 1998), germinating wheat seeds (Serrato et al., 2001)
and barley seed proteome (Kenji et al., 2003). Moreover,
the existence of several forms of thioredoxin h detected
in spinach leaves (Florencio et al., 1988), and wheat flour
(Johnson et al., 1987) supports the view that higher plants
possess multiple and divergent thioredoxin genes (Rivera-
Madrid et al., 1995). In this study, we present evidence
to show that the recombination protein, thioredoxin
h2 exhibits both DHA reductase and MDA reductase
activities.
mATeRIALS AND meThODS
Chemicals
Ascorbate, dehydroascorbate, electrophoresis grade
acrylamide and Bis (N, N¡¦-methylenediacrylamide),
TEMED (N, N, N¡¦, N¡¦-tetramethylethylenediamine) and
APS (ammonium persulfate) were from E. Merck Inc.
(Germany). Other chemicals and solvents were purchased
from Sigma Chemical Company (St. Louis, MO). The low
molecular weight kits for electrophoresis were obtained
from Amersham Pharmacia Biotech (Uppsala, Sweden).
expression of thioredoxin h2 in E. coli
Thioredoxin h 2 (Gene Bank accession number:
AY344228; Trx h2) was expressed in E. coli. The coding
sequence was amplified from Trx h2 cDNA using an
oligonucleotide (5¡¦-GAG AG G ATC CAA TGG GAG
GGG CT-3¡¦) with a BamHI site (underlined) at the putative
initial Met redisue, and an oligonucleotide (5¡¦-ATT TGA
AGC TTG ATT GAT GCT-3¡¦) with a HindIII site at the 3¡¦
end. The PCR fragment was subcloned in pGEM T-easy
vector, and the plasmid was then digested with BamHI
and HindIII and subcloned in pQE32 expression vector
(QIAexpress expression system, Qiagen). The resulting
plasmid, termed pQE-Trx h2, was introduced into E.
coli (M15). Cultures of the transformed E. coli (M15)
overexpressed a protein of the expected molecular mass,
which was purified by affinity chromatography in Ni-NTA
columns (Qiagen), according to Huang et al (Huang et al.,
2007).
DhA reductase activity assay
The DHA reductase activity of Trx h2 was assayed
according to the method of Trumper et al. (Trumper et al.,
1994) with some modifications. Ten milligrams DHA were
dissolved in 5.0 mL of 100 mM phosphate buffer with two
pH values (pH 6 and 7). The reaction was carried out at 30
¢XC by adding 100 £gL Trx h2 solution (100 £gg protein) to
0.9 mL DHA solution with or without 4 mM glutathione.
The increase of absorbance at 265 nm was recorded for 5
min. The non-enzymatic reduction of DHA in phosphate
buffer was measured in a separate cuvette at the same
time. A standard curve was plotted using 0.1-50 nmol AsA
(Jung et al., 2002; Washburn and Wells, 1999).
mDA reductase activity assay
The MDA reductase activity of Trx h2 was assayed
according to Hossain et al. (Hossain et al., 1984) by
following the decrease in absorbance at 340 nm due to
NADH oxidation. MDA free radicals were generated by
AsA oxidase (EC 1.10.3.3) in the assay system (Yamazaki
and Pette, 1961). The reaction mixtures contained 50 mM
phosphate buffer (pH 6 and 7, respectively), 0.33 mM
NADH, 3 mM AsA, AsA oxidase (0.9 U), and 200 £gL Trx
h2 solution (200 £gg protein) in a final volume of 1 mL.
Trx h2 solution was replaced with glutathione for controls.
Protein stainings of thioredoxin h2 in 15% SDS-
PAGe gels
Trx h2 were examined by protein staining in 15%
SDS-PAGE (sodium dodecylsulfate-polyacrylamide
gel electrophoresis) gels (Huang et al., 2004c; 2007).
Twenty microliter samples were mixed with 25 £gL
sample buffer containing 60 mM Tris buffer (pH 6.8), 2%
SDS, 25% glycerol and 0.1% bromophenol blue, with
2-mercaptoethanol (2-ME) in a final concentration of 14.4
mM, and heated at 100¢XC for 5 min for protein staining.
Coomassie brilliant blue G-250 was used for protein
staining (Hou et al., 2002). The protein concentration
of the supernatant was determined by the Bradford dye-
binding assay (Bio-Rad, Hercules, CA).
mDA reductase activity staining in 15% SDS-
PAGe gels
Trx h2 were examined for MDA reductase by activity
stainings in 15% SDS-PAGE gels. Diaphorase activity
staining for MDA reductase activity of Trx h2 was
according to the methods of Kaplan and Beutler (Kaplan
and Beutler, 1967) in a 15% SDS-PAGE gel. After
electrophoresis, the gel was washed with 25% isopropanol
in 10 mM Tris buffer (pH 7.9) twice to remove SDS before
activity staining.
Statistical Analysis. Means of triplicate were
calculated. Student¡¦s t test was used for comparison
between two treatments. A difference was considered to be
statistically significant when p < 0.05.
ReSULTS
effect of ph (6 and 7) on dehydroascorbate
reductase activity of thioredoxin h2
To express sweet potato Trx h2 in E. coli, the coding
sequence of Trx h2 was subcloned in a pQE-32 expression
vector so that sweet potato thioredoxin h was produced
with a 6x His-tag at the N-terminus. SDS-PAGE analysis
of crude extracts from transformed E. coli (M15)
showed a high level of a polypeptide with the expected
molecular mass (ca. 14 kDa). The expressed protein
was purified from crude extracts by Ni
2+
-chelate affinity
chromatography, which yielded highly purified His-tagged
thiredoxin h2 (Huang et al., 2004b).
pg_0003
HUANG et al. ¡X
Thioredoxin
h2
with both dehydroascorbate reductase and monodehydroascorbate reductase activities
3
The purified Trx h2 samples were used to examine DHA
reductase activity. Figure 1 shows AsA regeneration (.£D
265 nm) from DHA at both pH 6 and 7 with (A) or without
(B) glutathione. Figure 1A shows that Trx h2 exhibited
DHA reductase activity and was able to reduce DHA
back to AsA. The specific activities of DHA reductase
for Trx h2 in the presence of glutathione were 7.17 and
35.91 nmol AsA produced/min/mg protein at pH 6 and
7, respectively. However, in the absence of glutathione,
very low DHA reductase activities of Trx h2 were found
(Figure 1B): only 0.01 and 0.68 nmol AsA were produced/
min/mg protein at pH 6 and 7, respectively. Trx h2 acts as
a GSH-dependent DHA reductase (Figure 2), and the rate
of reduction was closely proportional to the concentration
of GSH. There was a significant increase in DHA activity
treated with 1, 2, 3 and 4 m£O GSH at pH 7 (p < 0.05). It
was reported that thioredoxin m and thioredoxin f from
spinach chloroplast and thioredoxin from E. coli exhibit
very low DHA reductase activities without glutathione
(Kobrehel et al., 1992).
effect of ph (6 and 7) on monodehydroascorbate
reductase activity of thioredoxin h.
MDA was reduced to AsA in coupling with NADH
oxidation (£GA340 nm) at pH 6 and 7 when Trx h2 was
used as MDA reductase. Trx h2 exhibited MDA reductase
activity at pH 6 and 7 (Figure 3), with higher activity at
pH 6 than pH 7 in our assay system. Trx h2 acts as a GSH-
dependent MDA reductase (Figure 3), and the rate of
reduction was closely proportional to the concentration of
GSH.
Protein and diaphorase activity stainings
in 15% SDS-PAGe gels for detection of
monodehydroascorbate reductase activity of
thioredoxin h2.
MDA reductase activity staining of Trx h2 was done for
Figure 1. Effect of pH (6 and 7) on dehydroascorbate reductase
activity. P urified recombinant protein of thioredoxin h2 was
with (A) or wi thout (B) 4 m M glutathione in the reac tion
mixtures. The reaction was carried out at 30¢XC by adding 100ƒn
£gL thioredoxin h2 solution (100 £gg protein, 100 mM phosphate
buffer, pH 7 and 6) to 0.9 mL DHA solution with or without 4
mM glutathione. Glutathione was used as a control. The increase
of absorbance at 265 nm was recorded for 5 min.
Figure 2. Dependence of dehydroascorbate reductase activity
of thioredoxin h2 on GS H concentration. The reaction was
carried out at 30¢XC by adding 100ƒn £gL thioredoxin h2 solution
(100 £gg protein, 100 mM phosphate buffer, pH 7) to 0.9 mL
DHA solution with different concentrations of glutathione. The
increase of absorbance at 265 nm was recorded for 5 min.
Figu re 3. Effect of pH (6 and 7) on monodehydroascorbate
reductase activity of thioredoxin h2. The reaction mixtures
contained 50 mM phos phate buffer (pH 6 and 7), 0.33 mM
NADH, 3 mM AsA, AsA oxidase (0.9 U), and 200 £gL ƒn
thioredoxin h2 solution (200 £gg protein) in a final volume of 1
mL. Thioredoxin h2 solution was replaced with glutathione for
controls.
pg_0004
4
Botanical Studies, Vol. 49, 2008
equipment to avoid oxidative stress. In plant extracts, a
glutathione-dependent DHA reductase activity which will
recycle DHA to ascorbate has been observed (Hossain
et al., 1984). An increase of DHA reductase activity
and an accumulation of DHA have been frequently
implied as biochemical indicators of oxidative stress in
plant metabolism (Wise, 1995; Hung et al., 2005) but a
characterization of DHA reductase has remained elusive
because of rapid loss of enzyme activity (Hou et al., 1997;
1999).
The thioredoxin system is vital for chloroplast
metabolism because redox control of at least 12 different
enzymes is achieved by the reductive cleavage of
regulatory disulfide bridges in these target enzymes
(Buchanan, 1991). Trx h2 thiol-disulfide interchanges
were found during DHA reduction to regenerate AsA.
Thionin was reported to have intermolecular disulfide
linkages with other proteins (Pinerio et al., 1995). Thiol
groups are central to most redox-sensitive processes in the
cell, and their redox state controls cellular processes such
as growth, differentiation, and apoptosis. Intracellular thiol
homeostasis is maintained by the thioredoxin systems,
which utilize reducing equivalents from NADPH to reduce
both protein and low molecular weight disulfides.
MDA reductase purified from potato was shown to
contain thiol groups in its catalytic sites (Leonardis et al.,
1995). Fernando et al. (1992) found that thioredoxin can
act as a radical scavenger and facilitate the regeneration of
oxidatively damaged proteins and Trx h2 might contribute
to its antioxidant activities against hydroxyl and peroxyl
radicals (Huang et al., 2004b). When AsA is the sole
hydrogen donor, the AsA oxidase can produce MDA
(Yamazaki and Pette, 1961). Nonenzymatic oxidations of
AsA also produce MDA when cells suffer from oxidative
stress (Heber et al., 1996). Taking the above results into
consideration, we constructed a reduction scheme of both
DHA and MDA to AsA catalyzed by the Trx h2 of sweet
potato roots. DHA and MDA can be reduced to regenerate
AsA by Trx h2 in order to prevent oxidative damage to
cytosols of sweet potato storage roots.
Acknowledgment. The authors want to thank the
China Medical University for the financial support
(CMU95-211).
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DISCUSSION
This is the first report showing that Trx h2 displays both
DHA reductase and MDA reductase activities with some
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In many physiological studies DHA reductase is
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Figu re 4. Protein (A) and diaphorase activity (B) stainings in
15% SDS-PAGE gels for detection of monodehydroascorbate
reductase activity of ƒnthioredoxin h2. The experiments were done
twice and a repres entative one is s hown. ¡¥M¡¦ represents the
molecular weight marker and 10 £gg protein was loaded in each
well.
pg_0005
HUANG et al. ¡X
Thioredoxin
h2
with both dehydroascorbate reductase and monodehydroascorbate reductase activities
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HUANG et al. ¡X
Thioredoxin
h2
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7
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