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

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).

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.

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

unique characteristics.

In many physiological studies DHA reductase is

regarded as one of the chloroplast enzymes involved in

the protection against oxidative stress. A specific DHA

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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.

HUANG et al. ¡X

Thioredoxin

h2

with both dehydroascorbate reductase and monodehydroascorbate reductase activities

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7