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Plant Physiology 132:2045-2057 (2003) © 2003 American Society of Plant Biologists Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1Laboratoire Génome et Développement des Plantes, Université de Perpignan, Unité Mixte de Recherche Centre National de la Recherche Scientifique 5096, 52 avenue de Villeneuve, 66860 Perpignan, France (C.B., J.-P.d.S., Y.M.); and Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, Université Louis Pasteur, 12 rue du Général Zimmer, 67084 Strasbourg, France (E.H.M., G.B.)
The Arabidopsis type II peroxiredoxin (PRXII) family is composed of six different genes, five of which are expressed. On the basis of the nucleotide and protein sequences, we were able to define three subgroups among the PRXII family. The first subgroup is composed of AtPRXII-B, -C, and -D, which are highly similar and localized in the cytosol. AtPRXII-B is ubiquitously expressed. More striking is the specific expression of AtPRXII-C and AtPRXII-D localized in pollen. The second subgroup comprises the mitochondrial AtPRXII-F, the corresponding gene of which is expressed constitutively. We show that AtPRXII-E, belonging to the last subgroup, is expressed mostly in reproductive tissues and that its product is addressed to the plastid. By in vitro enzymatic experiments, we demonstrate that glutaredoxin is the electron donor of recombinant AtPRXII-B for peroxidase reaction, but the donors of AtPRXII-E and AtPRXII-F have still to be identified.
Plants generate reactive oxygen species (ROS) such as the superoxide anion, hydrogen peroxide, or the hydroxyl radical as by-products of electron transport chains in chloroplast and mitochondria, photorespiration in the peroxisome, and cell wall oxidases and peroxidases (Dat et al., 2000  ). ROS are
necessary for plants, because they participate in signal transduction
(Karpinski et al., 1999;
Orozco-Cardenas et al., 2001;
Mullineaux and Karpinski,
2002) and play a role in response to pathogen attack
(Wojtaszek, 1997;
Bolwell, 1999;
Dat et al., 2000). However,
biotic or abiotic stress may promote ROS generation and break the redox
balance of the cell. To protect macromolecules such as lipids, proteins, or
nucleic acids from damage caused by ROS, cells contain a large variety of
antioxidant enzymes that include catalase, superoxide dismutase, ascorbate-
and glutathione-dependent peroxidases, and the more recently described
peroxiredoxin (PRX) family. The PRX family was first described as alkyl
hydroperoxide reductase C (Jacobson et
al., 1989) and later as thiol-specific antioxidant in Brewer's
yeast (Saccharomyces cerevisiae) and Escherichia coli
(Chae et al., 1994). In
contrast to other peroxidases, PRX enzymes do not have redox cofactors such as
metal or prosthetic groups. They reduce hydrogen peroxides and alkyl peroxides
to water and alcohols, respectively, by using reducing equivalents. These
reducers are derived specifically from thiol-containing donor molecules such
as thioredoxin (TRX; Chae et al.,
1994; Kwon et al.,
1994; Kang et al.,
1998), glutaredoxin (GRX; Rouhier et al.,
2001,
2002), and the flavin
containing AhpF, a subunit of the Salmonella typhimurium alkyl
hydroperoxide reductase highly similar to TRX reductase
(Jacobson et al., 1989;
Tartaglia et al., 1990).
Members of the PRX family have now been identified in a wide variety of
organisms ranging from archae and eubacteria to eukaryotes, including
vertebrates and plants.
All PRX proteins contain a conserved Cys in their N-terminal part and some
of them possess a second conserved Cys residue. The strictly conserved Cys
residue is oxidized during the mechanism of ROS scavenging
(Schroder and Ponting, 1998
In plants, the four types of PRX have been described
(Baier and Dietz, 1996 In this paper, we describe the most recently discovered PRXII family. In Arabidopsis, it is composed of six members that define three different subgroups. Using RT-PCR and plant reporter gene studies, we present the differential expression patterns of AtPRXII. We also report the detection of AtPRXII proteins in different organs and in three distinct cellular compartments. We discuss the in vitro activity characteristics of AtPRXII and demonstrate that at least one of them is reduced by the GRX system.
Characterization of Arabidopsis Type II PRX Gene and Protein Sequences
Searching for PRX homologs encoded by the nuclear genome of Arabidopsis, we
found 10 potential open reading frames (ORFs). To build a phylogenetic tree,
we used in addition all PRX sequences available for human, Brewer's yeast, and
plants (Fig. 1). On this
phylogenetic tree, built using the DARWIN program
(Gonnet et al., 1992
Homologs of the Brewer's yeast AHP1 protein, the last discovered yeast PRX
(Jeong et al., 1999 AtPRXII-B and AtPRXII-C are present as a tandem repeat. AtPRXII-A is on the same BAC F12P19 at a distance of approximately 10 kb, whereas AtPRXII-D is also on chromosome I, but further away. AtPRXII-E and AtPRXII-F are located on chromosome III. The coding sequences of AtPRXII-A, -B, -C, and -D are interrupted by two introns at the same positions, whereas AtPRXII-E has no intron. The AtPRXII-F coding sequence is interrupted by three introns at positions unrelated to those of AtPRXII-A, -B, -C, and -D. Thus, based on the exon/intron structure, the AtPRXII family can be divided into three subgroups.
Due to an error in the Arabidopsis genome annotation,
AtPRXII-D was previously described as a pseudogene
(Horling et al., 2002
It is not known if the AtPRXII-A protein is correctly predicted because no EST or complete cDNA are available at the present time. Therefore, this protein is not further analyzed in this study. AtPRXII-B, -C, and -D are highly similar, showing between 93% to 99% identity. They share about 60% identity with the amino acid sequence of AtPRXII-E without the N-terminal extension, whereas they only share 50% similarity with the predicted mature form of AtPRXII-F. Thus, the amino acid sequence of AtPRXII allowed us to define three subgroups corresponding to those already defined by the exon/intron structures of the genes. We were able to find rice homologs of each sub-group, suggesting that this organization is common to all higher plants (Fig. 1).
To test biochemical activities of AtPRXII, we produced His-tagged
recombinant proteins using the bacterial-expressing vector pET16b. Because the
amino acid sequences of AtPRXII-B, -C, and -D are so similar, we decided to
restrict our biochemical study to AtPRXII-B. AtPRXII-E was cloned as a
truncated form,
To test the in vitro peroxidase activity of these different AtPRXII, we
used the classical assay of plasmid DNA protection against ROS generated by
the Fenton reaction induced by this metal-catalyzed system. Dithiothreitol
(DTT) and FeCl3 were incubated with 1 µg of plasmid DNA at room
temperature in the presence or absence of 20 µM of recombinant
proteins. After 5 h of incubation, the DNA mixed without PRX was completely
degraded, as was DNA incubated with bovine serum albumin (BSA). In contrast,
DNA incubated with AtPRXII-B or
The reaction mechanism of 2Cys-PRX includes homodimer formation. In
contrast, the activity of type II PRXs analyzed so far is associated with
monomers. We tested the ability of the recombinant AtPRXII to form dimers.
Recombinant proteins were analyzed directly after purification under
nonreducing conditions or were incubated under reducing conditions with 5%
(v/v)
To further investigate the thiol dependence of the type II AtPRX peroxidase
activity, we followed the oxidation of NADPH by the TRX or GRX system. The TRX
system consists of Arabidopsis TRX reductase
(Jacquot et al., 1994
Rabbit antibodies raised against AtPRXII-B and To get insights into the subcellular localizations of the AtPRXII, we performed immunoblots against subcellular fractions of Arabidopsis protoplasts. The purity of each subcellular fraction was tested using antibodies raised against proteins specifically located in the different organelles: the large subunit of the light-harvesting complex II from C. reinhardtii for the chloroplast, TRX h (AtTRXh3) for the cytosol, pyruvate dehydrogenase for the mitochondrion, and catalase for the peroxisome. No contamination was detected in chloroplast and cytosol fractions (Fig. 6, lines C and E). However, a very weak contamination by chloroplasts in the mitochondrial fraction can be observed (Fig. 6, line D). Anti-AtPRXII-E serum reveals a 19-kD band in total protoplast and chloroplast fractions (Fig. 6, line B). A very faint signal is also observed in the mitochondrial fraction, which is most probably due to the weak contamination of this fraction by chloroplasts. The chloroplast localization of AtPRXII-E was confirmed by import experiments of the radiolabeled protein into purified organelles. No import could be detected into potato (Solanum tuberosum) mitochondria, whereas processing of the major 29-kD protein, obtained by in vitro coupled transcription/translation, was observed after its incubation under light with pea chloroplasts (data not shown). A signal of 19 kD resistant to added proteinase K appeared at the same position as the Arabidopsis protein immunodetected in chloroplast extracts. Thus we concluded that AtPRXII-E is only located in the chloroplasts.
Using the serum raised against AtPRXII-B, signals were obtained in protoplast and cytosol fractions but not in those of mitochondria or chloroplasts (Fig. 6, line A). As shown using the serum raised against catalase (Fig. 6, line F), peroxisomes copurify with the mitochondrial fraction, whereas soluble peroxisomal proteins contaminate the cytosolic fraction. Because no signal is detected by the AtPRXII-B serum in the mitochondrial fraction, we conclude that AtPRXII-B and probably AtPRXII-C and -D are cytosolic proteins and are not located in the peroxisomes.
Expression of AtPRXII-B,-C,-D, -E, and -F in plant tissues was analyzed by RT-PCR. For each gene, two primers were designed for transcript amplification. Amplified products were cloned and sequenced to check for the specificity of amplification, except for AtPRXII-F. This demonstrated that each couple of primers is highly specific.
The expression of each gene was analyzed by reverse transcription followed
by radioactive PCR (Fig. 7).
This allowed us to amplify the sequence of interest and the control gene,
actin 2 (GenBank accession no. U41998), which is supposed to be
uniformly expressed in all tissues, in the same tube. Because the
Act2 gene is not expressed in dry seeds, we used primers designed to
amplify EM1 mRNA (Carles et al.,
2002
AtPRXII-B mRNA is detected in all tissues but mostly in reproductive tissues such as buds, flowers, siliques, and seeds. AtPRXII-B is also strongly expressed in callus and cell suspensions. Concerning the AtPRXII-C transcripts, the strongest signals were obtained with buds and flowers, whereas a very faint signal is detected in all other tissues, except in roots. AtPRXII-D mRNA is present exclusively in buds and flowers. A signal corresponding to the AtPRXII-E transcripts was obtained with all tissues, but with a high predominance in buds, siliques, seeds, cell suspensions, and plantlets. AtPRXII-F transcripts are detected in all tissues without any significant difference of intensity (Fig. 7). Thus, the genes encoding PRXs have distinct tissue expression patterns.
To get more insight into the expression patterns of AtPRXII genes,
we analyzed their promoter activities using transgenic Arabidopsis plants
carrying AtPRXII-B to -E
promoter-
Concerning pAtPRXII-B::GUS plants, we did not obtain consistent results with the different lines. One line exhibited blue staining in vascular tissues of stem and roots, whereas another line was stained in blue exclusively in young anthers. Some lines were not stained at all. No line gave a staining pattern coherent with the RT-PCR data. Most probably the 1,465 bp of the promoter did not contain all of the information responsible for in vivo expression of AtPRXII-B gene. Looking at pAtPRXII-E::GUS plants, we observed blue staining in stamen of young flowers, the embryo sac of young seeds, and the albumen of older seeds from green or yellow siliques (Fig. 8B). These results are consistent with the expression pattern we obtained by RT-PCR. However, we did not observe blue staining in 10-d-germinating plantlets, whereas transcripts were detected by RT-PCR (Fig. 7). This may be due to a difference of stability of the AtPRXII-E transcripts in plantlets. The AtPRXII-E is weakly expressed, but the corresponding stable transcripts accumulate to a high level and thus are detectable by RT-PCR.
Because AtPRXII-B, -C, and -D differ only by 0.06 kD from one another
(Table I), we used
two-dimensional electrophoresis to distinguish AtPRXII-B from the AtPRXII-C
and -D proteins, using their predicted pI difference of 0.15. The mature form
of AtPRXII-E predicted by PSORT
(http://psort.nib-b.ac.jp;
Nakai, 2000
We first performed a one-dimensional western blot from an SDS-PAGE loaded with exactly 10 µg of proteins from the different tissues. Using the serum raised against AtPRXII-B, a stronger signal was observed with extracts from buds, flowers, and seeds than with proteins from other tissues (Fig. 9B). Probing the same membrane with the serum raised against AtPRXII-E (Fig. 9D), we observed the presence of AtPRXII-E in an approximately equal quantity in each tissue that was tested, except for roots where a weaker signal was seen. The serum also reacts with proteins of higher Mr that fit exactly the signals showed by anti-AtPRXII-B serum with the same membrane (Fig. 9D).
With all tissue extracts, one or several signals were obtained on two-dimensional gels with the serum raised against AtPRXII-B (Fig. 9A). These different signals may correspond to the AtPRXII-B, -C, and -D proteins or to modified isoforms of AtPRXII-B, -C and/or -D. Using serum raised against AtPRXII-E, two types of signal were obtained with two-dimensional blots (Fig. 9C): strong signals most probably corresponding to the mature AtPRXII-E, and weaker signals (indicated in Fig. 9C by a B) present at exactly the same position as those obtained with serum raised against AtPRXII-B and then corresponding to AtPRXII-B to -D. AtPRXII-E signals were observed with all tissues, leading to the conclusion that AtPRXII-E as well as AtPRXII-B and/or AtPRXII-C and/or AtPRXII-D are present in all plant tissues. The position of the AtPRXII-E spots, compared with the ones of AtPRXII-B to -D, coincides with the molecular mass and pI of the predicted mature AtPRXII-E (Table I; Fig. 9).
Higher Plants Present Three Subgroups of Type II PRX Genes
All eukaryotic PRXs can be classified in four groups, on the basis of their
sequences, as shown in the tree of Figure
1. This classification fits well with our knowledge on the
mechanisms of peroxide reduction which are different in each group. Among the
PRXs, the type II PRXs are less studied, probably because the peroxidase
activity of this family of proteins was only recently identified in yeast
(Jeong et al., 1999
We have analyzed in detail the expression pattern and protein accumulation
of three cytosolic PRXIIs (AtPRXII-B, -C, and -D). AtPRXII-B
is expressed in all tissues, and the protein also accumulates in all tissues.
This pattern may well correspond to the defense function that is frequently
associated with PRXs. Recently, Horling et al.
(2003
More striking is the almost exclusive expression in pollen of the two
AtPRXII-C and -D genes. To our knowledge, it is the
first report of PRX expression localized in flowers. Until now plant PRXs were
mainly characterized in leaves and seeds
(Baier and Dietz, 1996
The gene encoding plastidial PRX AtPRXII-E is expressed constitutively but
is overexpressed in anther tapetum and during seed formation, suggesting that
the function of this protein is not limited to chloroplasts, but is also very
active in other types of plastids. In particular, elaioplats are plastids
present in anther tapetum and contain mainly globuli of neutral esters
(Wu et al., 1997
The cytosolic AtPRXII-B and -C, the plastidial AtPRXII-E, and mitochondrial
AtPRXII-F are able to scavenge ROS in the presence of DTT (Horling et al.,
2002
The plastidial AtPRXII-E cannot be efficiently reduced by any of these
systems. This is not surprising because, on the basis of the Arabidopsis
genome, no gene was found to encode a GRX with a transit peptide. The lack of
activity in the presence of the cytosolic TRX system is more surprising,
because the substrate specificity of TRXs is rather low in vitro. For example,
a C. reinhardtii cytosolic TRX was reported to be an excellent
electron donor for a chloroplastic 2-Cys PRX from Arabidopsis
(Goyer et al., 2002
Plant Materials and Growth Conditions Arabidopsis ecotype Columbia seeds were sterilized by incubating them in 70% (v/v) ethanol for 10 min and then sown on one-half-diluted Murashige and Skoog medium supplemented with 1% (w/v) Glc, 0.5 g L-1 MES, and 0.8% (w/v) agar or directly in soil. Seeds were cold treated for 3 to 4 d at 4°C, then germinated and grown under continuous light at 22°C. Arabidopsis cell suspensions were maintained in Murashige and Skoog medium supplemented with 3% (w/v) Suc, 50 µg L-1 kinetin, and 500 µg L-1 naphthaleneacetic acid and subcultured every 14 d. Calli were grown for 1 month on the same medium solidified with 0.8% (w/v) agar.
The AtPRXII-B promoter region was isolated from genomic
DNA (ecotype Columbia) by PCR walking
(Devic et al., 1997
Total RNA was extracted from frozen cell suspensions, calli, and each plant
organ—siliques and seeds excepted—as described
(Kay et al., 1987
As a control for genomic DNA contamination, PCR was carried out on RNA not treated by reverse transcriptase, with each couple of primers. No amplification was observed. Furthermore, different couples of primers we used are able to amplify genomic DNA leading to the production of DNA fragments of higher Mr (612, 1,390, 770, 1,010, and 1,503 bp for Act2, AtPRXII-B, AtPRXII-D, AtPRXII-E, and AtPRXII-F, respectively). But such an amplification was never observed.
The AtPRXII-B ORF from the ATG codon to the stop codon,
and the AtPRXII-E ORF from the codon coding for the amino
acid A71 to the stop codon were amplified and cloned downstream from, and in
phase with, a sequence coding for His tag in the pET16b plasmid. The
construction with the incomplete reading frame of AtPRXII-E is called
To overproduce AtPRXII in Escherichia coli, BL21(DE3) strains were
cotransformed with pSBET (Schenk et al.,
1995
For one-dimensional electrophoresis, proteins were extracted from different
frozen plant organs according to the protocol described by Carles et al.
(2002
Immunodetections were done as described by Mouaheb et al.
(1998
Arabidopsis protoplasts were prepared from 3- to 4-d-old suspension cell
cultures as previously described (Sakamoto
et al., 2000
Concerning in vitro import into isolated chloroplast and mitochondria, the
precursor proteins were synthesized from the corresponding cDNA clones by
coupled in vitro transcription/translation (TNT kit, Promega, Madison, WI) in
the presence of [35S]Met. Chloroplasts were isolated from leaves of
10-d-old pea seedlings and import assays were carried out as described
previously (Bruce et al.,
1994
Metal-catalyzed oxidation DNA cleavage protection assays were performed as
previously described (Klimowski et al.,
1997
AtPRXII GRX-dependent or TRX-dependent peroxidase activity was assayed as
follows: After 5 min of preincubation, the reaction was initiated by the
addition of 1 mM H2O2 to 0.5 mL of 0.1
M potassium phosphate buffer at pH 7 containing 2 mM
EDTA, 0.5 mM NADPH (Sigma-Aldrich), different concentrations of
AtPRXII, and the GRX system composed of 0.5 unit of glutathione reductase
(Roche Diagnostics), 0.5 mM GSH (Sigma-Aldrich), and 10
µM Arabidopsis GRX AtGRX1, or the TRX system composed of 10
µM Arabidopsis TRX AtTRXh2 and 0.2 µM
Arabidopsis TRX reductase AtNTRB. The consumption of NADPH was followed
spectrophotometrically at 340 nm at 22°C. The TRX insulin disulfide
reduction assays were performed as described
(Laloi et al., 2001
We thank Dr. C. Laloi for the gift of recombinant NTRB, Dr. L. Verdoucq for pET-AtPRXII-B construct, Y. Chartier for technical help, Dr C. de Vitry for the gift of anti-light-harvesting complex II serum, and Dr. J.-P. Reichheld and Dr. R. Cooke for critical reading of the manuscript. Received February 22, 2003; returned for revision March 14, 2003; accepted April 28, 2003.
1 C.B. was the recipient of a fellowship from the Centre National de la Recherche Scientifique and from the Région Languedoc Roussillon.; E.H.M. was the recipient of a fellowship from the French Ministère de la Recherche et de la Technologie.  * Corresponding author; e-mail ymeyer{at}univ-perp.fr; fax 33–4–68–66–84–99.
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12711-12716 This article has been cited by other articles:
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TOP
ABSTRACT
RESULTS
70AtPRXII-E, lacking the 70 N-terminal amino acids
corresponding to the putative transit peptide. 
-mercaptoethanol and then submitted to SDS-PAGE
(
-32P]dCTP and 20
µM for the three other dNTPs. Fifteen cycles were performed to
amplify AtPRXII transcripts. One-third of the reaction was run on a
5% (w/v) acrylamide/bis-acrylamide gel. Radioactive amplifications were
visualized and quantified through PhosphorImager (Storm 640, Molecular
Dynamics, Sunnyvale, CA). The sequences of the primers used for RT-PCR
experiments are indicated in 
