Structural diversity and transcription of class III peroxidases from
Arabidopsis thaliana
Karen G. Welinder
1,2
, Annemarie F. Justesen
1
, Inger V. H. Kjærsga
˚
rd
1
, Rikke B. Jensen
1
,
Søren K. Rasmussen
3
, Hans M. Jespersen
1
and Laurent Duroux
2
1
Department of Protein Chemistry, University of Copenhagen, Denmark;
2
Department of Biotechnology, Aalborg University,
Denmark;
3
Plant Genetics, Risø National Laboratory, Denmark
Understanding peroxidase function in plants is complicated
by the lack of substrate specificity, the high number of genes,
their diversity in structure and our limited knowledge of
peroxidase gene transcription and translation. In the present
study we sequenced expressed sequence tags (ESTs) enco-

provide a common platform for combining knowledge of
peroxidase structure and function relationships obtained in
various species.
Keywords: EST; expression analysis by RT-PCR; peroxi-
dase gene annotation; peroxidase structure; propeptides.
Peroxidase enzymes have challenged chemists and biologists
for more than 70 years and have been used in a great
number of analytical applications [1]. The majority of
peroxidases contain an extractable heme (Fe
3+
protopor-
phyrin IX) center, whereas others contain a cytochrome c
type heme, a selenium center or a vanadium center.
Peroxidases react first with a peroxide to yield highly
oxidizing intermediates with redox potentials up to
1000 mV and thereafter with a variety of organic or
inorganic reducing substrates, which are often oxidized to
form radicals. Peroxidase activity was detected early in
horseradish roots (reviewed in [1]), which is still the major
source of commercial heme peroxidases. In addition,
peroxidases have been isolated from a variety of plant,
animal, fungal and bacterial sources. The bacterium
Escherichia coli expresses a single intracellular heme peroxi-
dase with dual catalase–peroxidase activities [2], a finding
confirmed by its genome sequence [3]. Mitochondrial yeast
cytochrome c peroxidase, chloroplast and cytosol plant
ascorbate peroxidases are rather similar in amino acid
sequence to the bacterial enzymes, and they are collectively
referred to as class I peroxidases [4]. These intracellular
peroxidases appear to function as protective peroxide

Prior to the present study it was known that
horseradish contained at least nine different genes for
class III peroxidases [11]. With this background, it seemed
ideal to study the entire repertory of plant peroxidase
genes in the model plant Arabidopsis thaliana,which
belongs to the same botanical family, taking advantage of
the expressed sequence tag (EST) sequencing programs in
progress [12–14], as well as the results of the Arabidopsis
genomic sequencing project [15]. Here we report the
complete sequencing and mRNA expression analyses of
class III Arabidopsis peroxidase transcripts mostly
obtained from the EST projects, and the predicted
protein structures derived from all 73 Arabidopsis peroxi-
dase genes [16].
MATERIALS AND METHODS
DNA sequencing and gene annotation
BLAST
and Entrez services at the National Center for
Biotechnology Information ()
[17,18] were used to search databases (nonredundant and
dbEST). EST clones were obtained from the Arabidopsis
Biological Resource Center, Ohio State University [12,13],
Genome Systems (Genome Systems Inc, St Louis, USA),
and the Kasuza Institute [14]. Plasmid DNA purification
and sequencing were performed as described previously [19]
and both strands were sequenced.
Genes encoding class III peroxidases in Arabidopsis
were searched for in the Munich Information Center for
Protein Sequences (MIPS) [20] and The Institute for
Genomic Research (TIGR) [21] annotated databases using

substitution
matrices [28] on truncated sequences corresponding to
residues 1–305 of mature HRPC. A first alignment was
done with all sequences to obtain similarity clusters. An
improved alignment was built using the profile alignment
mode of
CLUSTALX
. First, a group of sequences highly
similar to horseradish peroxidase C (HRPC) was aligned
taking into account the secondary structure assignments for
HRPC (default settings in
CLUSTALX
). This group of aligned
sequences was then used as a core onto which clusters of
sequences were added sequentially. Finally, minor manual
adjustments were made to exclude an excessive number of
gaps.
In calculating the pairwise distances, the sequence length
was defined as all matched residues, not counting gaps.
Calculation of pairwise distances and isoelectric points
used only aligned full-length sequences, which were trun-
cated to start at the position corresponding to the
N-terminal pyroglutamate residue of mature HRPC, and
ending at the position corresponding to HRPC residue
N305 [29].
Plant material and RNA purification
A. thaliana seeds, ecotype Columbia were kindly provided
by F. Floto, and cell suspension culture by O. Mattsson,
both at the Department of Plant Physiology, University of
Copenhagen. Plants were grown in plastic containers on

)were
tested to find the optimal conditions at which the primers
were specific. When possible, the primers were designed to
anneal in the 5¢ sequence encoding the signal peptide or in
the 3¢-UTR. Primer sets were tested for specificity in a
PCR, performed on a mixture of cDNA clones encoding
all the peroxidases investigated, including and excluding
the clone encoding the peroxidase for which the primers
were designed. RT-PCR analyses were performed twice
for each peroxidase using two different reverse transcribed
reactions for each time point and organ. As a control of
the quality of the mRNA, RT-PCR was performed with
primers specific for the elongation factor-1a (ef-1a)[19].
The RT-PCR products were analyzed on a 1% (w/v)
agarose gel.
6064 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Digital expression analysis
Transcription profiles were inferred from peroxidase EST
counts, abstracted from TIGR A. thaliana Gene Index [30]
(AtGI release version 6, May 2001) using ÔperoxidaseÕ as a
keyword for the search. Each Tentative Consensus (TC)
accession was verified and assigned to a unique peroxidase
gene [15,20]. For each accession, the number of ESTs per
library was counted. EST libraries (TIGR codes indicated
by ¢#¢) were grouped according to organ: 1, root Columbia,
#5336 [14], root-1 and -2 Col0 Columbia, #2336 and #2337
(Genome Systems, Inc.); 2, seedling hypocotyl CD4-13, -14,
-15 and -16, #NH28, #NH25, #NH26 and #NH27 [12]; 3,
rosette-1, -2 and -3 Col0 Columbia, #2338, #2340 and
#2341 (Genome Systems, Inc.); 4, above-ground organs two

strands and the putative peroxidases called AtP1 to AtP38.
The sequences have been deposited at GenBank or EMBL
databases under the accession numbers listed in Table 1.
Additional sequences of Arabidopsis peroxidase transcripts
were obtained from the literature and our own work,
AtPCa, -Cb, -Ea, -N, -A2, -RC (original names retained,
except for RCIIIa). Recent large-scale Arabidopsis cDNA
sequencing by the Riken Genomic Sciences Center, Yoko-
hama, Japan, and Ceres Inc., Malibu, California, has
currently brought the total of nonredundant peroxidase
transcripts up to 57, AtP39 to AtP51. These 57 transcripts
represent 58 genes, as two identical genes are represented by
AtP11 (Fig. 1; Table 1). The MIPS gene names are used for
the peroxidase genes for which no transcripts have been
observedsofar.
Analysis of the Arabidopsis genome [15] revealed a total
of 73 full-length class III peroxidase genes, two pseudo-
genes, and six fragments spread rather evenly on the five
Arabidopsis chromosomes[16;L.DurouxandK.G.
Welinder, unpublished observations]. Introns were localized
and their phase determined. Results are reported in Table 1,
and intron locations mapped to the protein sequences in
Fig. 1 (highlighted in reverse print). Introns 1, 2 and 3 are
predominant.
The peroxidase-encoding DNA sequences have been
analyzed thoroughly and annotated as in [23]. Table 1
provides an overview of all peroxidase genes and their
introns, the percentage adenine content of 5¢-UTRs,
predicted initiating Met, lengths of preproperoxidases and
ER-signal peptides, and calculated isoelectric points of the

2+
ions are in blue; cysteine residues involved in
disulfide bridges 11–91, 44–49, 97–301 and 177–209 are in yellow; an
invariant ion-pair motif are on a grey background; and putative
N-glycosylated triplets are in green. Unusual residues are highlighted
on a yellow background. Residue 1 (Z) in HRPC is pyroglutamate, a
modification that is likely for all AtPs starting with glutamine
(Q). Predicted N-terminal ER-targeting signals have been removed
(Table 1; Supplementary material, Fig. S1) with alternative predic-
tions for AtP32 and AtP1 indicated in brackets. Some AtPs show
N-terminal extensions relative to HRPC residue 1, referred to as NX
propeptides in the text. C-terminal extensions, CX propeptides, are
shown in italics, and are not thought to be present in mature peroxi-
dase. Intron positions in the corresponding genes are indicated by
residues in reversed print, phase 0 introns between two marked resi-
dues, phase 1 and 2 introns within a single residue. Two genes marked
by (?) are unlikely to form stable proteins. At4g16270 ? encodes a
21-residue insert after intron 1 at HRPC position 48. At4g33870 ? has
an unusual intron 2 at position 122, and an extra intron at position
236, both of which give rise to abnormal sequences (marked in yellow).
Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6065
twosetsofcDNAsforAtP1,AtP1aandAtP1b,withthree
conserved nucleotide mismatches, and two sets for AtP2,
AtP2a and AtP2b, with 19 mismatches and three deletions.
AtP1b and AtP2a are identical in sequence to the genes
At4g21960 and At2g37130, respectively. The nucleotide
differences result in one amino acid substitution within the
6066 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 1. (Continued).
Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6067

Fig. 2 in the same color as in Fig. 1 for reference. The
structures of peanut peroxidase C1 [36], 67% identical to
AtP49, barley grain peroxidase BP1 [37], 56% identical to
AtP4, and recombinant mature AtPN [38], AtPA2 [39,40],
and soybean peroxidase SBP [41], 61% identical to AtPA2
and 60% identical to AtPEa, have also been determined by
X-ray crystallography. All showed the same active site
structure and very similar protein folds, except for BP1 that
is inactive above pH 5, and at pH 5.5, 7.5 and 8.5 has a
distorted loop of 21 residues [37]. This appears to be a
special feature of BP1.
Active site residues of the plant peroxidase superfamily
[4], shown in red in Fig. 1, include the catalytic distal Arg38,
and His42 hydrogen-bonded to Asn70. In addition, the
carbonyl of Pro139 accepts a hydrogen bond from reducing
substrates and thereby becomes a determinant of peroxidase
substrate specificity [39,40]. At the proximal site of the
heme, His170 is coordinated to heme Fe
3+
and hydrogen
bonded to Asp247 [42]. Many active site mutants have been
designed for HRPC with the purpose of studying the
function of the individual side chains (reviewed in [10,43]).
Proximal His and Asp are both invariant in Fig. 1. At the
distal site, the most significant substitutions occur in the
74% identical AtP50 and At5g24070 proteins, where Phe41-
His42 is replaced by Tyr-Ser. The substitution of distal
histidine will result in a different reaction mechanism. The
change of Asn70, found in seven peroxidases, can cause a
significant change in the enzyme kinetics [43].

Length
(o/p)
A%
(o/p)
Start
Met score
Length
(aa) Length Score
At3g49120 AtPCb X71794 123 001 50/54 28/26 0.481 353 30 0.722 8.8
At3g49110 AtPCa AY049304 123 001 49/53 29/28 0.468 354 31 0.798 8.4
At3g32980 AtP16
X98777 123 001 44/48 39/35 0.478 352 29 0.727 7.7
At4g08770 AtP38
AF452387 123 001 11/51 55/49 0.682 346 22 0.899 8.1
At4g08780 123 001 –/52 –/46 0.534 346 22 0.900 8.1
At2g38380 AtPEa
AF452388 123 001 59/62 36/34 0.830 349 29 0.629 6.0
At2g38390 AtP34 AF452385 123 001 45/49 33/35 0.844 349 29 0.655 8.7
At5g06730 AtP29
Y11794 123 001 57/66 46/44 0.692 358 31 0.581 4.8
At5g06720 AtPA2
X99952 123 001 48/75 42/39 0.757 335 30 0.333 4.8
At5g19880 AtP42 (100990) 123 001 64/64 34/34 0.760 329 23 0.736 5.0
At5g19890 AtPN
X98453 123 001 67/69 45/45 0.647 321 21 0.978 6.4
At5g58390 AtP44 (124846) 12- 00- 81/83 43/42 0.293 316 19 0.957 9.9
At5g58400 12- 00- –/63 –/56 0.516 325 28 0.732 9.6
At5g05340 AtP49 AY065270 123 001 56/59 38/36 0.817 324 21 0.525 8.9
At1g14540 AtP46 AI996783
a

a
12- 00- –/36 –/47 0.542 336 24 0.736 5.3
At2g22420 AtP25
Y11790 12- 00- 70/92 49/48 0.739 329 20 0.572 5.0
At1g49570 AtP5
X98809 123 001 33/54 33/35 0.706 344 21 0.464 5.6
At1g68850 AtP23
Y11789 123 001 56/74 43/39 0.613 336 20 0.929 5.1
At4g16270? 123 101 –/66 –/42 0.618 21 0.737
At1g71695 AtP4
X98773 12- 00- 54/54 65/65 0.779 358 31 0.515 8.4
At5g42180 AtP17
X99096 123 001 70/74 43/43 0.864 317 22 0.869 9.2
At5g51890 AtP27
Y11792 12- 00- 66/66 44/44 0.886 322 24 0.803 9.4
At4g33420 AtP32
AF451951 123 001 57/57 42/42 0.627 314 25 0.385 5.8
At4g33870? 1n3n 0202 –/79 –/42 0.452 24 0.207
At5g64100 AtP3
X98808 ) 23 ) 01 61/64 51/52 0.860 331 23 0.870 9.1
At5g64110 AtP45 AY065173 ) 23 ) 01 84/89 52/53 0.745 330 24 0.588 6.1
At5g64120 AtP15
X99097 ) 23 ) 01 56/61 45/43 0.740 328 23 0.618 8.2
At5g39580 AtP24
Y11788 ) 23 ) 01 52/83 50/47 0.920 319 22 0.755 8.7
At2g41480 123 001 –/39 –/49
0.415 328 26 0.233 6.6
At1g77100 123 001 –/26 –/27
0.249 319 22 0.990 5.0
At4g25980 1 – 0 – –/63 –/21 0.479

X98928 123 001 40/64 43/41 0.823 329 25 0.846 9.4
At3g49960 AtP21
X98807 123 001 48/78 23/24 0.793 329 25 0.732 9.4
At4g30170 AtP8
X98855 123 001 81/84 38/38 0.650 325 25 0.804 9.4
At2g18980 AtP22
Y08781 123 001 6/23 33/39 0.193 323 23 0.921 9.6
At5g14130 AtP20
X98806 12- 00- 39/97 59/42 0.726 330 30 0.828 4.9
At5g40150 AtP26
Y11791
a
– – –/151 –/21 0.881 328 27 0.909 8.6
At3g28200 AtP41 AY034973 – – 12/15 25/20 0.719 316 19 0.641 9.2
At5g47000 AtP43 AY093131 – – 167/170 26/26
0.782 331 25 0.824 6.8
At4g17690 – – –/99 –/32 0.821 326 20 0.987 8.4
At1g24110 – – –/362 –/39 0.160 326 20 0.453 6.1
At2g34060 AtP51 AY080602
a
12- 00- –/18 –/39 0.355 346 31 0.259 9.1
At3g17070 AtP40 (155041) 1–3 0–1 53/130 42/32 0.568 339 28 0.735 4.8
At1g30870 AtP30 AA067592 1 – 0 – 50/57 54/54 0.729 349 22 0.526 7.7
At2g24800 123 001 –/186 –/30 0.494 329 29 0.793 5.0
At4g31760 AtP48 AI999763
a
123 001 –/365 –/32 0.658 326 26 0.332 4.6
At4g21960 AtP1
X98189 123 001 70/76 39/38 0.905 330 27 0.387 8.1
At2g37130 AtP2

AtP19 39 39 4041 42 404343414336394241414443433765AtP19
AtP11 38 38 3842 39 39404243403541433843424140364847AtP11
AtP12 38 37 3942 41 4141434541354242414544464439545654AtP12
AtP33 39 38 4039 40 424342424336444439404343403750545474AtP33
AtP9 39 40 40 41 42 37 38 38 35 37 33 39 43 37 38 39 37 42 38 40 38 41 37 35 AtP9
AtP10 38 38 3839 40 3938383736333941373637374037403840393974AtP10
AtP21 38 38 3840 41 413739363633394338373939423638383840377283AtP21
AtP8 38 38 39 41 41 38 40 39 38 38 32 41 39 35 37 40 42 43 37 41 40 41 39 37 65 65 64 AtP8
AtP22 40 40 3942 43 4041423938334241383740424336404141413963656484AtP22
AtP20 39 39 3938 39 403840353736424340384341423841424142405755545557AtP20
AtP26 38 37 3838 38 37393936343542364236373740333636383936444345434544AtP26
AtP1 34 34 34 34 36 34 36 36 34 32 31 36 33 34 31 34 33 37 31 32 32 33 35 34 34 33 33 33 34 35 37 AtP1
AtP2 33 33 33 32 33 32 39 38 36 32 33 34 32 34 28 32 32 38 31 32 32 33 34 34 32 32 32 33 35 35 39 57 AtP2
Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6073
site has two negatively charged aspartates and one or two
hydroxy side chains as ligands, except that AtP4 and AtP33
have one glutamate substituting for an aspartate. The Ca
2+
sites of the proximal domains of the AtP50 and At5g24070
proteins have only one negatively charged aspartate, and
might bind a monovalent cation similar to some class I
ascorbate peroxidases [5].
The presence of four disulfide bridges linking HRPC
cysteine residues, 11–91, 44–49, 97–301, and 177–209 are
conserved in class III peroxidases only, and highlighted in
dark yellow color in Fig. 1. The last Cys301 of AtP27 is
changed to a threonine. Therefore, only three disulfide
bridges can exist in this putative peroxidase, presumably
resulting in decreased stability.
A buried salt bridge motif, Asp99-Arg123, is an

to glycosylation, in particular the highly variable loop
between helices F¢ and F¢¢. The eight N-linked glycans of
HRPC have been experimentally verified [46]. O-linked
glycans have never been seen in a plant peroxidase. Triplets
containing X ¼ proline, or followed by a proline residue,
are not glycosylated in peroxidases that we have sequenced
[29,47,48], or in other proteins [49]. The latter statistical
study also found a decrease in glycosylated triplets towards
the C-terminus. The only glycan seen in barley peroxidase
BP1 is found at the Asn-Cys-Ser triplet, residues 300–302,
near the C-terminus of the mature peroxidase, however,
31 amino acids before the C-terminus of the properoxidase
existing during glycan attachment in the ER [48]. This
glycan is most likely present in the similar AtP4 (Fig. 1).
Triplets overlapping with the distal active site residue
corresponding to Asn70 of HRPC, or with Ca
2+
ligands
that are buried in the folded structure, are unlikely to be
glycosylated in a functional peroxidase, and have been
excluded in Fig. 1. Again BP1 provides an experimental
example of a nonglycosylated triplet at position Asn70 [48].
The majority of the peroxidases carry one or two putative
glycans. Seven appear to be nonglycosylated. Therefore, the
high number of glycans found in HRP C, E and A types is
unusual among class III peroxidases. Since glycans are
large, those close to substrate-binding residues (near
Pro139) are likely to affect substrate access and reaction
dynamics, due to a dampening of backbone motion [40].
Half of the putative mature AtPs are likely to start with a

marked. The structural elements are highlighted in the same colors as
in Fig. 1. (By courtesy of A. Henriksen, Carlsberg Research Center,
Denmark.)
6074 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002
only the first 34 amino acids serve as the ER-signal.
Therefore, it appeared that more than 30 amino acids had
been removed by proteolysis. Whether this was an artefact
of tissue culture conditions, or can also occur in planta,is
unknown. In the case of BP1, purified from barley grain, the
mature protein is indeed extended by seven residues at the
N-terminus (Ala-Glu-Pro-Pro-Val-Ala-Pro-) compared to
HRPC [48]. In BP1 the prolines might protect against
proteolysis. Vacuolar targeting of an NX propeptide is
possible. Thus, the protein sporamin contains the vacuolar
targeting sequence Asn-Pro-Ile-Arg-Leu at its N-terminus, a
sequence that if moved to the C-terminus still provided
vacuolar localization [54]. The observation of an NX
propeptide in some peroxidases is novel, and its role has
not been analyzed experimentally.
Some peroxidases show a C-terminal extension, a CX
propeptide, indicated in italics in Fig. 1, which appears to
target for vacuolar import. The function of a CX propeptide
was first discussed for barley grain peroxidase BP1, because
the cDNA clone encoded an additional 22 residues
preceding the stop codon as compared with the amino acid
sequence of the mature protein [48]. The import is associ-
ated with removal of the propeptide since the purified
mature proteins HRPC [46], HRPE5 [50] and barley grain
peroxidase BP1 terminates before these propeptides. Vac-
uolar location of barley grain peroxidase BP2 has been

known anionic horseradish peroxidases HRPA2 and
HRPA1, respectively.
Table 2 shows the pairwise amino acid sequence identities
among the 33 putative mature peroxidases subjected to
RT-PCR expression analysis and illustrates the tremendous
evolutionary divergence among the Arabidopsis peroxidases.
(All pairs are shown in Supplementary Table S3). Only a
few clusters show greater than 70% amino acid sequence
identity, which might be considered as a lower limit for
potentially related biochemical function. Such clusters are
boxed in Fig. 1.
Expression of AtP transcripts
Peroxidases show very limited substrate specificity in
general. Therefore, the biological functions of Arabidopsis
peroxidases were approached by expression studies. The
temporal and spatial expression of mRNA coding for
Arabidopsis peroxidases was analyzed by RT-PCR. This
method can differentiate between similar genes contrary to
the methods based on hybridization. RT-PCR is very
sensitive, however, it is not a quantitative method. Speci-
ficity was obtained using unique primer sets designed to
discriminate known peroxidase genes in combination with
individually optimized annealing conditions as outlined in
the methods section. One primer was preferentially placed in
the poorly conserved 5¢-or3¢-UTR (Supplementary mater-
ial, Table S1). The primer specificity has been checked
against the 73 Arabidopsis peroxidase genes. Only the
primers for AtP11 cannot discriminate between the identical
AtP11.1 and AtP11.2 genes. The PCR fragment sizes were
generally between 450 and 950 bp. Experiments were done

indicates that the level of transcription of peroxidase
genes varies tremendously, from zero to 181 ESTs.
Twenty-nine of the 49 different AtP ESTs have been seen
> 5 times. We consider libraries > 10 000 and EST
counts > 5 as significant semiquantitative indicators of
expression level [59,60], despite the fact that some of the
Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6075
libraries were normalized [14]. Groups 1, root, 4, Ôabove
groundÕ or aerial, 6, green silique, 7, germinating seed, 9,
ÔvariousÕ (dominated by the PRL2 library [12]), and ÔtotalÕ
shown in Table 4 were considered.
In general, the RT-PCR and EST counts agree well
regarding expression. However, the RT-PCR sensitivity was
optimized and very high. This can be seen by comparing the
expression results of whole plant and root in Tables 3 and 4.
Both methods support a constitutive expression of AtP1,
AtP2, AtP9, AtP17, AtP4 and AtPCb. It is noteworthy that
these AtPs represent highly diverse structures (Fig. 1,
Table 2) and properties. Three peroxidase transcripts,
AtP1, AtP16 and AtPCb, have been seen more than 100
times. RT-PCR did not differentiate expression levels
among organs of AtP16, while EST counts indicated a
high, but not exclusive, preference for root. AtP16 and
AtPCb are 91% identical in amino acid sequence (Table 2)
and probably catalyze identical reactions, if they are
expressed in the same cell type.
In root the AtPs accounted for an impressive 2.2% of
ESTs, and 35 different AtPs have already been seen in root
(Table 4). Both RT-PCR and EST counts (Tables 3 and 4)
demonstrated root preference of AtP22, AtP3, AtP11,

AtP17 PRL2 + + + + + + 22
AtP32 PRL2 + + + + + + + 2
AtP15 PRL2 + 35 + + + + 18
AtP24 seedling + + + + + + 7
AtP4 PRL2 6,15,35 + + + + + 27
AtPCb cDNA 6,15,35,59 + + + + + + 101
AtP8 PRL2 + + + + + + + 37
AtP26 PRL2 6,15,35,59 35,59 + + + + 3
AtPCa gene 15,35,59 + + + 2
AtPEa PRL2 6,15,35,59 + + + 62
AtP13 PRL2 + + + + 13
AtP31 silique + + + + + 2
AtP23 PRL2 6,15,35,59 + + 7
AtPN PRL2 6,15,35,59 + + + 6
AtPA2 cell culture + + + 3
AtP18 PRL2 3,6,15,35 + + 1
AtP22 PRL2 6,15,35,59 + + 6
AtP3 PRL2 + + 53
AtP11 PRL2 + + 31
AtP12 PRL2 + + 13
AtP5 PRL2 35 35 8
AtP33 seedling 6,15,35 15,35 1
AtP6 PRL2 3,6,15 35 12
AtP10 PRL2 3,6,15,35 15,35 11
AtP21 PRL2 3,6,15,35 15,35 9
AtP20 PRL2 6,15,35,59 + 3
AtP14 PRL2 No transcipts detected 2
AtP19 PRL2 No transcipts detected 24
6076 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002
There is no simple correlation between gene transcrip-

has no consensus TATA-box. The AtP1 protein has not
been detected despite a 38–39% A content of the 5¢-UTR.
However, studies of recombinant AtP1 showed a very low
specific peroxidase activity and absorption spectra typical of
cytochrome b
5
-type heme coordination, independent of pH
(I.V.H. Kjærsga
˚
rd and K.G. Welinder, unpublished results).
Therefore, AtP1 will stain poorly for peroxidase activity and
might not be detected.
The A-, C-, and E-type peroxidase proteins are abundant
in horseradish root, and traditionally named according to
pI, with A being the most acidic and E the most basic
[1,50,63]. The transcripts for AtPC-type, AtP16 (129 ESTs)
and AtPCb (101 ESTs), are the most abundant of all in
Arabidopsis root, contrary to AtPCa (2 EST), AtP38 (6
ESTs) and At4g08780 (0 ESTs). The gene for AtPCa is
followed in tandem by AtPCb on chromosome 3, whereas
AtP16 is single (gene numbers and names in Table 1;
protein similarity in Fig. 1). The genes for AtP38 and
At4g08780 are also in tandem, however, in inverted
orientation. The two E-types of genes also appear in
tandem, AtPEa (62 ESTs) is followed by AtP34 (7 ESTs).
Botharewellexpressedinroot.AllC-andE-type
transcripts encode a vacuolar targeting CX signal (Fig. 1).
The biological functions of the abundant cationic C- and
E-type peroxidases are unknown.
AtP38 appears to be the ortholog of HRPC2 (91% amino

coding for AtP16, AtP13, and AtP22. Reaction products are compared
to a standard of DNA bands of known sizes (last lane) by agarose gel
electrophoresis. The predicted sizes of the amplification products are
560 bp for AtP16, 420 bp for AtP13, 574 bp for AtP22, and 474 bp for
ef-1a. The larger size products represent amplification of traces of
genomic DNA. Reactions including reverse transcriptase are indicated
by (+), controls without by (–); a control without RNA is negative in
all cases (penultimate lane). Expression of mRNA encoding ef-1a was
used as a control of the quality of the RNA preparation, of the reverse
transcription reaction and for expression reference.
Ó FEBS 2002 73 peroxidases from Arabidopsis (Eur. J. Biochem. 269) 6077
Table 4. Numbers of Arabidopsis EST sequences, AtP sequences and the percentages expressed in different organs. Arabidopsis EST libraries were
grouped according to organ as outlined in Materials and methods, and the occurrence of the different AtPs counted. Counts of peroxidase ESTs.
The TIGR TC accession numbers (release v 6.0) are given. Some (splice-) variant forms are included, which have separate TC numbers (not listed).
(–) indicates no name or TC number. The AtPs are sorted according to apparent abundance in the various tissues.
Name TC number
EST library group
Total
1
Root
2
Hypocotyl
3
Rosette
4
Above ground
5
Flower bud
6
Silique

AtP34 TC115445 5 27
AtP23 TC116168 2 5 7
AtP24 TC122224 4 21 7
AtP27 TC122135 2 3 2 7
AtP22 TC104843 3 1 2 6
AtP41 TC122514 5 1 6
AtP38 TC116195 4 26
AtPN TC104410 4 26
AtP7 TC104292 1 34
AtPA2 TC122775 1 1 13
AtP29 TC104523 2 13
AtP20 TC110922 33
AtP26 TC105068 1 1 1 3
AtPCa TC121267 2 2
AtP32 TC117759 1 12
AtP31 TC123560 1 1 2
AtP14 TC111256 22
AtP46 TC112253 1 1
AtP30 – 11
AtP18 TC116977 11
AtP36 TC122998 1 1
AtP25 TC117275 11
AtP28 TC105819 11
AtP47 TC125273 1 1
AtP39 TC106126 1 1
AtP48 TC112957 11
AtP33 TC125109 11
AtP43 TC124099 1 1
6078 K. G. Welinder et al.(Eur. J. Biochem. 269) Ó FEBS 2002
and only 26 from liquid cultures or plants exposed to saline

In general we found that the Arabidopsis repertory of
class III peroxidase genes accounted very well for peroxid-
ases from other Brassicaceae, whereas Solanaceae, including
potato, tomato and tobacco, Fabaceae, including soybean
and peanut, and Poaceae, including barley and rice, have
additional groups and subgroups of paralogous peroxidase
genes, as illustrated by the few examples mentioned
throughout this paper. A phylogenetic analysis of class III
peroxidases will appear in a separate paper.
ACKNOWLEDGEMENTS
This research was supported by the Danish Agricultural and Veterinary
Research Council (5.23.26.10–1 to KGW and SKR), the Danish
Natural Science Research Council (9502825 to KGW), and the
European Commission (FMRX CT98 02000 to KGW). We are
grateful to Dr Anette Henriksen, Carlsberg Research Center, for
preparing Fig. 2.
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