Acta Physiol Plant (2017) 39:57
DOI 10.1007/s11738-017-2356-2
ORIGINAL ARTICLE
Immunological evidence for the presence of peroxiredoxin in pea
leaf peroxisomes and response to oxidative stress conditions
Francisco J. Corpas1 • José R. Pedrajas2 • José M. Palma1 • Raquel Valderrama2
Marta Rodrı́guez-Ruiz1 • Mounira Chaki2 • Luis A. del Rı́o1 • Juan B. Barroso2
Received: 8 August 2016 / Revised: 12 January 2017 / Accepted: 14 January 2017
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2017
Abstract Peroxiredoxins (Prxs) constitute a group of thiol-
specific antioxidant enzymes which are present in bacteria,
yeasts, and in plant and animal cells. Although Prxs are
mainly localized in the cytosol, they are also present in
mitochondria, chloroplasts, and nuclei, but there is no
evidence of the existence of Prxs in plant peroxisomes.
Using soluble fractions (matrices) of peroxisomes purified
from leaves of pea (Pisum sativum L.) plants, the
immunological analysis with affinity-purified IgG against
yeast Prx1 revealed the presence of an immunoreactive
band of about 50 kDa. The apparent molecular mass of the
peroxisomal Prx was not sensitive to oxidizing and
reducing conditions what could be a mechanism of pro-
tection against the oxidative environment existing in per-
oxisomes. Postembedment, EM immunocytochemical
analysis with affinity-purified IgG against yeast Prx1
antibodies, confirmed that this protein was present in the
peroxisomal matrix, mitochondria, and chloroplasts. In pea
Communicated by E. Schleiff.
Electronic supplementary material The online version of this
article (doi:10.1007/s11738-017-2356-2) contains supplementary
material, which is available to authorized users.
& Francisco J. Corpas
javier.corpas@eez.csic.es
1
Group of Antioxidants, Free Radicals and Nitric Oxide in
Biotechnology, Food and Agriculture, Department of
Biochemistry, Cell and Molecular Biology of Plants, Estación
Experimental del Zaidı́n, CSIC, Profesor Albareda 1,
18008 Granada, Spain
2
Group of Biochemistry and Cell Signaling in Nitric Oxide,
Department of Biochemistry and Molecular Biology,
University of Jaén, Campus ‘‘Las Lagunillas’’, 23071 Jaén,
Spain
•
plants grown under oxidative stress conditions, the protein
level of peroxisomal Prx was differentially modulated,
being slightly induced by growth of plants with 50 lM
CdCl2, but being significantly reduced by treatment with
the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The
presence in the matrix of peroxisomes of a protein
immunorelated to Prx of about 50 kDa, which is in the
range of molecular mass of the dimeric form of other Prxs,
opens new questions on the molecular properties of Prxs,
but also on their function in the metabolism of reactive
oxygen and nitrogen species (ROS/RNS) in these plant cell
organelles, where they could be involved in the regulation
of hydrogen peroxide and/or peroxynitrite.
Keywords Peroxiredoxin
Peroxisomes
Pea leaves

Pisum sativum
Oxidative stress
Nitrosative stress
Introduction
Peroxiredoxins (Prxs) constitute a new group of thiol-
specific antioxidant enzymes, also termed thioredoxin-de-
pendent peroxidases, which can be found in different
organisms, including bacteria, yeasts, plants, and mammals
(Dietz 2003, 2011; Wood et al. 2003; Rouhier and Jacquot
2005; Perkins et al. 2015). Prxs function as antioxidant
enzymes by their peroxidase activity (ROOH ? 2e- ?
ROH ? H2O), where hydrogen peroxide (H2O2), perox-
ynitrite (ONOO-), and other organic hydroperoxides
(ROOH) are reduced and detoxified (Wood et al. 2003;
Trujillo et al. 2007). Peroxiredoxins belong to a multige-
netic family, and there are four isoforms in bacteria
(Rouhier and Jacquot 2005), five in yeast (Park et al. 2000),
eight in Arabidopsis (Rouhier and Jacquot 2005; Horling
et al. 2002; Bhatt and Tripathi 2011), and six in mammals
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57 Page 2 of 12
(Wood et al. 2003). Although in plants, Prxs are mainly
localized in the cytosol, and they have also been found in
mitochondria, chloroplasts, and nuclei (Wood et al. 2003;
Rouhier and Jacquot 2005; Bhatt and Tripathi 2011).
All Prxs contain one or two essential cysteines in conserved
sequences. In higher plants, they have been classified into four
main groups: 1-Cys Prx, which has one conserved cysteine;
2-Cys Prx; Prx Q; and Prx type II, which have two catalytic
cysteines (Horling et al. 2002; Dietz et al. 2006; Lamkemeyer
et al. 2006; Bhatt and Tripathi 2011). In Arabidopsis, there are
four Prxs that seem to be localized in chloroplasts (2-Cys Prx
A and B, Prx II E, and Prx Q), one in mitochondria (Prx II F),
and there are three Prxs that have no apparent targeting signals
and their subcellular localization could be either the cytosol or
the nucleus (Dietz et al. 2006; Bhatt and Tripathi 2011). Thus,
among the eight Prxs expressed in Arabidopsis thaliana, the
molecular mass shows a broad range, being 24 kDa for the
1-Cys Prx, 24–29.5 kDa for the three types of 2-Cys Prxs (A,
B, and Prx Q), and 17–63 kDa for the six components (A to F)
of the Prxs type II (Dietz 2003). Interestingly, in mammals, it
has been described an atypical 2-Cys peroxiredoxin desig-
nated as PRDX5 which in human cells can be present in the
cytosol, nucleus, mitochondria, and also in peroxisomes
(Wood et al. 2003). This PRDX5 can reduce alkyl hydroper-
oxides or peroxynitrite (ONOO-) using either cytosolic or
mitochondrial thioredoxins (Knoops et al. 2011).
Peroxisomes are cell organelles characterized by their
single membrane and a prominent oxidative metabolism
(Tabak et al. 1999; del Rı́o et al. 2002; Baker and Graham
2002; Reumann et al. 2007; del Rı́o 2011; Corpas 2015). In
plants, peroxisomes participate in different metabolic
pathways, including photorespiration cycle, fatty acid b-
oxidation, the glyoxylate cycle, and the metabolism of
ureides (Corpas et al. 1997; Igamberdiev and Lea 2002; del
Rı́o et al. 2002), which point out that plant peroxisomes are
metabolically interconnected with other subcellular com-
partments (Igamberdiev and Lea 2002; Minorsky 2002;
Reumann and Weber 2006; Hu et al. 2012; del Rı́o 2013).
During the last decade, different evidences were obtained
showing that in peroxisomes, both reactive oxygen species
(ROS) and reactive nitrogen species (RNS) are generated.
Some of these molecules, such as H2O2 or nitric oxide
(NO), can participate as signal molecules in the commu-
nication between peroxisomes and other subcellular com-
partments (del Rı́o et al. 2006; del Rı́o 2011; Corpas et al.
2013a, 2017; del Rı́o and López-Huertas 2016). In this
sense, recently, it has been shown that plant peroxisomes
can produce ONOO- which could participate in the
mechanism of response to heavy-metal stress (Corpas and
Barroso 2014, 2016).
Although peroxiredoxins have been localized in yeast
and animal peroxisomes (Horiguchi et al. 2001; Knoops
et al. 1999; Walbrecq et al. 2015), no peroxiredoxin has
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Acta Physiol Plant (2017) 39:57
been clearly identified in plant peroxisomes so far. The
goal of this work is to explore the potential existence of this
enzyme in plant peroxisomes. Thus, the occurrence of Prx
was investigated in matrix proteins from peroxisomes
purified from pea (Pisum sativum L.) leaves and the results
indicated the presence of a protein immunorelated with
peroxiredoxins with a molecular mass of about 50 kDa,
whose expression was differentially modulated under
oxidative stress conditions.
Materials and methods
Yeast mitochondrial 1-Cys Prx protein (Prx1):
expression, purification, and production
of polyclonal antibodies
Recombinant mitochondrial Prx1 from S. cerevisiae (ac-
cession number YBL064C) was expressed in Escherichia
coli and obtained according to the procedure previously
described by Pedrajas et al. (2000).
For antibody production, two New Zealand White rab-
bits were immunized with the recombinant Prx1 protein as
described by Harboe and Ingild (1983). Affinity-purified
antibodies were obtained by the use of CNBr-activated
Sepharose-4B column (Amersham Biosciences), where the
recombinant Prx1 protein was coupled. Whole serum was
loaded on this column and washed with 50 mM Tris–HCl
buffer, pH 8.0, containing 2 M NaCl. Then, the antibody
was eluted with 0.58% (v/v) acetic acid containing 0.15 M
NaCl, which was rapidly neutralized with 2 M Trizma.
Finally, the IgG fraction was purified using the Econo-Pac
Serum IgG Purification Column prefilled with DEAE Affi-
Gel blue gel from Bio-Rad according to the manufacturer
recommendations.
Plant material and growth conditions
Plants of Arabidopsis thaliana, lettuce (Lactuca sativa L.,
var ‘‘Maravilla’’) and pepper (Capsicun annuum L., Cali-
fornia type) were grown on soil plus vermiculite (1:3) in a
growth chamber at 22 °C, 16-h photoperiod, and irradiance
of 100–120 lmol m-2 s-1 for 27, 29, and 45 days,
respectively. Pea (Pisum sativum L., cv. Lincoln) seeds
were processed as previously described by Corpas et al.
(2008). For cadmium treatment, healthy 14-day-old pea
plants were used and exposed to 50 lM CdCl2 for 14 days
(Romero-Puertas et al. 2004a). In the case of the herbicide
2,4-D, this was applied by spraying leaves with a 22.6 mM
solution and pea plants were further grown for 4–7 days,
and then, leaves were collected (Romero-Puertas et al.
2004b).
Acta Physiol Plant (2017) 39:57
Preparation of leaf crude extracts
Leaves of healthy plants from the four species were
frozen in liquid N2 and ground in a mortar with a
pestle. The powder was suspended in a homogenizing
medium containing 50 mM Tris–HCl, pH 7.8, 0.1 mM
EDTA, 0.2% (v/v) TritonX-100, and 10% (v/v) glyc-
erol. Leaf extracts were centrifuged at 17,0009g for
25 min, and the supernatants were used for the different
assays.
Organelle purification and separation
of peroxisomal matrix proteins
Purification of peroxisomes from pea leaves was performed
by combination of differential and sucrose density-gradient
(35–60%, w/w) centrifugations, as previously described by
López-Huertas et al. (1995).
Peroxisomal matrix proteins were obtained according to
Corpas et al. (1998). Briefly, whole peroxisomes were
broken by osmotic shock and membrane fractions were
separated by centrifugation at 120,000g for 30 min and
supernatants, containing the matrices, were concentrated
by ultrafiltration using a PM-10 membrane (Amicon).
Under these conditions, a concentration of the peroxisomal
soluble fractions of about 0.1 lg of protein ll-1 was
obtained.
Enzyme assays
Catalase activity was determined by measuring spec-
trophotometrically the disappearance of H2O2 (Aebi 1984).
Fumarase activity was determined by measuring the for-
mation of fumarate, according to the method of Walk and
Hock (1977).
Electrophoretic methods and immunoblot analyses
Protein samples were analyzed by SDS-PAGE on 4–20%
gradient precast Mini-Protean TGX gels, and stained with
Bio-Safe Coomassie G-250 Stain (Bio-Rad). For immu-
noblot analyses, the polypeptides were transferred to
PVDF membranes using a Trans-Blot Turbo Transfer
System (Bio-Rad). Then, the membranes were probed
with rabbit IgG against yeast Prx1 (dilution 1:1000 and
1:5000). For immunodetection, an affinity-purified goat
anti-rabbit IgG horseradish peroxidase conjugate (Bio-
Rad) (dilution 1:10,000) and an enhanced chemilumi-
nescence kit (ClarityTM Western ECL Substrate, Bio-Rad)
were used. Chemiluminescence was detected using a
Molecular ImagerÒ Gel DocTM XR documentation sys-
tem. Band intensity was quantified using the ImageJ 1.45
software.
Page 3 of 12 57
Stability of peroxisomal matrix proteins
under oxidizing and reducing conditions
Peroxisomal matrix proteins were exposed to different
oxidizing and reducing conditions to evaluate their stabil-
ity. Thus, the matrix proteins were preincubated at room
temperature for 15 min in the presence of either 5 mM
DTT and 10 mM N-ethylmaleimide (NEM) which can
bind to the sulfhydryl group of Cys and avoid disulfide
bond formation, or 5 mM H2O2. Then, samples were
heated either at 95 °C for 5 min or at 98 °C for 10 min.
The proteins were individualized by SDS-PAGE as
described previously, and gels were stained with silver
according to Corpas et al. (1994).
Alternatively, the gels were transferred onto PVDF
membranes (Immobilon P, Millipore Corp., Bedford, MA),
such as it has been described previously.
Electron microscopy and immunocytochemistry
Pieces from leaves of approximately 1 mm2 were cut,
fixed, dehydrated, embedded in LR White resin, and pro-
cessed as previously described by Corpas et al. (1994). The
sections were then incubated separately for 2 h with puri-
fied IgGs against yeast Prx1, as primary antibody (dilution
1/1). Pre-immune serum was used as control. Grids were
washed with several drops of TBST and incubated for 1 h
with goat anti-rabbit IgGs conjugated to 15 nm gold par-
ticles (British BioCell International) diluted 1/40 in TBST
buffer, as secondary antibody. Finally, grids were washed
with TBST and distilled water. Sections were post-stained
in 2% uranyl acetate for 7 min and examined with a Zeiss
EM 10C transmission electron microscope (Jena,
Germany).
Sequence analysis
The pairwise alignment was done with ClustalW at http://
www.ebi.ac.uk with the default settings in the slow mode.
The amino acid (aa) identity is high between members
within a Prx clade ([75%) but low between members of
different clades (\30%). Phylogenetic analyses were con-
ducted using the MEGA software version 6.0 (http://www.
megasoftware.net) (Chevenet et al. 2006; Dereeper et al.
2008).
Other assays
Protein concentration was determined with the Bio-Rad
Protein Assay (Hercules, CA), using bovine serum albumin
as standard. Total chlorophyll (a ? b) concentration
(lg ml-1) was obtained by measuring the optical density at
665 and 649 nm and using the formula: total
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57 Page 4 of 12 Acta Physiol Plant (2017) 39:57

chlorophyll = (6.45 9 A665) ? (17.72 9 A649) (Vernon (A) SDS-PAGE

1960). The density of the gradient fractions was calculated kDa
from the refractive index of the fractions, which was 97.4 -
measured at room temperature using a refractometer 66.2 -
(Atago, Japan). The intensity of bands was quantified using 45.0 -
the ImageJ 1.45 software. To estimate the statistical sig-
nificance of differences, the data were analyzed by the
31.0 -
Student’s t test. - Prx1
(28 kD)

21.0 -
Results
14.0 -
Purification of recombinant mitochondrial 1-Cys 1 2 3 4 5
Prx protein (Prx1p) in E. coli and characterization
(B) Immuno-blot
of the purified IgG fraction against the Prx1

Figure 1a shows the Coomassie blue staining of an SDS-

PAGE, where the different steps in the purification of
recombinant Prx1p expressed in E. coli were analyzed,
including total proteins of an E. coli extract in induced
culture (Fig. 1a, lane 2), flow-through (Fig. 1a, lane 3),
purified His-tag-Prx1p (Fig. 1a, lane 4), and purified Prx1p
without His-tag after thrombin treatment (Fig. 1a, lane 5).
Thus, it was obtained a purified Prx1 of 28 kDa which was
used as an antigen to immunize two New Zealand White
rabbits.
Figure 1b represents the immunoblot analysis of the IgG
fractions obtained from each rabbit using wild type (lanes 1
and 2) and null S. cerevisiae cells (Fig. 1b, lanes 3–4). The
IgG fractions of rabbit #1 recognized a single band of
28 kDa which corresponded to the expected size of the
Prx1 in S. cevevisae (Fig. 1b, lane 1). However, in the
extract of null PRX1 S. cerevisiae cells, no immune-reac-
tive band was detected (Fig. 1b, lane 3). In the case of the
IgG fraction of rabbit #2, the presence of three
immunoreactive bands in the extract of wild-type cells was
observed, one with a molecular mass of 28 kDa and
another two bands with higher molecular masses (Fig. 1b,
lane 2). Nevertheless, in the extract of null PRX1 S.
cerevisiae cells, the band of 28 kDa was not detected
(Fig. 1b, lane 4) and only the two bands of higher molec-
ular mass were observed. On the basis of these results, the
IgG fraction of rabbit #1 was selected and used for all the
further experiments.
Cross reactivity of the antibody to yeast
mitochondrial Prx1 with higher plant extracts
The cross reactivity of the antibody against yeast Prx1 with
crude extracts of pea, Arabidopsis, pepper, and lettuce
leaves is shown in Fig. 2. Pea leaves showed several
immune-reactive bands of about 50 and 37 kDa, being the
latter the most prominent one. Arabidopsis leaves showed
P 1-
Prx1
(28 kD)
1 2 3 4
Wild-type Null PRX1
cells cells
Fig. 1 Purification of recombinant mitochondrial 1-Cys Prx protein
(Prx1 p) in Escherichia coli and characterization by immunoblot
analysis of purified IgG fraction against Prx1p in protein extracts of
wild type and null Saccharomyces cerevisiae cells. a SDS-PAGE
(15%) stained with Coomassie blue. Lane 1 molecular markers. Lane
2 total proteins of E. coli extract in induced culture. Lane 3 flow-
through. Lane 4 purified His-tag-Prx1p. Lane 5 purified Prx1p without
His-tag after thrombin treatment. b Immunoblot analysis of purified
IgG fractions of two immunized rabbits with recombinant Prx1
protein. Lanes 1 and 2 protein extracts of wild-type S. cerevisiae cells.
Lanes 3 and 4 protein extracts of null PRX1 S. cerevisiae cells. Lanes
1 and 3 were assayed with purified IgG against Prx1 from rabbit #1.
Lanes 2 and 4 were assayed with purified IgG against Prx1 from
rabbit #2
an immunoreactive polypeptide of approximately 37 kDa.
However, in leaves of pepper and lettuce, although the
polypeptide of 37 kDa was detected, other immune-reac-
tive bands with lower relative molecular masses of 20 and
25 kDa in pepper and 23 kDa in lettuce were also present.
Isolation of pea leaf peroxisomal fractions
Isolation of peroxisomes required two previous differential
centrifugations to remove other subcellular fractions, such as
chloroplasts and nuclei. As a result, the 12,000 g particulate
pellet, which mainly contains peroxisomes and

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Acta Physiol Plant (2017) 39:57 Page 5 of 12 57

(A) Protein staining (B) Immunoblot (A) 0.20 -1.4

Density (
(g · cm-3)
MM Pea Ara Pep Let MM Pea Ara Pep Let
kDa kDa
0.15
250 - 250 -

Protein
( g · l-1)
150 - 150 -
0.10 -1.2
100 - 100 -
75 - 75 -
0.05

)
50 - 50 -
37 - 37 - -1.0
100
25 - 25 -

Chlorophyll
20 - 20 - 75

ml-1)
15 - 15 -
10 - 10 - 50

( g·
25
Fig. 2 Cross reactivity of antibody against yeast Prx1 in leaf extracts
of different plant species, including pea (Pea), Arabidopsis (Ara),

(nmol fumarate · min-1 · ml-1)

pepper (Pep), and lettuce (Let). a SDS-PAGE (4–20%) analysis of 175
150 Mitochondria
leaf extracts stained with Coomassie Brilliant Blue G-250. b Im-

Fumarase
munoblot analysis of leaf extracts of different plant species using 125
purified IgGs against yeast Prx1 (dilution 1:1000). About 20 lg 100
proteins of leaf extracts were loaded in each lane 75
50
mitochondria, was loaded onto a discontinuous sucrose 25
density gradient. Figure 3a represents the characterization of 60

ml-1)
Peroxisomes
this gradient, where peroxisomes (fractions 5–8) were 50
·
identified by a major peak of catalase activity, a well-known
min-1
Catalase

40
marker enzyme of peroxisomes (Huang et al. 1983; Baker 30
( mol H2O2 ·

and Graham 2002). These peroxisomal fractions had also an 20

equilibrium density of 1.24 g cm-3, which is also a char-
acteristic property of peroxisomes in sucrose solutions
(López-Huertas et al. 1995; Corpas et al. 2008). The other
peaks of catalase activity (fractions 12 and 19) corresponded
to gradient fractions contaminated by catalase released from
broken peroxisomes. The peak of peroxisomes was well
separated from mitochondria (fractions 19–22) that were
recognized by the peak of fumarase activity (Walk and Hock
1977). Broken chloroplasts (fractions 22–24) were identified
by the chlorophyll peak. Considering that the goal of this
study was to evaluate the possible presence of a protein
immunorelated to peroxiredoxin in plant peroxisomes, all
gradient fractions were analyzed by immunoblot assay using
the purified IgGs against yeast Prx1 (Fig. 3b). The presence
of multiple immunoreactive bands was detected in the
fractions 22–24 corresponding mainly to broken organelles.
However, in the mitochondrial fraction and through the
other gradient fractions, two immunoreactive bands were
mainly observed. In fractions 6 and 7 (maximum of catalase
activity), those two immunoreactive bands were detected,
being the lower band the most prominent one. Finally, in
fractions 2 and 3, the highest molecular mass immunore-
active band was the most abundant one. The distinct
molecular masses observed among fractions are mainly due
to changes in mobility attributable to density differences of
the different fractions as a consequence of the sucrose
concentration.
10
1 3
botton
5 7 9 11 13 15 17 19 21 23 25 27
top
Fraction number
(B) Immuno-blot
Fraction number
kDa MM 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 2122 23 24 25 26 27
75-
50-
37-
25-
Fig. 3 Purification of peroxisomes from pea leaves. Cell organelles
were purified from 6-week-old pea leaves by differential and sucrose
density-gradient centrifugations. a Gradient fractions of 1.5 ml were
eluted from the bottom of the tube using a density-gradient
fractionator and were assayed for specific marker enzymes of
peroxisomes (catalase) and mitochondria (fumarase). Enzyme activity
was determined spectrophotometrically, as indicated in the ‘‘Materials
and methods’’. b Immuno-related protein to Prx was detected in the
gradient fractions by immunoblotting using purified IgGs against the
yeast Prx1 (dilution 1:500 for fractions 2–10 and 1:1000 for fractions
11–27). For immunoblot analysis, 30 ll of each fraction was used
Properties of the peroxisomal matrix protein
immunorelated to yeast peroxiredoxin
To get more information on the peroxisomal matrix pro-
teins immunorelated to peroxiredoxins, the peroxisomal
matrix proteins were obtained from purified peroxisomes,

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57 Page 6 of 12 Acta Physiol Plant (2017) 39:57

preincubated under reducing and oxidizing conditions, and
then were analyzed by SDS-PAGE.
Figure 4a shows the silver-stained SDS gel, where the
following treatments were applied: lane 1, peroxisomal
matrix proteins incubated with 10 mM DTT and heated at
95 °C for 5 min; lane 2, peroxisomal matrix proteins
incubated with 5 mM DTT for 15 min at room temperature
and then heated at 98 °C for 10 min; lane 3, peroxisomal
matrix proteins incubated with 5 mM H2O2 for 15 min at
room temperature and then heated at 98 °C for 10 min; and
lane 4, peroxisomal matrix proteins incubated with 10 mM
NEM for 15 min at room temperature and then heated at
98 °C for 10 min. Results obtained showed that the per-
oxisomal matrix proteins apparently were not affected
either by temperature (95 or 98 °C) or DTT (5 or 10 mM)
treatments. On the other hand, only few protein bands in
the range of 50–70 kDa seemed to disappear after the
treatment with H2O2 (oxidizing condition) or NEM (agent
that irreversibly blocks cysteine residues). Figure 4b shows
the corresponding blot probed with the antibody against
yeast Prx-1, where one immunoreactive band of about
50 kDa did not show any change of mobility under all the
different treatments. In this context, in a very recent study
of the electrophoretical behaviour of S. cerevisiae recom-
binant Prx1 protein under reducing conditions, a single
band of about 28 kDa was also found, but under non-
reducing conditions at least two bands of 28 and 56 kDa
appeared, being the last one the dimeric form of the protein
(see Pedrajas et al. 2016).
Immunoelectron microscopy analysis
To corroborate the presence in peroxisomes of the protein
immunorelated to Prx, its subcellular distribution in leaves
was analyzed by electron microscopy immunocytochem-
istry (Fig. 5). Using the purified IgG against yeast Prx1,
immunogold labelling was detected in chloroplasts, mito-
chondria, cytoplasm, and peroxisomes, specifically in the
peroxisomal matrix. A magnification of a peroxisome and a
mitochondrion is shown in Fig. 5b and c, respectively. No
significant labelling was observed in leaf sections probed
with pre-immune serum (Fig. 5d).
Protein level of peroxisomal Prx under oxidative
stress conditions
In previous works, it was demonstrated that treatment of
pea plants with the heavy-metal Cd and herbicide 2,4-D
induced oxidative stress in leaves and roots (Romero-
Puertas et al. 2004a, b; Rodrı́guez-Serrano et al. 2006;
Corpas et al. 2008). Under these abiotic stress conditions,
the oxidative metabolism of peroxisomes was affected

(A) Silver-stained SDS-gel (B) Anti-yeast Prx1

Tº (ºC) 95 98 98 98 95 98 98 98
Time (min) 5 10 10 10 5 10 10 10
DTT (mM) 10 5 - - 10 5 - -
H2O2 (mM) - - 5 - - - 5 -
NEM (mM) - - - 5 - - - 5
kDa
250 -

150 -
100 -
75 -

50 - - Prx
37 -

25 -
20 -
15 -
10 -

M 1 2 3 4 M 1 2 3 4

Fig. 4 Effect of redox state on the pea peroxisomal matrix protein
immunorelated to yeast peroxiredoxin 1 (Prx1). a Silver-stained SDS-
acrylamide gel (4–20%). b Immunoblot probed with a polyclonal
antibody to yeast Prx1 (dilution 1/5000). A total of 2.5 lg proteins
were loaded per lane. M molecular markers. Lane 1 peroxisomal
matrix proteins with 10 mM DTT and heated at 95 °C for 5 min.
Lane 2 peroxisomal matrix proteins incubated with 5 mM DTT for
15 min at room temperature and then heated at 98 °C for 10 min.
Lane 3 peroxisomal matrix proteins incubated with 5 mM H2O2 for
15 min at room temperature and then heated at 98 °C for 10 min.
Lane 4 peroxisomal matrix proteins incubated with 10 mM NEM for
15 min at room temperature and then heated at 98 °C for 10 min

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Acta Physiol Plant (2017) 39:57
(A)
M P
Ch
CW
P
(C)
Cy
M
Fig. 5 Immunocytochemical localization in pea leaves of the protein
immunorelated to yeast peroxiredoxin 1 (Prx1). The electron
micrographs are representative of thin sections of pea leaves showing
the immunolocalization of the protein immuno-related to ScPrx1. Cell
sections were probed with a purified IgG against yeast Prx1 (dilution
1:1). a Cell section showing the immunogold labelling of Prx in
chloroplasts, mitochondria and peroxisomes. b High magnification of
(Romero-Puertas et al. 1999, 2004b). For this reason, in
plants exposed to both stress conditions, the level of the
peroxisomal protein immunorelated to Prx was estimated
by Western blotting (Fig. 6a). Results showed that stress by
Cd increased slightly the level of peroxisomal Prx, whereas
herbicide 2,4-D produced the opposite effect, with a sig-
nificant decrease in the Prx level of about 60% (Fig. 6b).
To get information on the potential immune relationship
between yeast Prx and plant Prxs, it was compared the
amino acid sequence identity of the mature forms (without
targeting signal) of S. cerevisiae mitochondrial 1-Cys Prx
protein (YBL064C), used in this study, with four repre-
sentative Prx types of A. thaliana, including 2-CysPrx
(At3g11630), PrxQ (At3g26060), 1-CysPrx (At1g48130),
and PrxIIC (At1g65970). This analysis is presented in
Table 1 and its corresponding phylogenetic tree in the
supplemental Fig. 1. The highest identity score (41%) was
obtained with A. thaliana 1-CysPrx (At1g48130), and this
identity score was even higher than that observed between
the own Prxs from Arabidopsis.
Page 7 of 12 57
(B)
(D)
M
M
Ch P
CW Ch
cell section showing the immunolocalization of Prx in peroxisomes.
c High magnification of cell section showing the immunolocalization
of Prx in mitochondria. d Cell section probed with pre-immune
serum. The inset shows a detail with one peroxisome without
immunogold labelling. Arrows indicate 15-nm gold particles. Ch
chloroplast, CW cell wall, Cy cytoplasm, M mitochondrion, P perox-
isome. Bar 0.5 lm
Discussion
In mammals and the yeast Candida boidinii, a peroxisomal
protein of 20 kDa (PMP20) which has a peroxisomal tar-
geting signal type 1 (PTS1) and belongs to the peroxire-
doxin family was identified (Yamashita et al. 1999;
Horiguchi et al. 2001). This protein has thiol-peroxidase
activity, which can remove H2O2, and has been proposed to
participate against oxidative stress in peroxisomes (Ya-
mashita et al. 1999). In mammalian cells, by immunoblot
analysis of peroxisomal fractions, a peroxiredoxin (sub-
class Prx V or PRDX5) was detected (Seo et al. 2000;
Knoops et al. 2011). Prxs belonging to this subclass are
considered atypical 2-Cys Prxs, because they are mono-
meric enzymes, while the typical 2-Cys and 1-Cys Prxs are
homodimeric (Wood et al. 2003). In the human and rat
protein AOEB166, which is part of the peroxiredoxin
family, analysis of their C-terminal domains allowed to
identify the presence of a peroxisomal targeting sequence
123
57 Page 8 of 12 Acta Physiol Plant (2017) 39:57

(A) Anti-yeast Prx1 peroxisomes purified from pea leaves was investigated. In
peroxisomal fractions, the measurement of peroxiredoxin
activity, based on the reduction of H2O2, is unreliable due
to the strong interferences produced in the assay by cata-
lase and ascorbate peroxidase, two enzymes present in
Prx peroxisomes that catalyze the removal of H2O2, and also
the reducing compounds ascorbate and glutathione (Yam-
aguchi et al. 1995; Bunkelmann and Trelease 1996; Jimé-
(B) nez et al. 1997; del Rı́o et al. 2002). For this reason, in this
800 work, we have used an immunological approach to study
Arbitrary units

* the putative presence of peroxiredoxin in leaf peroxisomes.

400 Thus, an antibody against Sacharomyces cerevisiae 1-Cys
* Prx (Prx1) which is present in mitochondria (Pedrajas et al.
0
2000, 2016) was prepared from the recombinant protein
ControlCdControl2,4-D
and used for this study.
Fig. 6 a Representative immunoblot analysis of leaf peroxisomal In general, the commonly known plant Prxs have
matrix from pea plants treated with CdCl2 and herbicide 2,4- molecular masses in the range 17–29 kDa (Dietz 2003).
dichlorophenoxyacetic acid (2,4-D). Different batches of pea plants However, there are some putative Prxs with unusual
were grown with 50 lM CdCl2 and treated with 22.6 mM 2,4-D, as
described in ‘‘Materials and methods’’. Peroxisomal matrices were
molecular masses. For example, a putative peroxisomal
purified from pea leaves by differential and sucrose density-gradient protein of Arabidopsis thaliana (At1g65990) deduced by
centrifugation (see ‘‘Materials and methods’’). SDS-PAGE was bioinformatic sequence analysis shows a theoretical
carried out on 12% polyacrylamide gels, and the protein blot was molecular mass of 62.7 kDa (Horling et al. 2002; Dietz
developed with a polyclonal antibody to yeast Prx1 (dilution 1/2000).
A total of 3 lg proteins were loaded per lane. b Relative quantifi-
et al. 2006), and in pea nodules, a new Prx isoform with a
cation of the intensity of bands using ImageJ program of at least three molecular mass of 68 kDa has been detected using an
independent experiments. Asterisks indicate that differences from antibody to Arabidopsis Prx II C (Groten et al. 2006). This
control values were statistically significant at P \ 0.05 Prx was more abundant in young nodules and its content
decreased with nodule senescence (Groten et al. 2006). In
Table 1 Amino acid sequence identity of the mature forms (without plants, some Prxs, such as 2-Cys Prx A and B, have the
targeting signal) between S. cerevisiae mitochondrial 1-Cys Prx
ability to form dimers upon oxidation (Horling et al. 2002;
protein (YBL064C) and four representative Prx types of A. thaliana,
including 2-CysPrx (At3g11630), PrxQ (At3g26060), 1-CysPrx Lee et al. 2015). For example, the Arabidopsis plastidial
(At1g48130), and PrxIIC (At1g65970) 2-Cys peroxiredoxin (2-Cys PRX) under various physio-
SeqA Name Length SeqB Name Length Score
logical temperatures, osmotic and light stress conditions
can undergo different overoxidation levels and changes in
1 YBL064C 241 2 At3g11630 200 27.0 its oligomerization status (Cerveau et al. 2016).
1 YBL064C 241 3 At3g26060 160 23.75 In our work, in leaf extracts of different plant species,
1 YBL064C 241 4 At1g48130 216 41.2 the antibody to yeast Prx-1 recognized a prominent band of
1 YBL064C 241 5 At1g65970 162 12.96 37 kDa and another band of about 50 kDa in pea leaves.
2 At3g11630 200 3 At3g26060 160 33.75 Arabidopsis and pepper showed an immunoreactive band
2 At3g11630 200 4 At1g48130 216 27.5 of 37 kDa, which were less abundant in lettuce. However, a
2 At3g11630 200 5 At1g65970 162 7.41 weaker immunoreactive band of approximately 25 kDa
3 At3g26060 160 4 At1g48130 216 21.25 was also observed in all the crude extracts of leaves
3 At3g26060 160 5 At1g65970 162 5.0 assayed. To evaluate the potential presence of any of these
4 At1g48130 216 5 At1g65970 162 17.9 immunoreactive bands in peroxisomes, pea plants were
selected, since previously we have carried out abundant
The pairwise alignment was done with ClustalW at http://www.ebi.ac.
uk with the default settings in the slow mode. The amino acid (aa) research work on peroxisomes from this plant species
identity is high between members within a Prx clade ([75%) but low (Corpas et al. 1993, 1998, 2008; Distefano et al. 1997;
between members of different clades (\30%) López-Huertas et al. 1999; Barroso et al. 1999).
When the sucrose density gradients designed for the
(PTS1) suggesting the occurrence of this protein in animal isolation of pea leaf, peroxisomes were analyzed, mainly,
peroxisomes (Knoops et al. 1999, 2011). two immunoreactive bands of about 50 and 37 kDa were
However, in plants, there is no information on the detected throughout the different gradient fractions (2–22).
presence of peroxiredoxins in peroxisomes, and in this In the case of the fractions with higher catalase activity
work, the potential occurrence of peroxiredoxin in (fractions 6–7) which correspond to intact peroxisomes

123
Acta Physiol Plant (2017) 39:57
(Fig. 3), the most prominent band was that of 37 kDa.
However, when the soluble fractions of peroxisomes (ma-
trices) were analyzed, only the immunoreactive band of
50 kDa was detected (Fig. 4).
To discard the possibility that the molecular mass
observed for the peroxisomal protein immunorelated to Prx
could be the consequence of oligomerization processes, as
has been reported for other Prxs (Wood et al. 2003; Bar-
ranco-Medina et al. 2009), matrix proteins from purified
peroxisomes were analyzed by SDS-PAGE under non-re-
ducing conditions, where samples prior to electrophoresis
were exposed to reducing and oxidizing conditions
(Fig. 4a, b). Results obtained showed that under all treat-
ments (temperature, reducing and oxidizing conditions, and
cystein blockage), the immunoreactive band of about
50 kDa did not change in mobility and was in the range of
molecular mass of the dimeric form of other Prxs, such as
the yeast mitochondrial Prx-1, which has a molecular mass
of about 54 kDa (Pedrajas et al. 2016). Considering the
characteristic oxidative metabolism of peroxisomes, this
could be a mechanism of protection against changes in
redox conditions that could affect the Prx activity of these
organelles.
The electron microscopy immunogold labelling tech-
niques are one of the main tools in cell biology for the
accurate localization of macromolecules in cell compart-
ments (Fahimi et al. 1996). Using this technique, different
enzymes have been localized in plant peroxisomes,
including several peroxisomal membrane proteins (PMPs)
(Corpas et al. 1994, 2000), ascorbate peroxidase (Bunkel-
mann and Trelease 1996), superoxide dismutases (Sandalio
et al. 1997; Corpas et al. 1998; del Rı́o et al. 2003), NADP-
dehydrogenases (Corpas et al. 1998, 1999), nitric oxide
synthase (Barroso et al. 1999), monodehydroascorbate
reductase (Leterrier et al. 2005), glutathione reductase
(Romero-Puertas et al. 2006) and xanthine oxidoreductase
(Corpas et al. 2008), among others. The EM immunocy-
tochemical results described in this work confirm the
occurrence of a protein immunorelated to Prx in different
cell compartments of pea leaves, including chloroplasts,
mitochondria, and cytoplasm, and demonstrate the pres-
ence of this Prx-related protein in pea leaf peroxisomal
matrices. Moreover, these results are in agreement with the
presence in pea leaves of a chloroplastic 2-Cys peroxire-
doxin (Bernier-Villamor et al. 2004) and a mitochondrial
type II peroxiredoxin (Barranco-Medina et al. 2006; 2007).
In rat liver cells, the immunolocalization of Prx showed
that this enzyme was distributed in the cytoplasm, nuclei,
mitochondria, and peroxisomes, and had a molecular mass
of 23 kDa (Immenschuh et al. 2003).
Peroxiredoxins have been shown to be involved in the
H2O2 detoxification in chloroplasts and mitochondria, in
DNA protection in nuclei, chloroplasts and mitochondria,
Page 9 of 12 57
and in pathogen defence, among other functions. More-
over, the expression of some Prxs under oxidative stress
conditions has also been reported (Horling et al.
2002, 2003; Haslekas et al. 2003; Sakamoto et al. 2003;
Finkemeier et al. 2005; Sevilla et al. 2015), and some Prxs
can also function as molecular chaperones, such as 1-Cys
Prx during seed germination and plant development of Ch
inese cabbage under stress conditions (Kim et al. 2011).
In previous studies with plants under cadmium and 2,4-D
stress, it has been shown that these abiotic conditions
affected in different degrees the peroxisomal oxidative and
nitrosative metabolism (McCarthy et al. 2001; Leterrier et al.
2005; Romero-Puertas et al. 2006; McCarthy et al. 2011;
Corpas and Barroso 2014). For this reason, we selected these
stresses to evaluate the expression of this protein immuno
related to Prx-1. Cadmium stress increased slightly the
protein level of peroxisomal Prx, whereas herbicide 2,4-D
caused a significant reduction of its protein level, indicating
the existence of a differential regulation of Prx biosynthesis
under these two oxidative stress-inducing conditions.
Plant peroxisomes have the capacity to generate reactive
oxygen species (ROS) and reactive nitrogen species (RNS)
signal molecules (del Rı́o et al. 2006; del Rı́o 2011; San-
dalio et al. 2013; Corpas and Barroso 2014; del Rı́o and
López-Huertas 2016; Corpas et al. 2017), but also have a
complex battery of antioxidative enzymes distributed in the
matrix and membrane of these cell organelles (Corpas and
Trelease 1998; Jiménez et al. 1997; Palma et al. 2006; del
Rı́o et al. 2002, 2006; López-Huertas and del Rı́o 2014;
Leterrier et al. 2005, 2016). On the other hand, considering
that some Prxs are regulated by oxidation and nitrosylation
(Dietz et al. 2006), the plant peroxisomal protein
immunorelated to Prx could have a role in modulating the
levels of ROS and RNS signal molecules which are pro-
duced in peroxisomes and released into the cytosol (del Rı́o
et al. 2006; del Rı́o and López-Huertas 2016). In leaf
peroxisomes from plants under cadmium and lead stress
situations, it has been recently reported the overproduction
of peroxynitrite (ONOO-), a powerful oxidant and also
strong protein nitrating agent (Corpas and Barroso
2014, 2016). On the other hand, it is known that some
peroxiredoxin isoforms possess peroxynitrite reductase
activity that detoxifies ONOO- in bacteria and plant sys-
tems (Bryk et al. 2000; Romero-Puertas et al. 2007).
Consequently, the presence of a Prx in peroxisomes could
also represent a mechanism of protection of peroxisomal
proteins against ONOO--mediated tyrosine nitration which
can provoke losses of function in enzymes, such as has
been reported recently for the peroxisomal NADH-depen-
dent hydroxypyruvate reductase (Corpas et al. 2013b) and
catalase (Chaki et al. 2015).
In summary, the immunological and immunocyto-
chemical data reported in this work provide evidence that
123
57 Page 10 of 12
the pea peroxisomal matrix contains a protein immunore-
lated to peroxiredoxin, which has an unusual molecular
mass of about 50 kDa, but in the range of molecular masses
of the dimeric form of other Prxs (Pedrajas et al. 2016),
which did not change under reducing and oxidizing con-
ditions and whose protein level is differentially modulated
under oxidative stress conditions.
Author contribution statement This work was concep-
tualized originally by FJC and JBB. Experiments were
performed by all authors, and FJC wrote the manuscript.
Acknowledgements This work was supported by ERDF-cofinanced
grants from the Ministry of Science and Innovation (BIO2012-33904)
and Junta de Andalucı́a (research groups CVI 192 and CVI 286).
Present research for FJC and JMP is supported by the ERDF-cofi-
nanced grant AGL2015-65104-P and for JBB the BIO2015-66390-P
grant both from the Ministry of Economy and Competitiveness
(MINECO), Spain. The electron microscopy assays were carried out
at the Centre of Scientific Instrumentation of the University of
Granada. The valuable technical assistance of Mr. Carmelo Ruı́z-
Torres is acknowledged.
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