Plant Cell Physiol. 47(7): 984–994 (2006)
doi:10.1093/pcp/pcj071, available online at www.pcp.oxfordjournals.org
ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
The Expression of Different Superoxide Dismutase Forms is Cell-type
Dependent in Olive (Olea europaea L.) Leaves
Francisco J. Corpas 1, *, Ana Fernández-Ocaña 2, Alfonso Carreras 2, Raquel Valderrama 2,
Francisco Luque 2, Francisco J. Esteban 2, Marı́a Rodrı́guez-Serrano 1, Mounira Chaki 2, José R. Pedrajas 2,
Luisa M. Sandalio 1, Luis A. del Rı́o 1 and Juan B. Barroso 2
1
Departamento de Bioquı´mica, Biologı´a Celular y Molecular de Plantas, Estación Experimental del Zaidı´n (EEZ), CSIC, Granada, Spain
Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Área de Bioquı´mica y Biologı´a
Molecular, Universidad de Jae´n, Spain
2
Introduction
Superoxide dismutase (SOD) is a key antioxidant
enzyme present in prokaryotic and eukaryotic cells as a
first line of defense against the accumulation of superoxide
radicals. In olive leaves, the SOD enzymatic system was
characterized and was found to be comprised of three
isozymes, an Mn-SOD, an Fe-SOD and a CuZn-SOD.
Transcript expression analysis of whole leaves showed that
the three isozymes represented 82, 17 and 0.8% of the total
SOD expressed, respectively. Using the combination of laser
capture microdissection (LCM) and real-time quantitative
reverse transcription–PCR (RT–PCR), the expression of
these SOD isozymes was studied in different cell types of olive
leaves, including spongy mesophyll, palisade mesophyll,
xylem and phloem. In spongy mesophyll cells, the isozyme
proportion was similar to that in whole leaves, but in the other
cells the proportion of expressed SOD isozymes was different.
In palisade mesophyll cells, Fe-SOD was the most abundant,
followed by Mn-SOD and CuZn-SOD, but in phloem cells
Mn-SOD was the most prominent isozyme, and Fe-SOD was
present in trace amounts. In xylem cells, only the Mn-SOD
was detected. On the other hand, the highest accumulation of
superoxide radicals was localized in vascular tissue which was
the tissue with the lowest level of SOD transcripts. These data
show that in olive leaves, each SOD isozyme has a different
gene expression depending on the cell type of the leaf.
Superoxide dismutases (SODs; EC 1.15.1.1) are a
family of metalloenzymes that catalyze the disproportionation of superoxide (O2 E ) radicals into H2O2 and O2, and
are a first line of defense against the toxic effects of
superoxide radicals produced in different cellular compartments (Fridovich 1986, Halliwell and Gutteridge 2000). In
general, there are three types of SOD, containing either Mn,
Fe, or Cu plus Zn as prosthetic metals (Fridovich 1986).
In higher plants, SOD isozymes have been localized in
different cell compartments. Mn-SOD is present in mitochondria and peroxisomes (Baum and Scandalios 1981, del
Rı́o et al. 1983, Palma et al. 1986, Sandalio and del Rio
1987, Bowler et al. 1994, Corpas et al. 1998, del Rio et al.
2003). Fe-SOD has been found mainly in chloroplasts (Salin
1988, Asada 1994) but has also been detected in peroxisomes (Droillard and Paulin 1990), and CuZn-SOD has
been localized in cytosol, chloroplasts, peroxisomes and
apoplast (Sandalio and del Rı́o 1987, Sandalio and del Rı́o
1988, Salin 1988, Kanematsu and Asada 1991, Ogawa et al.
1996, Ogawa et al. 1997, Sandalio et al. 1997, Corpas et al.
1998, del Rı́o et al. 2002). The number and type of SOD
isozymes can change depending on the plant species, age of
development and environmental conditions (Bridges and
Salin 1981, Bowler et al. 1994, Kliebenstein et al. 1998,
Alscher et al. 2002), and there are also cases of plants, such
as sunflower, with only one type of isoform, a CuZn-SOD
(Corpas et al. 1998).
Plant SODs have been studied under many aspects,
including phylogenetic distribution, biochemical and
molecular properties, structure and function, enzyme
regulation, gene organization and expression, subcellular
localization, role in abiotic and biotic stress, etc. (Bridges
and Salin 1981, del Rı́o et al. 1983, Tsang et al. 1991,
Bowler et al. 1994, Bueno et al. 1995, Allen et al. 1997,
Corpas et al. 1998, Kliebenstein et al. 1998, Alscher et al.
2002, Fink and Scandalios 2002). However, in higher
plants, there is still very little information on the specific
function of each SOD isoenzyme (Mn-SOD, Fe-SOD
Keywords: Olive (Olea europaea L.) — Palisade mesophyll
— Phloem — Spongy mesophyll — Superoxide dismutase
(SOD) — Xylem.
Abbreviations: BSA, bovine serum albumin; CLSM, confocal
laser scanning microscopy; DHE, dihydroethidium; LCM, laser
capture microdissection; RT–PCR, reverse transcription–PCR;
SOD, superoxide dismutase.
Sequence data from this article have been deposited in the
EMBL/GenBank data libraries under accession numbers
AF427107 for Mn-SOD, AY168776 for Fe-SOD and AF426829
for CuZn-SOD.
* Corresponding author: E-mail: [email protected]; Fax, +34-958-129600.
984
Gene expression of SODs in olive leaf cells
A. Native-PAGE
CuZn-SODs
I- (34%)
Relative % Trasmittance
and CuZn-SOD) in cells of different tissues. It is necessary
to obtain deeper insights into the relationship between
cellular localization and specific function of each SOD
isoenzyme.
The olive tree (Olea europaea L.) is an important crop
in Southern Europe with a strong economic impact in the
agricultural industry of these countries because the olive
fruit is used to obtain the oil of choice for the
Mediterranean diet (Owen et al. 2000). However, there are
few studies on the metabolism of reactive oxygen species
(ROS) in olive trees and, to our knowledge, there is no
biochemical and molecular information on SODs and their
subcellular localization in leaves of this plant species
(Valderrama et al. 2006).
In this work, using the combination of laser
capture microdissection (LCM) and quantitative reverse
transcription–PCR (RT–PCR), the gene expression of
the three SOD isozymes identified in olive leaves was
analyzed. The existence of a differential transcript expression of SODs depending on the leaf cell type and the
presence of Mn-SOD in the vascular tissue of leaves was
demonstrated.
985
Fe-SOD
(33%)
II- (18%)
Mn- SOD
(15%)
−
+
B. Immuno blot
kDa
M
97.4
66.2
45.0
Results
31.0
Biochemical and molecular analysis of the SOD isozymes of
olive leaves
In crude extracts of olive leaves, the total SOD specific
activity was 1.7 U mg1 protein. The analysis of the SOD
activity by native PAGE showed the presence of four
isozymes which were identified with specific inhibitors
(CN and H2O2) as a Mn-SOD, an Fe-SOD and two
CuZn-SODs (I and II), which represented 15, 33 and 52%
of the total SOD activity, respectively (Fig. 1A). The
analysis by Western blot with specific antibodies against
each type of SOD showed subunit molecular masses of
27 kDa for Mn-SOD, 25 kDa for Fe-SOD and 17 kDa for
CuZn-SOD (Fig. 1B).
On the basis of the information from the gene
databank on the different plant SOD isozymes, oligonucleotides for conserved regions of each SOD isozyme
were designed (see Table 1). Thus, a partial clone for each
SOD isozyme was obtained. The olive leaf Mn-SOD
(accession No. AF427107) showed an identity of 83%
with the Mn-SOD of Glycine max, and 81% with the
Mn-SODs of Lycopersicon esculentum, Prunus persica and
Nicotiana plumbaginifolia. The olive leaf Fe-SOD (accession
No. AY168776) showed an identity of about 80% with the
Fe-SODs of Capsicum annuum, N. plumbaginifolia and
L. esculentum. The olive leaf CuZn-SOD (accession No.
AF426829) had identities of about 85% with the CuZnSODs of C. annuum, L. esculentum, Solanum tuberosum and
21.5
- Mn-SOD
- Fe-SOD
- CuZn-SOD
14.5
C. mRNA SODs
Arbitrary Units
0.04
0.02
Mn-SOD
Fe-SOD
CuZn-SODc
Fig. 1 Identification of the SOD isozymes present in olive leaves.
(A) Activity of SOD isozymes. SODs were separated by native
PAGE on 10% (w/v) polyacrylamide gels, and gels were stained by
the photochemical nitroblue tetrazolium method. Gels were
analyzed using a Gel Doc system (BioRad) coupled with a highly
sensitive CCD camera, and band intensities were expressed as
relative transmittance (T) units. (B) Western blot of SODs of olive
leaf extracts probed with antibodies against: pea Mn-SOD
(1 : 2,000 dilution), Fe-SOD and spinach CuZn-SOD (1 : 3,000
dilution). Proteins (30 mg per lane) were separated by 15% SDS–
PAGE and transferred onto a PVDF membrane. (C) Transcript
analysis (arbitrary units) of the SOD isozymes by real-time
quantitative RT–PCR. Data are the mean SEM of at least three
different experiments.
986
Table 1
Gene expression of SODs in olive leaf cells
Oligonucleotides used for the cloning and real-time quantitative RT–PCR analysis of the three SOD isozymes
Name
cDNA cloning
Mn-SOD-f
Mn-SOD-r
CuZn-SOD-f
CuZn-SOD-r
Fe-SOD-f
Fe-SOD-r
Quantitative-PCR
Mn-SOD-f1
Mn-SOD-r1
CuZn-SOD-f1
CuZn-SOD-r1
Fe-SOD-f1
Fe-SOD-r1
RNA 18S-f1
RNA 18S-r1
Oligonucleotide sequence (50 to 30 )
Product size (bp)
ACM MGA ARC ACC AYC ARACTTA
TGM ARG TAG TAG GCA TGY TCC CA
CCT GGA CTT CAT GGC TTC CAT
TCT TCC GCC AGC GTT TCC AGT G
TYC ACT GGG GKA AGC AYC A
TCM ARR TAG TAA GCA TGC TCC CA
435
AGT CAA GTT GCA GAG TGC AAT CAA GTT C
CAA AGT GAT TGT CAA TAG CCC AAC CTA AAG
GGC TGT ATG TCA ACT GGA CCT CAT TTC A
TGT CAA CAA TGT TGA TAG CAG CGG TG
AAC AAG CAA ATA GCC GGA ACA GAA CTA AC
AGA AAT CGT GAT TCC AGA CCT GAG CAG
TTT GAT GGT ACC TGC TAC TCG GAT AAC C
CTC TCC GGA ATC GAA CCC TAA TTC TCC
144
312
435
140
128
274
‘f’ and ‘r’ correspond to forward and reverse oligonucleotides, respectively. For the degenerated oligonucleotides: M ¼ A,C; R ¼ A,G;
Y ¼ C,T; K ¼ G,T.
Populus tremuloides. As shown in Fig. 1C, the mRNA
expression analysis of SODs in olive leaves by quantitative
RT–PCR using specific oligonucleotides (Table 1) showed
the maximum expression for Fe-SOD (82.5%), followed by
CuZn-SOD (16.7%) and Mn-SOD (0.8%).
LCM of different cell types of olive leaves and gene
expression of the SOD isozymes
Fig. 2A–L shows representative pictures of the
appearance of the leaf tissues before, during and after the
use of the LCM method to obtain cell types from four olive
leaf tissues, including spongy and palisade mesophyll, xylem
and phloem. The selected cells were used as starting
material to obtain the corresponding RNAs, which were
used for the gene expression analysis of Fe-SOD, CuZnSOD and Mn-SOD, by real-time quantitative PCR using
specific primers (see Table 1). The mRNA content of each
isozyme in the cells of spongy mesophyll, palisade
mesophyll, phloem and xylem tissues is shown in Fig. 2M.
Mn-SOD was the only isoform which was present in all cell
types. The highest expression of Mn-SOD was observed in
palisade mesophyll cells followed by spongy mesophyll cells,
xylem and phloem cells. On the other hand, Fe-SOD was
only detected in palisade and spongy mesophyll. The
highest expression of Fe-SOD was observed in palisade
mesophyll cells, followed by spongy mesophyll and phloem
cells. CuZn-SOD was only detected in spongy and palisade
mesophyll cells.
Immunocytochemical localization of Fe-SOD, Mn-SOD and
CuZn-SOD in olive leaves
Electron micrographs of thin sections of olive leaves
showing the specific subcellular localization of Fe-SOD,
Mn-SOD and CuZn-SOD in spongy mesophyll and xylem
cells are shown in Fig. 3. Fe-SOD was localized in
chloroplasts (Fig. 3A), but the immunogold labeling of
Mn-SOD was present in mitochondria of spongy mesophyll
cells (Fig. 3B) and in xylem cells (Fig. 3C). CuZn-SOD was
present in different cell compartments, including amorphous electron-dense structures in the cytosol, chloroplasts
and peroxisomes, and crystalline bodies in nuclei (Fig. 3D
and E). The pre-immune serum did not show any significant
labeling (data not shown).
Immunohistochemical localization of Mn-SOD in olive leaves
by CLSM
Considering that the mRNA of Mn-SOD was the only
SOD transcript present in the four cell types, the protein
expression of Mn-SOD was also analyzed by immunohistochemical analysis at the cellular level using an antibody
against pea Mn-SOD which recognizes a single band of 27 kDa
in crude extracts of olive leaves (Fig. 1B). The appearance
under the optical microscope of an olive leaf section showing
its different tissues is presented in Fig. 4A. A representative
picture of the immunolocalization of Mn-SOD in olive leaf
sections analyzed by confocal laser scanning microsopy
(CLSM) is shown in Fig. 4B. The green fluorescence, which is
attributable to the Mn-SOD, was observed in all cell types
Gene expression of SODs in olive leaf cells
A
987
C
B
Sm
50 µm
D
E
F
Pm
50 µm
G
H
I
Xy
100 µm
J
K
L
Ph
100 µm
M
Arbitraty Units
mRNA content (x10−5)
700
Mn-SOD
600
Fe-SOD
500
CuZn-SOD
400
300
200
100
0
Sm
Pm
Phloem
Xylem
Fig. 2 Visualization of laser capture microdissection (LCM) of cell types from olive leaves and transcript analysis of the SOD isozymes.
(A–C) Appearance of spongy mesophyll (Sm). (D–F) Appearance of palisade mesophyll (Pm). (G–I) Appearance of xylem (Xy).
(J–L) Appearance of phloem (Ph). A, D, G and J show the olive leaf tissues before LCM analysis. B, E, H and K show the delimited target
region. C, F, I and L show the appearance of the tissues after LCM analysis. (M) Real-time quantitative RT–PCR transcript analysis (arbitrary
units) of the three SOD isozymes in olive leaf cells from spongy (Sm) and palisade mesophyll (Pm), phloem and xylem. Data are
mean SEM of, at least, four independent RNA samples from leaf cells obtained by LCM.
988
Gene expression of SODs in olive leaf cells
B (Mn-SOD)
A(Fe-SOD)
M
CW
M
CW
CH
D (CuZn-SOD)
C (Mn-SOD)
Xy
CB
N
CH
1 µm
E (CuZn-SOD)
M
CH
Fig. 3 Immunogold electron microscopy localization of SODs in spongy mesophyll and xylem cells of olive leaves. Representative
electron micrographs of spongy mesophyll cells of olive leaves. The sections were incubated with antibodies against Fe-SOD (dilution
1 : 2,000), Mn-SOD (dilution 1 : 500) and CuZn-SOD (1 : 300 dilution). (A) Immunolocalization of Fe-SOD in spongy mesophyll cells.
(B) Immunolocalization of Mn-SOD in spongy mesophyll cells. (C) Immunolocalization of Mn-SOD in xylem cells. (D and E)
Immunolocalization of CuZn-SOD in spongy mesophyll cells. Arrows indicate 15 nm gold particles. *electron-dense structures in the
cytosol; CB, crystalline body; CH, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; Xy, xylem. Bars represent
1.0 mm in A, C, D and E; and 0.5 mm in B.
Gene expression of SODs in olive leaf cells
A
Pm
989
B
E
Sm
Xy
Ph
E
200 µm
50 µm
D
C
100 µm
80 µm
Fig. 4 Immunohistochemical localization of Mn-SOD in olive leaves and detection of superoxide radicals (O2 E ). (A) Appearance of an
olive leaf section under the optical microscope. (B) Cy2-streptavidin immunofluorescence (green color) attributable to the antibody against
Mn-SOD (dilution 1 : 200). The olive leaf section was analyzed by confocal laser scanning microscopy (CLSM). (C) Representative image
illustrating the CLSM detection of superoxide radicals (O2 E ) in olive leaf sections incubated for 1 h at 258C, in darkness, with 10 mM DHE,
where O2 E is detected by its bright green fluorescence. (D) Representative image of a leaf section pre-incubated with 1 mM TMP,
a superoxide scavenger, and then with 10 mM DHE. The orange-yellow color corresponds to the Chl autofluorescence. E, epidermis.
Ph, phloem. Pm, palisade mesophyll. Sm, spongy mesophyll. Xy, xylem.
except in epidermis cells, and the strongest fluorescence was
detected in the spongy mesophyll cells.
On the other hand, the accumulation of superoxide
radicals (O2 E ) in olive leaf sections was analyzed by CLSM
using the fluorescence probe dihydroethidium (DHE). The
green fluorescence, due to O2 E radicals, was mainly
localized in vascular tissue and epidermal cells (Fig. 4C).
The localization of superoxide accumulation in vascular
tissue is consistent with the results showing that this tissue
has the lowest level of SOD transcripts (Fig. 2M). When the
olive leaf sections were pre-incubated with 1 mM TMP
(a superoxide scavenger), a significant reduction of the
green fluorescence was observed (Fig. 4D).
Discussion
In this work using leaves of olive plants as a model, the
expression of the SOD genes in different cell types of leaf
tissues, including phloem, xylem, and palisade and spongy
mesophyll, was studied by LCM and quantitative RT–PCR.
In olive leaves, three SOD isozymes were found, Mn-SOD,
Fe-SOD and CuZn-SOD, and this isozyme pattern is
different from that found in olive pollen tubes, where only
four CuZn-SODs are present (Alché et al. 1998). The
occurrence of the three types of SOD has also been
described in other plant species such as Brassica campestris
(Bridges and Salin 1981), Pisum sativum (Sandalio et al.
2001, Gómez et al. 2004), Coffea arabica (Daza et al. 1993)
and N. plumbaginifolia (Van Camp et al. 1997), among
others. However, the existence of different patterns of SOD
isozymes has been reported in other plant species. CuZnSOD and Mn-SOD are present in Phaseolus vulgaris and
Vigna unguiculata (Corpas et al. 1991), CuZn-SODs
in
Helianthus
annuun
and
Hibiscus
esculentus
(Bridges and Salin 1981, Corpas et al. 1998), CuZn-SODs
and Fe-SODs in Ginkgo biloba, and Fe-SODs and Mn-SOD
in Nuphar luteum (Bridges and Salin 1981). All these cases
provide evidence of the heterogeneous distribution of SOD
990
Gene expression of SODs in olive leaf cells
isozymes in higher plant species, and suggest that each SOD
isoenzyme must have a specific function probably related to
its cellular and subcellular localization.
The comparison of results of the percentile activity of
SOD isozymes and their transcript expression showed clear
discrepancies. Whilst the highest activity corresponded to
CuZn-SODs (I þ II), Fe-SOD was the isozyme showing the
highest expression level. The reason for these differences
could be due to the fact that the activity determinations
were carried out in leaf crude extracts, and some isozyme(s)
bound to the membranes of different cell compartments
could have been lost in the pellet fraction after the
centrifugation of homogenates, with the subsequent
activity decrease. Some of these membrane-bound SODs
could be the Fe-SOD of thylakoids, the CuZn-SOD of
crystal bodies (Fig. 3) and the xylem Mn-SOD (Fig. 3C).
On the other hand, the mRNA level was evaluated by
quantitative RT–PCR using specific primers of the cDNAs
obtained, which corresponded to conserved regions of SOD
isozymes from plant origin. However, perhaps some of the
isozymes had different sequences in these regions and,
therefore, their corresponding mRNAs could not be
determined.
The subunit molecular mass of olive Mn-SOD is in the
range 24–27 kDa reported for Mn-SODs from other higher
plant species (Baum and Scandalios 1981, Hayakawa et al.
1985, Distefano et al. 1999), and is identical to the
molecular mass determined for the mitochondrial and
peroxisomal Mn-SOD of pea leaves (Palma et al. 1998,
del Rı́o et al. 2003).
Fe-SOD is the main isoenzyme expressed in photosynthetic
cells
LCM is a new tool in the study of cell type-specific
expression. This technique was designed to be used in
animal tissues (Emmert-Buck et al. 1996), and there are few
reports on its application in plant cells (Kerk et al. 2003,
Nakazono et al. 2003, Day et al. 2005). In this work, the
combination of LCM with quantitative PCR has been used
to establish the gene expression pattern of the SOD
isozymes of olive leaves. The results obtained indicated
that the Fe-SOD gene had the highest expression in whole
leaves and isolated cells from spongy and palisade
mesophyll. This could be correlated with the well known
presence of Fe-SOD in chloroplasts, which are one of the
most abundant organelles in photosynthetic cells. In
chloroplasts of tobacco leaves, Fe-SOD is the most
abundant isoenzyme and these organelles also contain a
CuZn-SOD which is expressed in low amounts (Van Camp
et al. 1997). This situation is similar to that reported in
this work for Fe-SOD and CuZn-SOD which were
immunolocalized in chloroplasts of spongy mesophyll cells
(Fig. 3A, E). The chloroplastic CuZn-SOD of olive leaves
could be involved in the response to oxidative stress in this
plant species, such as has been described in Arabidosis
whose chloroplasts contain one CuZn-SOD and three
Fe-SODs with a differential regulation under environmental
stimuli (Kliebenstein et al. 1998).
CuZn-SOD is mainly expressed in spongy mesophyll cells
CuZn-SOD is the most abundant SOD isozyme in
many plant species (Asada et al. 1980, Bridges and Salin
1981, Bowler et al. 1994, Schinkel et al. 2001, Alscher et al.
2002). In crude extracts of olive leaves the activity of the
two CuZn-SOD isozymes represented 52% of the total SOD
activity (Fig. 1A) and this was not correlated with the
mRNA expression data. The CuZn-SOD mRNA only
represents 6% of the total mRNA in photosynthetic cells
(Fig. 2M), and this SOD is not expressed in vascular tissues.
However, it should be mentioned that the transcription
analyses were done on the basis of the partial cDNA
obtained for CuZn-SOD and, therefore, a strict relationship
between the CuZn-SOD activity and its RNA expression
cannot be established.
In olive leaves, CuZn-SOD was localized in chloroplasts, cytosol, nuclei and peroxisomes. These subcellular
localizations have also been reported for CuZn-SODs in
other plants species (Kanematsu and Asada 1991, Bueno
et al. 1995, Ogawa et al. 1995, Ogawa et al. 1996, Sandalio
et al. 1997, Corpas et al. 1998, Kernodle and Scandalios
2001). The occurrence of CuZn-SOD in the nucleus has
been reported in spinach leaves (Ogawa et al. 1995), but the
presence of CuZn-SOD in nuclear crystalline inclusions of
olive leaves is most unusual in plant cells. The function of
CuZn-SOD in the nucleus could be the protection of DNA
against superoxide-derived oxidative damage. It is interesting to note the absence of CuZn-SOD in the vascular tissue
and extracellular space of olive leaves, because in other
plant species this is the only SOD isozyme present there
(Ogawa et al. 1996, Schinkel et al. 1998, Karlsson et al.
2005).
Mn-SOD is the only SOD expressed in vascular tissues
The gene expression pattern of Mn-SOD in olive leaves
was different from that of Fe-SOD. In photosynthetic cells
(palisade and spongy tissues), transcripts of Mn-SOD,
CuZn-SOD and Fe- SOD represent 21, 6 and 73% of the
total SOD transcripts, respectively (Fig. 2M). However,
Mn-SOD mRNA was the only SOD transcript which was
present in all cell types analyzed, including phloem and
xylem cells. At the cellular level, the immunolocalization of
Mn-SOD was in agreement with the gene expression data
because the green fluorescence was present in all cell types
of leaves. At the subcellular level, Mn-SOD was localized in
mitochondria where it could be involved in the control of
superoxide radicals generated in the mitochondrial electron
Gene expression of SODs in olive leaf cells
transport chain (Bowler et al. 1994, Jiménez et al. 1997,
Alscher et al. 2002). However, in leaves from other plant
species, such as peas, Mn-SOD is present in both
mitochondria and peroxisomes, and the isozymes of both
organelles are differentially expressed during leaf senescence
(del Rı́o et al. 2003).
On the other hand, the presence of Mn-SOD (gene and
protein) in olive vascular tissue is new because so far only
CuZn-SODs had been demonstrated to have an apoplastic
or extracellular localization (Ogawa et al. 1996, Schinkel
et al. 1998). In this respect, the Mn-SOD of vascular tissue
could be involved in the biosynthesis of lignin in olive
leaves, as was proposed for CuZn-SOD in spinach
hypocotyls (Ogawa et al. 1996, Ogawa et al. 1997), but
Mn-SOD could also participate in the antioxidant defense
of vascular tissue. In phloem sap of cucumber, the presence
of several antioxidative enzymes, including CuZn-SOD,
monodehydroascorbate reductase and peroxidase, has been
demonstrated recently (Walz et al. 2002, Walz et al. 2004).
The presence of the gaseous radical nitric oxide (NOE)
in the vascular tissues of roots, stems and leaves has been
reported (Corpas et al. 2004, Corpas et al. 2006) and in
these tissues NO could be involved in the cell wall
differentiation and xylem lignification (Ferrer and RosBarceló 1999, Gabaldón et al. 2005). It is known that NO
can react with O2 E radicals to form the powerful oxidant
peroxynitrite (ONOO) (Radi 2004). The accumulation
of superoxide radicals in vascular tissue of olive leaves
(Fig. 4C) suggests that the Mn-SOD present in that
tissue could have a regulatory role in the formation of
peroxynitrite that could be used in the programmed
cell death (PCD) reactions of the xylogenesis process
(Gabaldón et al. 2005).
In summary, the results obtained in this work show
that in olive leaves the Fe-SOD and CuZn-SOD genes were
only expressed in photosynthetic cells, and the maximum
expression corresponded to Fe-SOD. On the other hand,
the Mn-SOD gene was expressed in all cell types and this
was the only SOD present in vascular tissues where it could
perform a specific function. This indicates that in olive
leaves each SOD isozyme has a different gene expression
pattern depending on the cell type, and strongly suggests
that each isozyme could have a specific function depending
on its cellular and subcellular localization.
Materials and Methods
Plant material and growth conditions
Experiments were carried out with olive seeds (Olea europaea
L., cv. Manzanillo) provided by the World Bank of Germoplasm,
Departamento de Olivicultura y Arboricultura Frutal, CIFA,
Córdoba. Seedlings were grown in the dark at 138C for 15 d in an
embryo medium and then were transferred to a DKW medium
(Driver and Kumiyuki 1984). These cultures were grown in
991
a temperature-controlled chamber at 258C for another 51 d,
with a 16 h photoperiod under Sylvania Gro-Lux (Sylvania,
Westfield, IN, USA) lighting with a photon flux density of
130–140 mmol m2 s1. Then, plants were harvested and leaves used
for the preparation of crude extracts and RNA extraction.
Crude extracts of olive leaves
All operations were performed at 0–48C. Leaves were ground
to a powder in a mortar with liquid nitrogen, and were suspended
in 100 mM Tris–HCl buffer, pH 8.0 (1/4; w/v) containing 1 mM
EDTA, 1 mM EGTA, 0.1 M NaCl, 7% (w/v) polyvinyl polypyrrolidone (PVPP), 15 mM dithiothreitol (DTT), 15 mM phenylmethylsulfonyl fluoride (PMSF) and a commercial cocktail of
protease inhibitors (AEBSF, 1,10-phenantroline, pepstatin A,
leupeptin, bestatin and E-64) (Sigma, St. Louis, MO, USA).
Homogenates were filtered through one layer of miracloth
(Calbiochem, San Diego, CA, USA) and centrifuged at 3,000 g
for 5 min (Valderrama et al. 2006). For SOD activity and Western
blots, the supernatants were passed through NAP-10 columns
(Amersham-Biosciences, Piscataway, NJ, USA) that were equilibrated with 10 mM Na-phosphate buffer, pH 6.8, and eluted with
10 mM K-phosphate buffer, pH 7.8.
Production of antibodies to Fe-SOD and Mn-SOD
The service of polyclonal antibody production from a selected
peptide of Sigma-Genosys (Cambridge, UK) was used to obtain
the antibody against Fe-SOD. A peptide of 14 amino acids from
the C-terminus of the deduced amino acid sequence of the N.
plumbaginifolia Fe-SOD (accession No. P22302) was selected. The
peptide was SWEAVSSRLKAATA which corresponds to the
residues between Ser189 and Ala202. This peptide is conserved
among different plant Fe-SODs, is hydrophilic and contains one
predicted b-turn. The selected peptide was conjugated to a carrier
protein, the keyhole limpet hemocyanin (KLH) which is derived
from marine molluscs via the thiol group of a cysteine residue
added to the N-terminus of the selected peptide using MBS
(maleimidobenzoyl-N-hydroxysuccinimide ester) chemistry. Thus
the construction KLH-[C]-SWEAVSSRLKAATA was used for
the immunization of two rabbits according to the protocol of six
immunizations per rabbit (Sigma-Genosys, Cambridge, UK). For
the preparation of the antibody to pea leaf Mn-SOD, the enzyme
was purified to homogeneity from pea (P. sativum L.) leaves, as
described by del Rı́o et al. (1983), and the antibodies were prepared
in New Zealand rabbits by Immune Systems Ltd (Paignton, UK).
The IgG fraction of serum was isolated using an Econ-Pac Serum
IgG purification kit (Bio-Rad Laboratories, Hercules, CA, USA).
Both antisera were evaluated by Western blot using the
pre-immune sera as negative control.
Enzyme activity, electrophoretic methods and Western blot analyses
Total SOD activity (EC 1.15.1.1) was assayed according to
the ferricytochrome c method of McCord and Fridovich (1969).
SOD isozymes were separated by native PAGE on 10% acrylamide
gels and visualized by a photochemical method (Beauchamp and
Fridovich 1971). Quantification of the bands was performed using
a Gel Doc system (Bio-Rad Laboratories, Hercules, CA, USA)
coupled with a high sensitive CCD camera. Band intensity was
expressed as relative transmittance units. Polypeptides were
separated by 15% SDS–PAGE as described by Corpas et al.
(1998). For immunodetection, polyclonal antibodies against
cytosolic CuZn-SOD from spinach (1 : 3,000 dilution)
(Kanematsu and Asada 1989), pea Mn-SOD (1 : 2,000 dilution)
and Fe-SOD (1 : 2,000 dilution) were used with an enhanced
992
Gene expression of SODs in olive leaf cells
chemiluminescence kit (ECL-PLUS, Amersham Pharmarcia
Biotech) and were detected with a photographic film (Hyperfilm;
Amersham Pharmarcia Biotech).
Other assays
The protein concentration of samples was determined by the
method of Bradford (1976) with bovine serum albumin (BSA) as
standard.
RNA isolation and partial cDNA cloning of the three SOD isozymes
Total RNA was isolated from olive leaves with the TrizolÕ
reagent (Gibco-BRL, Life Technologies, Paisley, UK) as described
in the manufacturer’s manual, and RNA was quantified spectrophotometrically. First-strand cDNA was synthesized from 1 mg of
total RNA primed with 3.2 mg of random primer (pdN)6 and AMV
reverse transcriptase, using the first-strand cDNA synthesis kit
(Roche, Basel, Switzerland). Using SOD sequences from the data
bank, specific oligonucleotides in conserved domains of each SOD
isoform were designed (see Table 1) and by RT–PCR the following
partial cDNAs were obtained: 435 bp for Mn-SOD (accession No.
AF427107); 435 bp for Fe-SOD (accession No. AY168776); and
312 bp for CuZn-SOD (accession No. AF426829).
Laser capture microdissection (LCM)
An LCM system (P.A.L.M. Microlaser Technologies), connected to an Olympus IX-70 microscope, was used. By means of a
cryostat (2800 Frigocut E, Reichert-Jung, Vienna, Austria), a series
of olive leaf sections, 14–16 mm thick, were obtained. Cells from
palisade and spongy mesophyll, and vascular tissue (xylem and
phloem) were selected from 5–7 leaf sections. To confirm that the
samples were representative, this procedure was repeated at least
seven times for each tissue using 3–4 leaves from different plants
each time. These microdissected cells were catapulted on an
Eppendorf tube which contained 3 ml of mineral oil and 5 ml of
10 mM Tris–HCl buffer, pH 8.3, with 50 mM KCl and 50 U of
RNase inhibitors (Roche). These samples were previously treated
with a thermal shock of 958C for 5 min, and cooled on ice, and then
used directly for the first-strand cDNA synthesis, with the same kit
mentioned above.
Real-time quantitative RT–PCR
Real-time quantitative RT–PCR was performed in 20 ml of
reaction mixture, composed of 1 ml of different cDNAs and master
mix IQTM SYBRÕ Green Supermix with a final concentration of
0.5 U of hot-start iTaqTM DNA Polymerase (Bio-Rad
Laboratories, Hercules, CA, USA), 16 mM Tris–HCl buffer,
pH 8.4, 20 mM KCl, 0.16 mM each dNTPs, 2.4 mM MgCl2,
0.5 mM gene-specific primers (see Table 1) and SYBR Green I,
8 nM fluorescein, using a iCycler iQ system (Bio-Rad).
Amplifications were performed under the following conditions:
initial polymerase activation: 958C, 4 min; then 40 cycles at 958C,
30 s; 588C, 30 s; 728C, 1 min and a final extension at 728C for 7 min.
The primers (see Table 1) were designed to anneal at different
exons, at distances large enough to avoid the appearance of falsepositive bands caused by co-amplification of contaminating DNA,
in the partial cDNA previously obtained. An internal control of
18S rRNA (accession No. L49289) was used for the normalization
of results. For microdisected samples, identical conditions of realtime quantitative RT–PCR were used, but with 50 cycles. SOD
mRNA contents were measured from at least four batches of cells,
in three replicates each. In all experiments, controls without reverse
transcriptase were included.
Electron microscopy and immunocytochemistry
Olive leaf segments (1 mm2) were fixed, dehydrated and
embedded in LR White resin as previously described by Corpas
et al. (1998). Gold sections were mounted on nickel grids and were
incubated for 1.5 h in blocking solution composed of 10 mM Tris–
HCl buffer (pH 7.6), 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and
0.02% (w/v) NaN3 (TBST) containing 5% (w/v) fetal calf serum.
The sections were then incubated for 2 h with antibodies against
the following SODs: pea Mn-SOD (1 : 500 dilution), watermelon
CuZn-SOD (1 : 300 dilution) (Bueno et al. 1995) and Fe-SOD
(1 : 2,000 dilution). Pre-immune serum was used as control. The
sections were then incubated for 1 h with goat anti-rabbit IgG
conjugated to 15 nm gold particles diluted 1/40 in TBST buffer.
Sections were post-stained in 2% (v/v) uranyl acetate for 3 min and
examined in a Zeiss (Jena, Germany) EM 10C transmission
electron microscope.
Immunohistochemical localization of Mn-SOD by CLSM
Olive leaves from plants grown under optimal conditions
were cut into 4–5 mm pieces and fixed in 4% (w/v) p-formaldehyde
in 0.1 M phosphate buffer, pH 7.4 (PB), for 3 h at room
temperature. Then they were cryoprotected by immersion in 30%
(w/v) sucrose in PB overnight at 48C. Serial sections, 60 mm thick,
were obtained by means of a cryostat (2800 Frigocut E, ReichertJung, Vienna, Austria). Free floating sections were incubated
overnight at room temperature with an antibody to pea Mn-SOD
diluted 1 : 200 in 5 mM Tris–HCl buffer, pH 7.6, 0.9% (w/v) NaCl,
containing 0.05% (w/v) sodium azide, 0.1% (w/v) BSA and 0.1%
(v/v) Triton X-100 (TBSA-BSAT). After several washes with
TBSA-BSAT, sections were incubated with biotinylated goat
anti-rabbit IgG (Pierce, Rockford, IL, USA), diluted 1 : 1,000 in
TBSA-BSAT, for 1 h at room temperature. Then, sections were
washed again and incubated with Cy2-streptavidin (Amersham
Biosciences, Piscataway, NJ, USA), diluted 1 : 1,000 in TBSABSAT, for 1.5 h at room temperature. Controls for background
staining, which was usually negligible, were performed by replacing
the corresponding primary antiserum by pre-immune serum. Leaf
sections were examined with a confocal laser scanning microscope
(Leica TCS SL, Leica Microsystems, Wetzlar, Germany).
Detection of superoxide radicals by CLSM
Detection of superoxide radicals (O2 E ) in olive leaf sections
was carried out using the fluorophore DHE, according to the
method described by Rodrı́guez-Serrano et al. (2006). Olive leaf
segments of approximately 25 mm2 were incubated for 1 h at 258C,
in darkness, with 10 mM DHE prepared in 5 mM Tris–HCl buffer,
pH 7.4, and samples were washed twice with the same buffer for
15 min each. After washing, leaf sections were embedded in a
mixture of 15% acrylamide–bisacrylamide stock solution, as
described by Peinado et al. (2000), and 100 mm thick sections, as
indicated by the vibratome scale, were cut under 10 mM
phosphate-buffered saline (PBS). Sections were then soaked in
glycerol : PBS (containing azide) (1 : 1 v/v) and mounted in the
same medium for examination with a confocal laser scanning
microscope system (Leica TCS SL, Leica Microsystems, Wetzlar,
Germany), using standard filters and collection modalities for
DHE green fluorescence ( excitation 488 nm; emission 520 nm)
and Chl autofluorescence (Chl a and b, excitation 429 and
450 nm, respectively; emission 650 and 670 nm, respectively) as
orange. As a negative control, leaf sections were pre-incubated for
1 h at 258C, in darkness, with 1 mM tetramethylpiperidinooxy
(TMP), a scavenger of superoxide radicals, and then for 1 h at 258C
with 10 mM DHE (Rodrı́guez-Serrano et al. 2006).
Gene expression of SODs in olive leaf cells
Acknowledgments
M.R.-S. and M.C. acknowledge PhD fellowships from the
Ministry of Education and Science and University of Jaén,
respectively. This work was supported by a grant from the
CICYT, Ministry of Science and Technology (AGL2003-05524),
Universidad de Jaén (OA/2/2004) and Junta de Andalucı́a (groups
CVI 0286 and CVI 0192). Olive seeds were kindly provided by the
Departamento de Olivicultura y Arboricultura Frutal, Banco de
Germoplasma Mundial, CIFA, Córdoba. The valuable help of
Dr Araceli Barceló (CIFA, Churriana, Málaga) in setting up the
in vitro culture conditions of olive plants is appreciated. We
specially acknowledge Professor Kozi Asada (Fukuyama
University, Japan) for his generous donation of the antibody
against spinach CuZn-SOD. The valuable technical help of
Miss Emperatriz Córdoba for the maintenance of in vitro plant
cultures is also appreciated. Confocal laser scanning microscopy
analyses were carried out at the Technical Services of the
University of Jaén, and special thanks are given to Miss Nieves
de la Casa-Adán for her technical assistance. The electron
microscopy assays were carried out at the Centre of Scientific
Instrumentation of the University of Granada.
References
Alché, J.D., Corpas, F.J., Rodrı́guez-Garcı́a, M.I. and
del Rı́o, L.A. (1998) Identification and immunolocalization of
superoxide dismutase isoenzymes of olive pollen. Physiol. Plant.
104: 772–776.
Allen, R.D., Webb, R.P. and Schake, S.A. (1997) Use of transgenic
plants to study antioxidant defenses. Free Radic. Biol. Med. 23:
473–9.
Alscher, R.G., Erturk, N. and Heath, L.S. (2002) Role of
superoxide dismutases (SODs) in controlling oxidative stress in
plants. J. Exp. Bot. 53: 1331–41.
Asada, K. (1994) Production and action of active oxygen species in
photosynthetic tissues. In Causes of Photooxidative Stress and
Amelioration of Defense Systems in Plants. Edited by
Foyer, C.H. and Mullineaux, P.M. pp. 77–104. CRC Press,
Inc., Boca Raton, FL, USA.
Asada, K., Kanematsu, S., Okada, S. and Hayakawa, T. (1980)
Phylogenic distribution of three types of superoxide dismutase in
organisms and in cell organelles. In Chemical and Biochemical
Aspects of Superoxide and Superoxide Dismutase. Edited by
Bannister, J.V. pp. 136–153. Elsevier, Amsterdam.
Baum, J.A. and Scandalios, J.G. (1981) Isolation and characterization of the cytosolic and mitochondrial superoxide dismutase
of maize. Arch. Biochem. Biophys. 206: 249–264.
Beauchamp, C.O. and Fridovich, I. (1971) Superoxide dismutase:
improved assays and an assay applicable to acrylamide gels.
Anal. Biochem. 44: 276–287.
Bowler, C., Camp, W.V., Montagu, M.V. and Inzé, D. (1994)
Superoxide dismutase in plants. Crit. Rev. Plant Sci. 13:
199–218.
Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein–dye binding. Anal. Biochem. 72: 248–254.
Bridges, S.M. and Salin, M.L. (1981) Distribution of ironcontaining superoxide dismutases in vascular plants. Plant
Physiol. 68: 275–278.
Bueno, P., Varela, J., Giménez-Gallego, G. and del Rı́o, L.A.
(1995) Peroxisomal copper, zinc superoxide dismutase.
993
Characterization of the isoenzyme from watermelon cotyledons.
Plant Physiol. 108: 1151–1160.
Corpas, F.J., Barroso, J.B., Carreras, A., Quiros, M., León, A.M.,
et al. (2004) Cellular and subcellular localization of endogenous
nitric oxide in young and senescent pea plants. Plant Physiol.
136: 2722–33.
Corpas, F.J., Barroso, J.B., Carreras, A., Valderrama, R.,
Palma, J.M., León, A.M., Sandalio, L.M. and del Rı́o, L.A.
(2006) Constitutive arginine-dependent nitric oxide synthase
activity in different organs of pea seedlings during plant
development. Planta 224: 246–254.
Corpas, F.J., Sandalio, L.M., Palma, J.M., Leidi, E.O.,
Hernández, J.A., Sevilla, F. and del Rı́o, L.A. (1991)
Subcellular distribution of superoxide dismutase in leaves of
ureide-producing leguminous plants. Physiol. Plant. 82: 285–291.
Corpas, F.J., Sandalio, L.M., del Rı́o, L.A. and Trelease, R.N.
(1998) Copper–zinc superoxide dismutase is a constituent
enzyme of the matrix of peroxisomes in the cotyledons of oilseed
plants. New Phytol. 138: 307–314.
Day, R.C., Grossniklaus, U., Richard, C. and Macknight, R.C.
(2005) Be more specific! Laser-assisted microdissection of plant
cells. Trends Plant Sci. 10: 397–406.
Daza, M.C., Sandalio, L.M., Quijano-Rico, M. and del Rı́o, L.A.
(1993) Isoenzyme pattern of superoxide dismutase in coffee
leaves from cultivars susceptible and resistant to the rust
Hemileia vastatrix. J. Plant Physiol. 141: 521–526.
del Rı́o, L.A., Corpas, F.J., Sandalio, L.M., Palma, J.M.,
Gómez, M. and Barroso, J.B. (2002) Reactive oxygen species,
antioxidant systems and nitric oxide in peroxisomes. J. Exp. Bot.
53: 1255–1272.
del Rı́o, L.A., Lyon, D.S., Olah, I., Glick, B. and Salin, M.L.
(1983) Immunocytochemical evidence for a peroxisomal localization of manganese superoxide dismutase in leaf protoplasts
from a higher plant. Planta 158: 216–224.
del Rı́o, L.A., Sandalio, L.M., Altomare, D.A. and Zilinskas, B.A.
(2003) Mitochondrial and peroxisomal manganese superoxide
dismutase: differential expression during leaf senescence. J. Exp.
Bot. 54: 923–933.
Distefano, S., Palma, J.M., McCarthy, I. and del Rı́o, L.A. (1999)
Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves. Planta 209: 308–313.
Droillard, M.J. and Paulin, A. (1990) Isozymes of superoxide
dismutase in mitochondria and peroxisomes isolated from petals
of carnation (Dianthus caryophyllus) during senescence. Plant
Physiol. 94: 1187–1192.
Emmert-Buck, M.R., Bonner, R.F., Smith, P.D., Chuaqui, R.F.,
Zhuang, Z., Goldstein, S.R., Weiss, R.A. and Liotta, L.A. (1996)
Laser capture microdissection. Science 274: 998–1100.
Ferrer, M.A. and Ros-Barceló, A. (1999) Differential effects of
nitric oxide on peroxidase and H2O2 production by the xylem of
Zinnia elegans. Plant Cell Environ. 22: 891–97.
Fink, R.C. and Scandalios, J.G. (2002) Molecular evolution and
structure–function relationships of the superoxide dismutase
gene families in angiosperms and their relationship to other
eukaryotic and prokaryotic superoxide dismutases. Arch.
Biochem. Biophys. 399: 19–36.
Fridovich, I. (1986) Superoxide dismutases. Adv. Enzymol. Relat.
Areas Mol. Biol. 58: 61–97.
Gabaldón, C., Gómez Ros, L.V., Pedreño, M.A. and
Ros-Barceló, A. (2005) Nitric oxide production by the differentiating xylem of Zinnia elegans. New Phytol. 165: 121–130.
Gómez, J.M., Jiménez, A., Olmos, E. and Sevilla, F. (2004)
Location and effects of long-term NaCl stress on superoxide
994
Gene expression of SODs in olive leaf cells
dismutase and ascorbate peroxidase isoenzymes of pea (Pisum
sativum cv. Puget) chloroplasts. J. Exp. Bot. 55: 119–30.
Halliwell, B. and Gutteridge, J.M.C. (2000) Free radicals in
biology and medicine. Oxford: Oxford University Press.
Hayakawa, T., Kanematsu, S. and Asada, K. (1985) Purification
and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts. Planta 166: 111–116.
Jiménez, A., Hernández, J.A., del Rı́o, L.A. and Sevilla, F. (1997)
Evidence for the presence of the ascorbate–glutathione cycle in
mitochondria and peroxisomes of pea leaves. Plant Physiol. 114:
275–84.
Karlsson, M., Melzer, M., Prokhorenko, I., Johansson, T. and
Wingsle, G. (2005) Hydrogen peroxide and expression of hipIsuperoxide dismutase are associated with the development of
secondary cell walls in Zinnia elegans. J. Exp. Bot. 56: 2085–93.
Kanematsu, S. and Asada, K. (1991) Chloroplast and cytosol
isozymes of CuZn-superoxide dismutase: their characteristic
amino acid sequences. Free Radic. Res. Commun. 12–13: 383–90.
Kernodle, S.P. and Scandalios, J.G. (2001) Structural
organization, regulation, and expression of the chloroplastic
superoxide dismutase Sod1 gene in maize. Arch. Biochem.
Biophys. 391: 137–47.
Kerk, N.M., Ceserani, T., Tausta, S.L., Sussex, I.M. and
Nelson, T.M. (2003) Laser capture microdissection of cells
from plant tissues. Plant Physiol. 132: 27–35.
Kliebenstein, D.J., Monde, R.A. and Last, R.L. (1998) Superoxide
dismutase in Arabidopsis: an eclectic enzyme family with
disparate regulation and protein localization. Plant Physiol.
118: 637–50.
McCord, J.M. and Fridovich, I. (1969) Superoxide dismutase: an
enzymic function for erythrocuprein (hemocuprein). J. Biol.
Chem. 244: 6049–6055.
Nakazono, M., Qiu, F., Borsuk, L.A. and Schnable, P.S. (2003)
Laser-capture microdissection, a tool for the global analysis of
gene expression in specific plant cell types: identification of genes
expressed differentially in epidermal cells or vascular tissues of
maize. Plant Cell 15: 583–96 (erratum in: Plant Cell 15, 1049).
Ogawa, K., Kanematsu, S. and Asada, K. (1996) Intra- and extracellular localization of ‘cytosolic’ CuZn-superoxide dismutase in
spinach leaf and hypocotyls. Plant Cell Physiol. 37: 790–799.
Ogawa, K., Kanematsu, S. and Asada, K. (1997) Generation of
superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their
association with lignification. Plant Cell Physiol. 38: 1118–1126.
Ogawa, K., Kanematsu, S., Takabe, K. and Asada, K. (1995)
Attachment of CuZn-superoxide dismutase to thylakoid membranes at the site of superoxide generation (PSI) in spinach
chloroplasts: detection by immuno-gold labelling after
rapid freezing and substitution method. Plant Cell Physiol. 36:
565–572.
Owen, R.W., Giacosa, A., Hull, W.E., Haubner, R., Wurtele, G.,
Spiegelhalder, B. and Bartsch, H. (2000) Olive-oil consumption
and health: the possible role of antioxidants. Lancet Oncol. 1:
107–112.
Palma, J.M., López-Huertas, E., Corpas, F.J., Sandalio, L.M.,
Gómez, M. and del Rı́o, L.A. (1998) Peroxisomal manganese
superoxide dismutase: purification and properties of the isozyme
from pea leaves. Physiol. Plant. 104: 720–726.
Palma, J.M., Sandalio, L.M. and del Rı́o, L.A. (1986) Manganese
superoxide dismutase and higher plant chloroplasts: a
reappraisal of a controverted cellular localization. J. Plant
Physiol. 125: 427–439.
Peinado, M.A., Torres, M.I., Thompson, R.P. and Esteban, F.J.
(2000) Immunolocalization of the HNK-1 epitope in the
autonomic innervation to the liver and upper digestive tract of
the developing rat embryo. Histochem. J. 32: 439–446.
Radi, R. (2004) Nitric oxide, oxidants, and protein tyrosine
nitration. Proc. Natl Acad. Sci. USA 101: 4003–4008.
Rhee, S.G., Kang, S.W., Jeong, W., Chang, T.S., Yang, K.S. and
Woo, H.A. (2005) Intracellular messenger function of hydrogen
peroxide and its regulation by peroxiredoxins. Curr. Opin. Cell
Biol. 17: 183–189.
Rodrı́guez-Serrano, M., Romero-Puertas, M.C., Zabalza, A.,
Corpas, F.J., Gómez, M., del Rı́o, L.A. and Sandalio, L.M.
(2006) Cadmium effect on the oxidative metabolism of pea
(Pisum sativum L.) roots. Imaging of ROS and NO production
in vivo. Plant Cell Environ. 29: 1532–1544.
Salin, M.L. (1988) Toxic oxygen species and protective systems of
the chloroplast. Physiol. Plant. 72: 681–689.
Sandalio, L.M., Dalurzo, H.C., Gómez, M., RomeroPuertas, M.C. and del Rı́o, L.A. (2001) Cadmium-induced
changes in the growth and oxidative metabolism of pea plants.
J. Exp. Bot. 52: 2115–2126.
Sandalio, L.M. and del Rı́o, L.A. (1987) Localization of superoxide dismutase in glyoxysomes from Citrullus vulgaris.
Functional implication in cellular metabolism. J. Plant Physiol.
127: 395–409.
Sandalio, L.M. and del Rı́o, L.A. (1988) Intraorganellar
distribution of superoxide dismutase in plant peroxisomes
(glyoxysomes and leaf peroxisomes). Plant Physiol. 88:
1215–1218.
Sandalio, L.M., López-Huertas, E., Bueno, P. and del Rı́o, L.A.
(1997) Immunocytochemical localization of copper, zinc superoxide dismutase in peroxisomes from watermelon (Citrullus
vulgaris Schrad.) cotyledons. Free Radic. Res. 26: 187–194.
Schinkel, H., Hertzberg, M. and Wingsle, G. (2001) A small family
of novel CuZn-superoxide dismutases with high isoelectric points
in hybrid aspen. Planta 213: 272–279.
Schinkel, H., Streller, S. and Wingsle, G. (1998) Multiple forms of
extracellular superoxide dismutase in needles, stem tissues and
seedlings of Scots pine. J. Exp. Bot. 49: 931–936.
Tsang, E.W., Bowler, C., Herouart, D., Van Camp, W.,
Villarroel, R., Genetello, C., Van Montagu, M. and Inzé, D.
(1991) Differential regulation of superoxide dismutases in plants
exposed to environmental stress. Plant Cell 3: 783–92.
Valderrama, R., Corpas, F.J., Carreras, A., GómezRodrı́guez, M.V., Chaki, M., Pedrajas, J.R., FernándezOcaña, A., del Rı́o, L.A. and Barroso, J.B. (2006) The
dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants.
Plant Cell Environ. 29: 1449–1459.
Van Camp, W., Inzé, D. and Van Montagu, M. (1997) The
regulation and function of tobacco superoxide dismutases.
Free Radic. Biol. Med. 23: 515–520.
Walz, C., Giavalisco, P., Schad, M., Juenger, M., Klose, J. and
Kehr, J. (2004) Proteomics of curcurbit phloem exudate
reveals a network of defence proteins. Phytochemistry 65:
1795–1804.
Walz, C., Juenger, M., Schad, M. and Kehr, J. (2002) Evidence for
the presence and activity of a complete antioxidant defence
system in mature sieve tubes. Plant J. 31: 189–197.
(Received February 19, 2006; Accepted May 26, 2006)