The Expression of Different Superoxide Dismutase Forms is Cell-type Dependent in Olive (Olea europaea L.) Leaves Francisco J. Corpas 1, *, Ana Ferna´ndez-Ocan˜a 2 , Alfonso Carreras 2 , Raquel Valderrama 2 , Francisco Luque 2 , Francisco J. Esteban 2 , Marı´a Rodrı´guez-Serrano 1 , Mounira Chaki 2 ,Jose´ R. Pedrajas 2 , Luisa M. Sandalio 1 , Luis A. del Rı´o 1 and Juan B. Barroso 2 1 Departamento de Bioquı´mica, Biologı´a Celular y Molecular de Plantas, Estacio ´n Experimental del Zaidı´n (EEZ), CSIC, Granada, Spain 2 Grupo de Sen ˜alizacio ´n Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), A ´ rea de Bioquı´mica y Biologı´a Molecular, Universidad de Jae´n, Spain 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. 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. Introduction Superoxide dismutases (SODs; EC 1.15.1.1) are a family of metalloenzymes that catalyze the disproportiona- tion of superoxide (O 2 E ) radicals into H 2 O 2 and O 2 , and are a first line of defense against the toxic effects of superoxide radicals produced in different cellular compart- ments (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 mito- chondria and peroxisomes (Baum and Scandalios 1981, del Rı´o 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 peroxi- somes (Droillard and Paulin 1990), and CuZn-SOD has been localized in cytosol, chloroplasts, peroxisomes and apoplast (Sandalio and del Rı´o 1987, Sandalio and del Rı´o 1988, Salin 1988, Kanematsu and Asada 1991, Ogawa et al. 1996, Ogawa et al. 1997, Sandalio et al. 1997, Corpas et al. 1998, del Rı´o 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 Rı´o 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 * Corresponding author: E-mail: [email protected]; Fax, +34-958-129600. 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]984 by guest on December 2, 2015 http://pcp.oxfordjournals.org/ Downloaded from
Transcript
The Expression of Different Superoxide Dismutase Forms is Cell-type
Dependent in Olive (Olea europaea L.) Leaves
Francisco J. Corpas1,*, Ana Fernandez-Ocana
2, Alfonso Carreras
2, Raquel Valderrama
2,
Francisco Luque2, Francisco J. Esteban
2, Marıa Rodrıguez-Serrano
1, Mounira Chaki
2, Jose R. Pedrajas
2,
Luisa M. Sandalio1, Luis A. del Rıo
1and Juan B. Barroso
2
1 Departamento de Bioquımica, Biologıa Celular y Molecular de Plantas, Estacion Experimental del Zaidın (EEZ), CSIC, Granada, Spain2 Grupo de Senalizacion Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al CSIC (EEZ), Area de Bioquımica y BiologıaMolecular, Universidad de Jaen, Spain
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.
Keywords: Olive (Olea europaea L.) — Palisade mesophyll
Sequence data from this article have been deposited in theEMBL/GenBank data libraries under accession numbersAF427107 for Mn-SOD, AY168776 for Fe-SOD and AF426829for CuZn-SOD.
Introduction
Superoxide dismutases (SODs; EC 1.15.1.1) are a
family of metalloenzymes that catalyze the disproportiona-
tion of superoxide (O2E�) radicals into H2O2 and O2, and
are a first line of defense against the toxic effects of
superoxide radicals produced in different cellular compart-
ments (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 mito-
chondria and peroxisomes (Baum and Scandalios 1981, del
Rıo 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 peroxi-
somes (Droillard and Paulin 1990), and CuZn-SOD has
been localized in cytosol, chloroplasts, peroxisomes and
apoplast (Sandalio and del Rıo 1987, Sandalio and del Rıo
1988, Salin 1988, Kanematsu and Asada 1991, Ogawa et al.
1996, Ogawa et al. 1997, Sandalio et al. 1997, Corpas et al.
1998, del Rıo 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 Rıo 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
Plant Cell Physiol. 47(7): 984–994 (2006)doi:10.1093/pcp/pcj071, available online at www.pcp.oxfordjournals.org� 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]
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 expres-
sion of SODs depending on the leaf cell type and the
presence of Mn-SOD in the vascular tissue of leaves was
demonstrated.
Results
Biochemical and molecular analysis of the SOD isozymes of
olive leaves
In crude extracts of olive leaves, the total SOD specific
activity was 1.7Umg�1 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, oligo-
nucleotides 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 CuZn-
SODs of C. annuum, L. esculentum, Solanum tuberosum and
B. Immuno blot
- Mn-SOD- Fe-SOD
- CuZn-SOD
97.466.2
45.0
31.0
21.5
14.5
kDa M
Mn-SOD Fe-SOD CuZn-SODc
0.04
0.02
Arb
itra
ry U
nit
s
C. mRNA SODs
A. Native-PAGE
Mn- SOD(15%)
Fe-SOD (33%)
I- (34%)
II- (18%)
CuZn-SODs
− +
Rel
ativ
e %
Tra
smit
tan
ce
Fig. 1 Identification of the SOD isozymes present in olive leaves.(A) Activity of SOD isozymes. SODs were separated by nativePAGE on 10% (w/v) polyacrylamide gels, and gels were stained bythe photochemical nitroblue tetrazolium method. Gels wereanalyzed using a Gel Doc system (BioRad) coupled with a highlysensitive CCD camera, and band intensities were expressed asrelative transmittance (T) units. (B) Western blot of SODs of oliveleaf extracts probed with antibodies against: pea Mn-SOD(1 : 2,000 dilution), Fe-SOD and spinach CuZn-SOD (1 : 3,000dilution). Proteins (30 mg per lane) were separated by 15% SDS–PAGE and transferred onto a PVDF membrane. (C) Transcriptanalysis (arbitrary units) of the SOD isozymes by real-timequantitative RT–PCR. Data are the mean� SEM of at least threedifferent experiments.
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 targetregion. C, F, I and L show the appearance of the tissues after LCM analysis. (M) Real-time quantitative RT–PCR transcript analysis (arbitraryunits) of the three SOD isozymes in olive leaf cells from spongy (Sm) and palisade mesophyll (Pm), phloem and xylem. Data aremean� SEM of, at least, four independent RNA samples from leaf cells obtained by LCM.
Fig. 3 Immunogold electron microscopy localization of SODs in spongy mesophyll and xylem cells of olive leaves. Representativeelectron micrographs of spongy mesophyll cells of olive leaves. The sections were incubated with antibodies against Fe-SOD (dilution1 : 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 thecytosol; CB, crystalline body; CH, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; Xy, xylem. Bars represent1.0 mm in A, C, D and E; and 0.5 mm in B.
except in epidermis cells, and the strongest fluorescence was
detected in the spongy mesophyll cells.
On the other hand, the accumulation of superoxide
radicals (O2E�) in olive leaf sections was analyzed by CLSM
using the fluorescence probe dihydroethidium (DHE). The
green fluorescence, due to O2E� 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 1mM 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 (Alche 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, Gomez 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. CuZn-
SOD 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
A
PhXy
Pm
Sm
E
E
50 µm
C
80 µm 100 µm
D
200 µm
B
Fig. 4 Immunohistochemical localization of Mn-SOD in olive leaves and detection of superoxide radicals (O2E�). (A) Appearance of an
olive leaf section under the optical microscope. (B) Cy2-streptavidin immunofluorescence (green color) attributable to the antibody againstMn-SOD (dilution 1 : 200). The olive leaf section was analyzed by confocal laser scanning microscopy (CLSM). (C) Representative imageillustrating the CLSM detection of superoxide radicals (O2E
�) in olive leaf sections incubated for 1 h at 258C, in darkness, with 10 mMDHE,where O2E
� is detected by its bright green fluorescence. (D) Representative image of a leaf section pre-incubated with 1mM 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.
transport chain (Bowler et al. 1994, Jimenez 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 Rıo 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 Ros-
Barcelo 1999, Gabaldon et al. 2005). It is known that NO�
can react with O2E� 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
(Gabaldon 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 europaeaL., cv. Manzanillo) provided by the World Bank of Germoplasm,Departamento de Olivicultura y Arboricultura Frutal, CIFA,Cordoba. Seedlings were grown in the dark at 138C for 15 d in anembryo medium and then were transferred to a DKW medium(Driver and Kumiyuki 1984). These cultures were grown in
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 of130–140 mmolm�2 s�1. Then, plants were harvested and leaves usedfor the preparation of crude extracts and RNA extraction.
Crude extracts of olive leaves
All operations were performed at 0–48C. Leaves were groundto a powder in a mortar with liquid nitrogen, and were suspendedin 100mM Tris–HCl buffer, pH 8.0 (1/4; w/v) containing 1mMEDTA, 1mM EGTA, 0.1M NaCl, 7% (w/v) polyvinyl poly-pyrrolidone (PVPP), 15mM dithiothreitol (DTT), 15mM phenyl-methylsulfonyl fluoride (PMSF) and a commercial cocktail ofprotease 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 gfor 5min (Valderrama et al. 2006). For SOD activity and Westernblots, the supernatants were passed through NAP-10 columns(Amersham-Biosciences, Piscataway, NJ, USA) that were equili-brated with 10mM Na-phosphate buffer, pH6.8, and eluted with10mM K-phosphate buffer, pH7.8.
Production of antibodies to Fe-SOD and Mn-SOD
The service of polyclonal antibody production from a selectedpeptide of Sigma-Genosys (Cambridge, UK) was used to obtainthe antibody against Fe-SOD. A peptide of 14 amino acids fromthe C-terminus of the deduced amino acid sequence of the N.plumbaginifolia Fe-SOD (accession No. P22302) was selected. Thepeptide was SWEAVSSRLKAATA which corresponds to theresidues between Ser189 and Ala202. This peptide is conservedamong different plant Fe-SODs, is hydrophilic and contains onepredicted b-turn. The selected peptide was conjugated to a carrierprotein, the keyhole limpet hemocyanin (KLH) which is derivedfrom marine molluscs via the thiol group of a cysteine residueadded to the N-terminus of the selected peptide using MBS(maleimidobenzoyl-N-hydroxysuccinimide ester) chemistry. Thusthe construction KLH-[C]-SWEAVSSRLKAATA was used forthe immunization of two rabbits according to the protocol of siximmunizations per rabbit (Sigma-Genosys, Cambridge, UK). Forthe preparation of the antibody to pea leaf Mn-SOD, the enzymewas purified to homogeneity from pea (P. sativum L.) leaves, asdescribed by del Rıo et al. (1983), and the antibodies were preparedin New Zealand rabbits by Immune Systems Ltd (Paignton, UK).The IgG fraction of serum was isolated using an Econ-Pac SerumIgG purification kit (Bio-Rad Laboratories, Hercules, CA, USA).Both antisera were evaluated by Western blot using thepre-immune sera as negative control.
Enzyme activity, electrophoretic methods and Western blot analyses
Total SOD activity (EC 1.15.1.1) was assayed according tothe ferricytochrome c method of McCord and Fridovich (1969).SOD isozymes were separated by native PAGE on 10% acrylamidegels and visualized by a photochemical method (Beauchamp andFridovich 1971). Quantification of the bands was performed usinga Gel Doc system (Bio-Rad Laboratories, Hercules, CA, USA)coupled with a high sensitive CCD camera. Band intensity wasexpressed as relative transmittance units. Polypeptides wereseparated by 15% SDS–PAGE as described by Corpas et al.(1998). For immunodetection, polyclonal antibodies againstcytosolic 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
chemiluminescence kit (ECL-PLUS, Amersham PharmarciaBiotech) and were detected with a photographic film (Hyperfilm;Amersham Pharmarcia Biotech).
Other assays
The protein concentration of samples was determined by themethod of Bradford (1976) with bovine serum albumin (BSA) asstandard.
RNA isolation and partial cDNA cloning of the three SOD isozymes
Total RNA was isolated from olive leaves with the Trizol�
reagent (Gibco-BRL, Life Technologies, Paisley, UK) as describedin the manufacturer’s manual, and RNA was quantified spectro-photometrically. First-strand cDNA was synthesized from 1mg oftotal RNA primed with 3.2mg of random primer (pdN)6 and AMVreverse transcriptase, using the first-strand cDNA synthesis kit(Roche, Basel, Switzerland). Using SOD sequences from the databank, specific oligonucleotides in conserved domains of each SODisoform were designed (see Table 1) and by RT–PCR the followingpartial cDNAs were obtained: 435 bp for Mn-SOD (accession No.AF427107); 435 bp for Fe-SOD (accession No. AY168776); and312 bp for CuZn-SOD (accession No. AF426829).
Laser capture microdissection (LCM)
An LCM system (P.A.L.M. Microlaser Technologies), con-nected to an Olympus IX-70 microscope, was used. By means of acryostat (2800 Frigocut E, Reichert-Jung, Vienna, Austria), a seriesof olive leaf sections, 14–16mm thick, were obtained. Cells frompalisade and spongy mesophyll, and vascular tissue (xylem andphloem) were selected from 5–7 leaf sections. To confirm that thesamples were representative, this procedure was repeated at leastseven times for each tissue using 3–4 leaves from different plantseach time. These microdissected cells were catapulted on anEppendorf tube which contained 3ml of mineral oil and 5 ml of10mM Tris–HCl buffer, pH8.3, with 50mM KCl and 50U ofRNase inhibitors (Roche). These samples were previously treatedwith a thermal shock of 958C for 5min, and cooled on ice, and thenused directly for the first-strand cDNA synthesis, with the same kitmentioned above.
Real-time quantitative RT–PCR
Real-time quantitative RT–PCR was performed in 20 ml ofreaction mixture, composed of 1ml of different cDNAs and mastermix IQTM SYBR� Green Supermix with a final concentration of0.5U of hot-start iTaqTM DNA Polymerase (Bio-RadLaboratories, Hercules, CA, USA), 16mM Tris–HCl buffer,pH 8.4, 20mM KCl, 0.16mM each dNTPs, 2.4mM 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, 4min; then 40 cycles at 958C,30 s; 588C, 30 s; 728C, 1min and a final extension at 728C for 7min.The primers (see Table 1) were designed to anneal at differentexons, at distances large enough to avoid the appearance of false-positive bands caused by co-amplification of contaminating DNA,in the partial cDNA previously obtained. An internal control of18S rRNA (accession No. L49289) was used for the normalizationof results. For microdisected samples, identical conditions of real-time quantitative RT–PCR were used, but with 50 cycles. SODmRNA contents were measured from at least four batches of cells,in three replicates each. In all experiments, controls without reversetranscriptase were included.
Electron microscopy and immunocytochemistry
Olive leaf segments (1mm2) were fixed, dehydrated andembedded in LR White resin as previously described by Corpaset al. (1998). Gold sections were mounted on nickel grids and wereincubated for 1.5 h in blocking solution composed of 10mM Tris–HCl buffer (pH7.6), 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20 and0.02% (w/v) NaN3 (TBST) containing 5% (w/v) fetal calf serum.The sections were then incubated for 2 h with antibodies againstthe following SODs: pea Mn-SOD (1 : 500 dilution), watermelonCuZn-SOD (1 : 300 dilution) (Bueno et al. 1995) and Fe-SOD(1 : 2,000 dilution). Pre-immune serum was used as control. Thesections were then incubated for 1 h with goat anti-rabbit IgGconjugated to 15 nm gold particles diluted 1/40 in TBST buffer.Sections were post-stained in 2% (v/v) uranyl acetate for 3min andexamined in a Zeiss (Jena, Germany) EM 10C transmissionelectron microscope.
Immunohistochemical localization of Mn-SOD by CLSM
Olive leaves from plants grown under optimal conditionswere cut into 4–5mm pieces and fixed in 4% (w/v) p-formaldehydein 0.1M phosphate buffer, pH 7.4 (PB), for 3 h at roomtemperature. Then they were cryoprotected by immersion in 30%(w/v) sucrose in PB overnight at 48C. Serial sections, 60mm thick,were obtained by means of a cryostat (2800 Frigocut E, Reichert-Jung, Vienna, Austria). Free floating sections were incubatedovernight at room temperature with an antibody to pea Mn-SODdiluted 1 : 200 in 5mM 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 withTBSA-BSAT, sections were incubated with biotinylated goatanti-rabbit IgG (Pierce, Rockford, IL, USA), diluted 1 : 1,000 inTBSA-BSAT, for 1 h at room temperature. Then, sections werewashed again and incubated with Cy2-streptavidin (AmershamBiosciences, Piscataway, NJ, USA), diluted 1 : 1,000 in TBSA-BSAT, for 1.5 h at room temperature. Controls for backgroundstaining, which was usually negligible, were performed by replacingthe corresponding primary antiserum by pre-immune serum. Leafsections 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 (O2E�) in olive leaf sections
was carried out using the fluorophore DHE, according to themethod described by Rodrıguez-Serrano et al. (2006). Olive leafsegments of approximately 25mm2 were incubated for 1 h at 258C,in darkness, with 10mM DHE prepared in 5mM Tris–HCl buffer,pH7.4, and samples were washed twice with the same buffer for15min each. After washing, leaf sections were embedded in amixture of 15% acrylamide–bisacrylamide stock solution, asdescribed by Peinado et al. (2000), and 100mm thick sections, asindicated by the vibratome scale, were cut under 10mMphosphate-buffered saline (PBS). Sections were then soaked inglycerol : PBS (containing azide) (1 : 1 v/v) and mounted in thesame medium for examination with a confocal laser scanningmicroscope system (Leica TCS SL, Leica Microsystems, Wetzlar,Germany), using standard filters and collection modalities forDHE green fluorescence (� excitation 488 nm; � emission 520 nm)and Chl autofluorescence (Chl a and b, � excitation 429 and450 nm, respectively; � emission 650 and 670 nm, respectively) asorange. As a negative control, leaf sections were pre-incubated for1 h at 258C, in darkness, with 1mM tetramethylpiperidinooxy(TMP), a scavenger of superoxide radicals, and then for 1 h at 258Cwith 10mM DHE (Rodrıguez-Serrano et al. 2006).
M.R.-S. and M.C. acknowledge PhD fellowships from theMinistry of Education and Science and University of Jaen,respectively. This work was supported by a grant from theCICYT, Ministry of Science and Technology (AGL2003-05524),Universidad de Jaen (OA/2/2004) and Junta de Andalucıa (groupsCVI 0286 and CVI 0192). Olive seeds were kindly provided by theDepartamento de Olivicultura y Arboricultura Frutal, Banco deGermoplasma Mundial, CIFA, Cordoba. The valuable help ofDr Araceli Barcelo (CIFA, Churriana, Malaga) in setting up thein vitro culture conditions of olive plants is appreciated. Wespecially acknowledge Professor Kozi Asada (FukuyamaUniversity, Japan) for his generous donation of the antibodyagainst spinach CuZn-SOD. The valuable technical help ofMiss Emperatriz Cordoba for the maintenance of in vitro plantcultures is also appreciated. Confocal laser scanning microscopyanalyses were carried out at the Technical Services of theUniversity of Jaen, and special thanks are given to Miss Nievesde la Casa-Adan for her technical assistance. The electronmicroscopy assays were carried out at the Centre of ScientificInstrumentation of the University of Granada.
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(Received February 19, 2006; Accepted May 26, 2006)
