Content uploaded by Luit Slooten
Author content
All content in this area was uploaded by Luit Slooten
Content may be subject to copyright.
Plant Physiol.
(1
996)
11
2:
1703-1 71
4
Enhancement
of
Oxidative Stress Tolerance
in
Transgenic
-
Tobacco Plants Overproducing Fe-Superoxide
Dismutase
in
Chloroplasts’
Wim
Van Camp, Katelijne Capiau, Marc Van Montagu, Dirk Inzé, and
Luit
Slooten*
Laboratorium voor Genetika, Department of Genetics, Flanders lnteruniversity lnstitute for Biotechnology,
Universiteit Gent, K.L. Ledeganckstraat
35,
8-9000 Gent, Belgium (W.V.C., M.V.M.,
D.I.);
Laboratoire Associé
de I’lnstitut National de Ia Recherche Agronomique, France
(D.I.);
and Laboratorium voor Biofysica, Faculteit
Wetenschappen, Vrije Universiteit Brussel, Pleinlaan
2,
1050
Brussels, Belgium (K.C., L.S.)
A
chimeric gene consisting of the coding sequence for chloro-
plastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana,
coupled to the chloroplast targeting sequence from the pea
ribulose-1,5-bisphosphate
carboxylase/oxygenase small subunit,
was expressed
in
Nicofiana fabacum cv Petit Havana
SR1.
Expres-
sion of the transgenic FeSOD protected both the plasmalemma and
photosystem
I1
against superoxide generated during illumination of
leaf discs impregnated with methyl viologen. By contrast, overpro-
duction of a mitochondrial MnSOD from
Nicotiana
plumbaginifolia
in
the chloroplasts of cv
SR1
protected only the plasmalemma, but
not photosystem
11,
against methyl viologen
(L.
Slooten,
K.
Capiau,
W.
Van Camp,
M.
Van Montagu,
C.
Sybesma, D. lnzé
[19951
Plant
Physiol
107: 737-750).
The difference
in
effectiveness correlates
with different membrane affinities of the transgenic FeSOD and
MnSOD. Overproduction of FeSOD does not confer tolerance to
HzO2, singlet oxygen, chilling-induced photoinhibition in leaf disc
assays, or to salt stress at the whole plant level.
In
nontransgenic
plants, salt stress led to a 2- to 3-fold increase in activity, on a
protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD,
ascorbate peroxidase, dehydroascorbate reductase, and glutathione
reductase. In FeSOD-overproducing plants under salt stress, the
induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed,
whereas induction of a water-soluble chloroplastic ascorbate per-
oxidase isozyme was promoted.
~
The photosynthetic electron transport chain contains at
the acceptor side of PSI a number of autooxidizable en-
zymes that reduce oxygen to superoxide (reviewed by
Badger, 1985; Asada and Takahashi, 1987; Asada, 1994).
These include Fd (Furbank and Badger, 1982; Hosein and
Palmer, 1983) and the Fe-S centers
X
or A/B in the aprotic
This work was supported by grants from the Vlaams Actie-
programma Biotechnologie (no. 067), the Belgian National Fund
for Scientific Research (no. 3.0104.90), the Belgian Programme on
Interuniversity Poles
of
Attraction (Prime Minister’s Office, Sci-
ence Policy Programming, no. 38), the International Atomic
Agency (no. 5285), and the International Human Frontier Science
Program (IHFSP RG 434/94M). W.V.C. is a postdoctoral fellow of
the Belgian National Fund for Scientific Research (Belgium). D.I. is
a Research Director of the Institut National de Ia Recherche
Agronomique (France).
*
Corresponding author; e-mail
Islooten8vnet3.vub.ac.be;
fax
32-2-6293389.
membrane interior of the PSI reaction center complex (Ta-
kahashi and Asada, 1988). The superoxide may mediate
cyclic electron flow around PSI (Asada, 1994) or it may
diffuse it to the stromal membrane surface, where it is
dismutated to oxygen and H,O, in nonenzymic and enzy-
mic reactions (see below). Recent evidence suggests that
superoxide and
H,O,
can also be produced by PSII during
high-light treatment of thylakoids or intact chloroplasts
(Landgraf et al., 1995). H,O, at
10
PM
inhibits
CO,
fixation
(Kaiser, 1979); at concentrations as low as
1
PM
it causes a
substantial inactivation of thiol-modulated Calvin cycle
enzymes (Buchanan, 1980, 1991; Takeda et al., 1995). Su-
peroxide can inactivate some metal-containing enzymes
such as the Fd-linked nitrate reductase, catalase, and per-
oxidases (for review, see Asada and Takahashi, 1987). But
the chief danger is that H,O, can react with reduced metal
ions, especially Fe, resulting in the formation of the hy-
droxyl radical. The hydroxyl radical initiates self-
propagating reactions leading to peroxidation of mem-
brane lipids, base mutations, breakage of DNA strands,
and destruction of proteins (Asada and Takahashi, 1987;
Halliwell, 1987; Bowler et al., 1992). In addition, there is
some evidence that part of the superoxide generated in
illuminated chloroplasts diffuses toward the thylakoid lu-
men. Because of the low pH in the lumen during illumina-
tion, the superoxide can be protonated in that compart-
ment, yielding the perhydroxyl radical, which, unlike the
superoxide anion, can initiate lipid peroxidation directly
(for review, see Asada and Takahashi, 1987). The condi-
tions leading to damage caused by active oxygen species
will be referred to as oxidative stress.
Formation of active oxygen species in the chloroplasts is
enhanced when carbon assimilation is inhibited. Oxidative
Abbreviations: APx, ascorbate peroxidase (EC
1.11.1.11);
AT,
3-amino-1,2,4-triazole; DHAR, dehydroascorbate reductase (EC
1.8.5.1); eosin,
2,4,5,7-tetrabromo-fluorescein;
F,/
F,,,,
quantum
efficiency for exciton trapping by the PSII reaction center in dark-
adapted material; GA3P-DH, NADP-dependent glyceraldehyde-3-
phosphate dehydrogenase (EC 1.2.1.13); GR, glutathione reductase
(EC 1.6.4.2); KCN, potassium cyanide; MDHAR, monodehy-
droascorbate reductase (EC 1.6.5.4); MV, methyl viologen; SOD,
superoxide dismutase (EC 1.15.1.1); trFeSOD, transgenic (overpro-
duced) FeSOD; trMnSOD, transgenic (overproduced) MnSOD.
1703
1704
Van
Camp
et al. Plant
Physiol.
Vol.
11
2,
1996
stress arises, for example, when high irradiance is com-
bined with chilling temperatures, drought, or heat (Bowler
et al., 1992). Salt stress is another condition that may give
rise to oxidative stress, as suggested by the increase in
activities of antioxidant enzymes in response to high salin-
ity, and by the correlation of salt tolerance with antioxidant
enzyme levels (Gossett et al., 1994; Olmos et al., 1994;
Hernandez et al., 1995; Sehmer et al., 1995).
An elaborate antioxidant system, a defense against oxi-
dative stress, is present in the chloroplasts. SOD catalyzes
the dismutation of superoxide into oxygen and H,O,.
SODs are classified, according to their metal cofactor, as
FeSOD, MnSOD, or Cu
/
ZnSOD. Chloroplasts generally
contain Cu/ZnSOD and, in a number of plant species,
FeSOD (Van Camp et al., 1994a). APx reduces H,O, to
water, with ascorbate as the electron donor. Chloroplastic
APx was recently found to occur in a stromal and a
membrane-bound form (Miyake and Asada, 1992). The
re-reduction of the reaction product of APx, monodehy-
droascorbate, proceeds along different pathways, depend-
ing on the type of APx involved. The reaction product of
stromal APx is reduced by NADPH, either directly or via
glutathione. The enzymes taking part in this so-called
ascorbate-glutathione cycle (Foyer and Halliwell, 1976; Na-
kano and Asada, 1981) are GR, DHAR, and MDHAR
(for reviews, see Asada and Takahashi, 1987; Halliwell,
1987). In contrast, monodehydroascorbate produced by
membrane-bound APx is re-reduced directly by Fd (Mi-
yake and Asada, 1994).
The overproduction of antioxidant enzymes provides a
way to study the role of these enzymes in the antioxidant
system, and to study the contribution of oxidative stress
tolerance to tolerance to physiological types of stress. Over-
production of SOD in the chloroplasts has been found to
result in enhanced oxidative stress tolerance in transgenic
tobacco (Bowler et al., 1991; Sen Gupta et al., 1993a; Foyer
et al., 1994; Van Camp et al., 1994b; Slooten et al., 1995),
alfalfa (McKersie et al., 1993), potato (Perl et al., 1993), and,
according to preliminary data, in cotton (Allen, 1995). Sim-
ilar results were obtained with overproduction of SOD in
the mitochondria of alfalfa (McKersie et al., 1993) and in
the cytosol of potato (Perl et al., 1993). In many of these
studies, oxidative stress tolerance was assessed in assays
based on the use of MV. This herbicide passes electrons
from various electron transport chains to oxygen, generat-
ing superoxide. During illumination, MV generates super-
oxide primarily in the chloroplasts (Halliwell, 1984; Slooten
et al., 1995) and thus simulates the oxidative stress compo-
nent of the environmental stresses. In addition, an en-
hanced tolerance to freezing stress in transgenic alfalfa
overproducing SOD in the chloroplasts has been reported
(McKersie et al., 1993), and transgenic tobacco overproduc-
ing SOD in the chloroplasts exhibits an enhanced tolerance
to chilling in the dark (Foyer et al., 1994) or in the light (Sen
Gupta et al., 1993a).
We have shown that expression of plant mitochondrial
MnSOD in the chloroplasts
of
transgenic Nicofiana
fabacum
cv SR1 and cv PBD6 reduces cellular damage generated by
treatment with MV (Bowler et al., 1991; Slooten et al., 1995)
or ozone (Van Camp et al., 1994b). In PBD6, overproduc-
tion of MnSOD had a clear protective effect on MV-induced
ion leakage and, albeit to a lesser extent, also on MV-
induced inactivation of the PSII reaction center. In SR1,
protection was observed with regard to ion leakage, but
not with regard to the PSII reaction center (Slooten et al.,
1995). Apparently, improvement in the antioxidant defense
was more pronounced at the plasmalemma than at
PSII,
in
spite of the fact that the overproduced SOD is located in the
chloroplasts. It is possible that the pathway from MV-
mediated superoxide generation at PSI to damage at PSII is
poorly accessible to plant mitochondrial MnSOD expressed
in the chloroplasts. This lack of protection of PSII might be
specific for overproduced plant mitochondrial MnSOD,
since chloroplastic overproduction of Cu/ ZnSOD in potato
(Perl et al., 1993) and of Escherickia coli MnSOD in tobacco
(Foyer et al., 1994) does alleviate the inhibition of photo-
synthesis by MV.
Here we report that overproduction of FeSOD in the
chloroplasts of
N.
tabacum
cv SR1 protects both the plas-
malemma and PSII from MV-induced damage. This dem-
onstrates that FeSOD provides better protection of chloro-
plasts than MnSOD. We argue that this is because FeSOD,
in contrast to MnSOD, is indigenous to the chloroplasts.
Functional differences between the FeSOD and MnSOD
enzymes of
E.
coli
have been reported with regard to pro-
tection of DNA and proteins (Hopkin et al., 1992). The data
presented in this study, to our knowledge, are the first to
demonstrate that in plant chloroplasts, FeSOD and MnSOD
have different protective properties that may be related to
their suborganellar location. We also show that overpro-
duction of FeSOD interferes with signal pathways, leading
to induction of cytosolic Cu/ZnSOD during salt stress. In
addition, overproduction of FeSOD leads to induction of
chloroplastic APx during salt stress.
MATERIALS AND METHODS
Ceneration
of
FeSOD-Overproducing Plants
To construct pEXSOD10, a PCR product was generated
with the 5' primer TCAAGTGCTGTAGATCTAAAC-
TACGTCCTC (positions 54-83 of pSOD10,
BglII
site intro-
duced) and the 3' primer ACACACAAAACGGATCCA-
CACTCAGAAAAG (complementary to positions 842-871
of pSOD10, BamHI site introduced) using pSODlO as a
template. pSODlO contains an FeSOD cDNA from Arabi-
dopsis
fkaliana
(Van Camp et al., 1990). The amplified frag-
ment was cloned in a dT-tailed, HincII-digested pUC18.
The fragment was re-excised by a BamHI-BglII digest and
cloned into the BamHI site of pKAHl (Bowler et al., 1991).
This generates an in-frame fusion of the chloroplast transit
peptide derived from the small subunit of Rubisco from
pea with the FeSOD mature protein. Compared with the
mature FeSOD expressed in A.
fkaliana,
the mature fusion
protein contains an additional Met after processing. The
chimeric construct was cloned as a ClaI-BamHI fragment
into the ClaI-BamHI-digested binary vector pGSJ780A
(Bowler et al., 1991). This generated the cassette for chlo-
roplastic FeSOD overproduction under control of the cau-
Tobacco Overproducing Fe-Superoxide Dismutase
1705
liflower mosaic virus 35s promoter. Transformation of
Nicotiana
tabacum
var Petit Havana SR1 with this cassette,
which was named pEXSOD10, was according to the meth-
ods of De Block et al. (1987).
Plant Material
Plants were grown in pots
on
peat-based compost con-
taining fertilizer and were watered with demineralized
water to which, after bolting,
1
vol
%
of a commercial
fertilizer (NPK 6-3-6) was added. Unless otherwise indi-
cated, the plants were grown in a 12-h light/12-h dark
cycle, with day and night temperatures of about 22 and
16"C, respectively. The light, provided by mercury-halogen
vapor lamps with
a
daylight spectrum (HQI-T, Osram,
Munich, Germany), had an intensity of approximately 135
pmol m-'
s-*.
Unless indicated otherwise, the experiments
were carried out
on
the seventh to ninth leaves from the
top at
10
to 12 weeks after sowing.
In some experiments the plants were transferred to a
growth chamber at
1
month after sowing (i.e. at the fifth-
leaf stage), at which time they were grown in a 12-h light
/
12-h dark cycle at 22/15"C. In the course of
8
d the light
intensity was gradually raised from 180 to 650 pmol m-'
s-*.
The light intensity remained at this leve1 during the
next 14 d, and the experiments were performed at the end
of this period.
In the salt-stress experiments, plants were grown at an
intensity of 65 pmol m-'s-', with day and night temper-
atures of about 25 and 20"C, respectively. The plants were
watered with demineralized water, to which 60 mM NaCl
was added from d 12 after sowing.
Assessment
of
Oxidative Stress Tolerance
Leaf discs of approximately 1.5 cm2 were preincubated
overnight with reagents that mediate the formation
of
toxic
oxygen species during subsequent illumination of the leaf
discs: MV generates superoxide (Bowler et al., 1991), AT
generates H,O, (see "Results"), and eosin generates singlet
oxygen (Knox and Dodge, 1985). The MV assays were
carried out as described by Slooten et al. (1995). In the case
of AT or eosin treatment, the leaf discs were placed in Petri
dishes containing 3 mL of aqueous solutions of these com-
pounds. The Petri dishes were put in a gas-tight, thermo-
statted container with a glass lid. The leaf discs were illu-
minated with white light from mercury-halogen vapor
lamps, filtered by
8
cm of water. The light intensity was 360
pmol m-' s-I. The temperature in the Petri dishes during
illumination was maintained at 18°C. After illumination
the leaf discs were incubated in the dark for at least 2 h to
ensure complete dark adaptation prior to measuring
F,/
F,,,
as a measure of the activity of the PSII reaction cen-
ters. This ratio gives the exciton trapping efficiency when
a11 photochemical traps are open (Genty et al., 1989). The
fluorescence measurements were made as described by
Slooten et al. (1995). The conductance of the floating solu-
tions was determined as a measure of ion leakage from the
leaf discs, which was due to lipid peroxidation of the cell
membranes (Slooten et al., 1995).
For the photoinhibition experiments, the leaf discs were
floated overnight
on
an aqueous solution of 75
p~
of the
chloroplast translation inhibitor chloramphenicol prior to
illumination. At this concentration chloramphenicol does
not act as a PSI electron acceptor (Okada et al., 1991).
Biochemical Assays
The basal medium for the preparation of leaf disc ex-
tracts for enzyme assays contained 50 mM potassium phos-
phate, 0.1% Triton X-100, 20% (w/v) SUC, 2% polyvinyl-
polypyrrolidone,
1
mM EDTA,
1
mM EGTA,
1
mM PMSF, 4
mM benzamidine, 4 mM caproic acid, and KOH to pH 7.6.
SUC and
polyvinylpolypyrrolidone
were omitted from the
basal medium used for the preparation of extracts for SOD
determinations. Whole-leaf extracts were prepared by
grinding leaf discs (0.2
g)
in an ice-cold mortar with 1.2 mL
of
basal medium supplemented with the following addi-
tions: for APx, GR, and MDHAR,
30
mM sodium ascorbate;
for GA3P-DH,
30
mM sodium ascorbate and 0.05% (w/v)
P-mercaptoethanol; for DHAR, 2 mM sodium ascorbate;
and for SOD, 0.05% (w/v) P-mercaptoethanol and 0.1
mg/mL BSA. The extracts were centrifuged for
30
min at
40,OOOg. The supernatants were stored on ice and used the
same day. The assays of APx, DHAR, GR, and MDHAR
were carried out as described previously (Slooten et al.,
1995). GA3P-DH was assayed as described by Stitt et al.
(1989). For these assays,
1
unit equals
1
pmol of substrate
converted per min.
APx activity on nondenaturing polyacrylamide gels was
detected as described by Mittler and Zilinskas (1993). SOD
activity was determined first in a solution assay, and then
after an activity staining following electrophoresis
on
non-
denaturing polyacrylamide gels (Bowler et al., 1991). The
gels were stained as described previously (Slooten et al.,
1995) and then scanned with a one-dimensional scanning
densitometer. The relative contribution of each of the SOD
isoforms to the overall activity was determined from the
contribution of the area under the corresponding peak of
the densitogram to the total area. The overall
SOD
activity
in leaf disc extracts was determined in the solution assay
with the same assay mixture. In the solution assay one unit
of SOD activity causes a half-maximal inhibition of the rate
of light-induced, riboflavin-mediated reduction of ni-
troblue tetrazolium. In both assays the activity was mea-
sured with and without 2 mM KCN, which causes a virtu-
ally complete inhibition of Cu/ZnSOD (Geller and Winge,
1984).
For
the preparation and washing of chloroplasts we
used the low-salt grinding and resuspension media de-
scribed by Cerovic and Plesnicar (1984), except that
P-mercaptoethanol was added to both media at
0.05%
(v/
v). Chloroplasts were prepared by grinding 4
g
of leaf
material for
3
s
in 40 mL of ice-cold grinding medium in a
blender (Waring). The homogenate was filtered through
four layers of Miracloth (Calbiochem) and centrifuged for
1.5 min at 600g at 4°C. The sedimented chloroplasts were
washed twice in 20 mL of resuspension medium, followed
by centrifugation as before.
1706
Van Camp et
al.
Plant
Physiol.
Vol.
11
2,
1996
Chlorophyll in whole-leaf extracts was determined as
described previously (Slooten et al., 1995). Protein was
estimated according to Bradford (1976) using BSA as a
standard.
A11
absorbance measurements were carried out
on a spectrophotometer (Cary 2300, Varian Techron, Mul-
grave, Victoria, Australia).
RESULTS
SOD
Activity Levels in Nonstressed Transgenic and
Control Plants
To overexpress chloroplastic FeSOD from
A.
thaliana
in
tobacco chloroplasts, we made a chimeric gene in which
the FeSOD coding region was fused in frame with the
coding sequence for the chloroplast transit peptide of the
Rubisco small subunit. The expression of this gene fusion
was driven by the 35s promoter from the cauliflower mo-
saic virus. Relatively high levels of FeSOD activity were
observed in leaf extracts of seven primary transformants.
Six of these lines, each containing a single copy of the
transgene, were made homozygous and selected for further
study. Most of the data shown below were obtained with
line SRl-14, but the results were confirmed with severa1
other lines.
Figure
1
shows the banding patterns observed after non-
denaturing electrophoresis of leaf extracts or chloroplasts
from mature leaves, followed by SOD activity staining. The
bands are numbered in order of increasing mobility. In leaf
extracts from nontransgenic plants (Fig. lA, dashed line),
three bands were observed after staining without KCN:
1
1
Figure
1.
Densitogram
of
the SOD-banding pattern of leaf extracts
(A
and
B)
and chloroplasts (C and D) from FeSOD-overproducing plants
of line
SR1-14
(solid lines) and control plants (dashed lines). The gels
were stained in the absence
(A
and
C)
or presence
(B
and D) of
2
mM
KCN. Migration
is
from left to right. Bands 1 and
2,
Engineered
FeSOD; band
3,
a mixture of cytosolic Cu/ZnSOD and endogenous
FeSOD; band
3’,
endogenous FeSOD; bands
4
and
5,
chloroplastic
Cu/ZnSOD.
Table
1.
SOD activities
in
leaf extracts from FeSOD-overproducing
and control plants
Mean
2
SE
(13 plants).
Activity
SR1 SR1-14
SOD
Species
unitshg
chlorophyll
MnSOD 0.62
?
0.20
0.52
?
0.1
7
FeSOD 1.88
2
0.1
9 13.85
2
0.63
Chloroplastic Cu/ZnSOD
5.23
k
1.33
3.67
k
0.79
Cytosolic Cu/ZnSOD
6.04
2
0.64
6.70
Ifr
0.80
band 3 contains both chloroplastic FeSOD and cytosolic
Cu/ZnSOD, and bands
4
and 5 represent chloroplastic
Cu/ZnSOD (Slooten et al., 1995). Preincubation with
KCN
prior to staining caused a complete inactivation
of
Cu/
ZnSOD; the remaining activity represents only FeSOD (Fig.
lB,
dashed line, band 3’). In chloroplast extracts from
nontransgenic plants (Fig. lC, dashed line), cytosolic Cu/
ZnSOD was absent, and band 3’ again corresponds to
chloroplastic FeSOD. Pretreatment with KCN abolished the
chloroplastic Cu/ ZnSOD activity, but had little effect on
the activity of chloroplastic FeSOD (Fig. lD, dashed line).
Extracts from immature leaves also contain a small, slow-
moving band corresponding to mitochondrial MnSOD (not
shown) (Slooten et al., 1995).
Leaf and chloroplast extracts from FeSOD-overproducing
plants (line SR1-14) exhibited
two
extra bands (nos.
1
and
2)
representing trFeSOD (Fig. 1, solid lines). Similar results
were obtained with other transgenic lines (not shown). In
the KCN-treated lanes, the endogenous FeSOD shows up as
a small shoulder at the fast-moving side of band 2 (Fig.
1,
B
and D, solid lines). A comparison with the corresponding
traces from nontransgenic plants indicates that FeSOD-
overproducing plants contained about one-half of the en-
dogenous FeSOD activity that was found in nontransgenic
plants. Thus, either trFeSOD leads to a suppression of the
endogenous FeSOD activity, or some of the endogenous
FeSOD forms heterodimers with trFeSOD.
We used the SOD activity gels to estimate the activities of
the different SOD isoforms, as described in ”Materials and
Methods.” The staining intensities of the bands on the
activity gels indicate that KCN, which was used to inacti-
vate Cu/ZnSOD, also caused an inhibition of both endog-
enous and overproduced FeSOD. This can be seen most
clearly in extracts prepared from isolated chloroplasts,
which do not contain cytosolic Cu/ZnSOD (Fig. 1, C and
D). The KCN inhibition amounted to
20
to 30% for both
endogenous and overproduced FeSOD. In the gels without
KCN, the contribution of cytosolic Cu/ZnSOD to band 3
was obtained by subtracting the contribution of FeSOD.
This contribution was estimated from the KCN-treated
gels, taking the observed KCN inhibition by FeSOD into
account. The results for line SR1-14 are shown in Table I. In
leaf extracts from SR1-14, we found an average of 13.8
units/mg chlorophyll of FeSOD, most of which represents
transgenic FeSOD (cf. Fig. 1). There was no significant
difference in activity of mitochondrial MnSOD and cytoso-
lic and chloroplastic Cu/ZnSOD between transgenic and
control plants.
Tobacco Overproducing Fe-Superoxide Dismutase
1707
Table
II.
KCN-insensitive
SOD
activity
in
leaf extracts from
differ-
ent transgenic lines overproducing FeSOD
Mean
t
SE
(four
plants,
unless indicated otherwise).
Line Activity
SR1
(control)
SR1-23-1
SR1-36-7
SR1-42-8
SR1-14
SR1-32-2
SR1-21-1
unitshg
chlorophyll
2.88
2
0.43
10.56
2
0.71
10.84
i
0.39
12.34
2
0.73
14.37
i
0.65"
17.21
?
0.85
17.32
i
1 .O4
a
13
plants.
The data shown in Table I indicate that the total activity
of chloroplastic SOD species (FeSOD and chloroplastic
Cu/ZnSOD) was, on a chlorophyll basis, in line SR1-14
approximately 2.5 times higher than in control plants.
However, chloroplastic Cu/ ZnSOD is quite variable in
activity; it occurs only in young, expanding leaves, and
previous experiments yielded no indication that it pro-
vides tolerance against light-dependent, MV-mediated
oxidative stress (Slooten et al., 1995). Therefore, the ratio
of FeSOD contents may be more relevant in this respect.
The chloroplastic FeSOD activity was, on a chlorophyll
basis, in line SR1-14 approximately 7.4 times higher than
in control plants.
The SOD activity observed in the presence of KCN rep-
resented mainly FeSOD (cf. Fig.
l),
sometimes with a minor
contribution by MnSOD (less than 10% of the total KCN-
insensitive activity, as indicated by the banding patterns on
the SOD activity gels). Table I1 shows the KCN-insensitive
activities observed in the different transgenic lines. The
difference in KCN-insensitive SOD activity between the
highest and the lowest expressor amounted to only approx-
imately 60%.
-A----
-
&
10
5
--o
Control
i
-0-
SRI -1
4
o
0.
0.0
0.5
1.0
1.5
2.0
Oxidative Stress Tolerance
in
Transgenic and
Control Plants
Plants overproducing FeSOD were clearly more tolerant
to MV than control plants. This
is
shown in Figure
2
for
young plants (9 weeks) and older plants (13 weeks) of line
SR1-14. These plants were grown at a light intensity of 135
pmol
m-'
s-'.
Older plants are considerably more tolerant
to MV than young plants, as indicated by the difference in
scales along the
x
axis. We reported previously that in var
SR1, overproduction of MnSOD provided protection
against MV-induced ion leakage (due to cell membrane
deterioration), but not against MV-induced inactivation of
PSII (Slooten et al., 1995). In contrast, overproduction of
FeSOD resulted in protection against both types
of
damage.
This is indicated by the fact that the extent of MV-induced
ion leakage (Fig.
2,
A and
B),
as well as the extent
of
the
MV-induced decrease in
F,/F,,,
(Fig. 2, C and D), were
lower in transgenic plants than in control plants. Similar
results were obtained with other transgenic lines (Fig. 3).
Furthermore, similar results were obtained with plants
grown at a light intensity of 650 pmol m-'
s-'
(Table 111),
although these plants were 15 to
30
times less sensitive to
MV than plants grown at 135 pmol
m-'
s-'.
AT inhibits catalase (Margoliash et al., 1960; Havir, 1992)
and causes an increase in the H,O, content of tobacco
leaves during illumination (Chen et al., 1993); at the same
time, it causes inactivation
of
endogenous, chloroplastic
APx. Apparently as
a
consequence of the resulting increase
in the concentration of
H,O,
in the chloroplasts, the
PSII
reaction center is inactivated. This is not accompanied by
any significant ion leakage from the leaf discs
(L.
Slooten,
K.
Capiau,
S.
Kushnir, M. Van Montagu, and D. Inzé,
unpublished data). Plants overproducing FeSOD were not
more tolerant to AT than control plants (Table IV).
Leaf discs impregnated with eosin generate singlet oxy-
gen during illumination (Knox and Dodge, 1985). Plants
overproducing FeSOD were not more tolerant to eosin than
Figure
2.
MV-induced conductance increase
(A
and
B)
and
MV-induced
decrease
in
FJF,,,
(ex-
pressed
as
a
percentage of no
MV)
(C
and
D)
in
leaf
discs
from transgenic
and
control
plants
at
9
weeks
(A
and
C)
or
14
weeks
(B
and
D)
after
sowing.
Conductance increase
was
expressed
in
microsiemens
cm-'
35
mg-'
fresh
weight.
Error
bars
indicate
SD
for
four
plants.
Open
symbols,
Control
plants;
solid
symbols,
SR1-14.
1708
Van Camp et al. Plant Physiol. Vol.
11
2,
1996
Figure
3.
MV-induced conductance increase
(A),
and MV-induced decrease in FJF,,,,, (ex-
pressed as a percentage of no MV)
(B)
in leaf
discs from transgenic and control plants at
11
weeks after sowing. The MV concentration was
2
p~.
Conductance increase was expressed in
microsiemens
cm-’
35
mg-’ fresh weight. Error
bars indicate
SE
for four plants. Numbers in the
figures indicate the significance
of
the difference
in mean values between transgenic and control
plants, as established with
a
two-sided
t
test.
control plants. It made no difference in this respect whether
the damage was assessed from the decrease in activity of
the PSII reaction center (Table IV), or from the increase in
the conductance of the floating solution (not shown).
In addition, plants overproducing FeSOD were not more
tolerant to chilling-induced photoinhibition than control
plants (Table
V).
It made no difference in this respect
whether the photoinhibitory treatment was carried out for
4
h at 360 pmol m-’s-’ or for 30 h at 30 pmol m-’s-*.
Membrane Affinity
of
trFeSOD and trMnSOD
We reported previously that in var SR1, overproduction
of MnSOD provided protection against MV-induced ion
leakage (due to cell membrane deterioration), but not
against MV-induced inactivation of PSII (Slooten et al.,
1995). In contrast, overproduction of FeSOD resulted in
protection against both types
of
damage. Jt seemed possi-
ble that this might be due to a different suborganellar
location of the overproduced MnSOD and FeSOD. Specif-
ically, these enzymes might have a different membrane
affinity.
To
test this hypothesis, we determined the activity
of the overproduced SODs and of a stromal marker en-
zyme, NADP-dependent GABP-DH, in washed chloro-
plasts from MnSOD-overproducing plants and from
FeSOD-overproducing plants. We made use of the obser-
vation that a considerable proportion of the chloroplasts
became leaky during isolation and lost their stromal con-
tent before washing. One would expect that electrostati-
cally membrane-associated enzymes would be washed out
to a lesser extent than stromal enzymes. Prior to assay, the
suspension was sonicated to the extent that the chloro-
plasts that until then had remained intact were disrupted
in a medium in which the enzymes were quantitatively
released. In addition, we measured the activity of
Table
111.
Enhancement
of
MV
tolerance
in
FeSOD-overproducing
plants
MV-induced conductance increase (in microsiemens cm-’
35
mg-’ fresh weight) and MV-induced decrease in FJF,,,,, (in percent-
age of no MV) in leaf discs from transgenic and control plants grown
ata light intensity of
650
pmol m-’s-’. The MV concentration was
14
p~.
Mean
-+
SE
for six plants.
Plants Conductance lncrease Decrease in
FJF,,,,,
SRI-14
1
.I6
2
0.49 10.12
rt
2.02
SR1 4.02
rt
1.41 24.15
-+
2.02
0.99
23-1
A
>
0.995
36-7
B
1
0.97 0.98
0.94
E
Y
5
‘=
20
g
10
i
.-
a?
VI
*
control
21-1 23-1 36-7
GA3P-DH and of the overproduced SODs in leaf extracts
prepared from the same leaf as the chloroplasts. A11 activ-
ities were expressed on a chlorophyll basis. For each en-
zyme, the recovery in the chloroplasts was calculated as the
ratio of activities in sonicated chloroplasts to that in leaf
extracts. The recovery of GA3P-DH was approximately
35%. We then calculated the ratio of recoveries of
GA3P-DH over the overproduced SOD. This ratio is ex-
pected to be
1
for a stromal enzyme and lower than
1
for a
membrane-associated enzyme. Finally, we calculated the
ratio of recoveries of FeSOD and MnSOD
(R,,,,,/R,,,,,)
as the ratio between RCA3P.DH/RMnSOD in MnSOD-
overproducing plants, and
RCA3P.DH
/
RFeSOD
in FeSOD-
overproducing plants, where R
is
the recovery
of
the en-
zyme indicated in the subscript. This method allowed us to
compare recoveries of FeSOD and MnSOD corrected for
differences in quality of the chloroplast preparation. This
procedure was repeated five times in independent experi-
ments. The results are shown in Table
VI.
Overproduced
MnSOD exhibited about the same recovery as GA3P-DH,
indicating that it behaves like a stromal enzyme. Overpro-
duced FeSOD exhibited higher recoveries, indicating elec-
trostatic binding to chloroplast membranes. This was espe-
cially clear from the average ratio of recoveries of FeSOD
and MnSOD, calculated as indicated above.
Effect
of
trFeSOD
on
lnduction
of
Antioxidant
Enzymes during Salt Stress
Plants respond to salt stress with increases in antioxidant
enzyme activities, indicating that oxidative stress is in-
volved in salt stress (Gossett et al., 1994; Hernandez et al.,
1995; Sehmer et al., 1995). To study the effect of FeSOD
overproduction on salt tolerance, we grew transgenic and
control plants under salt stress (see “Materials and Meth-
Table
IV. Lack
of
enhancement
of
AJ
and eosin tolerance
in
FeSO D-o verproducing pla
n
ts
Leaf discs were illuminated for
16
h in the presence of
5
mM AT,
or for
2
h in the presence of
0.1
mM eosin. For additional details, see
”Materials and Methods.” Mean
2
SE
(number of plants).
Decrease in
FJF,,,
AT
Eosin
Plants
%
of
dark
controls
SRI 31
2
1.4
(5)
44.1
2
2.5
(6)
SRI-14 30
t
2.6
(5)
47.7
2
3.0 (6)
Tobacco
Overproducing Fe-Superoxide Dismutase
1709
Table
V.
Lack
of
enhancement
of
tolerance to chilling-induced photoinhibition
in
FeSOD-
overproducing plants
Leaf discs floating
on
water containing 75
WM
chloramphenicol were illuminated
as
indicated in
closed Petri dishes.
FJF,,,,,
was measured 2 h after cessation of illumination. Mean
t
SE
(number of
plants).
Photoinhibitory Treatment Decrease in
FJF,,,
Liaht intensitv Duration Temoerature
SR1 SR1-14
pmol
m-z
5-l
%
of
dark control
39.1
t
1.3 (6)
30 31 h 4
"C
24.8
t
2.7 (4) 28.6
t
2.1 (4)
360 4h 7
"C
36.5
t
1.5 (6)
ods"). Salt-stressed plants accumulated considerably less
biomass, in terms of fresh weight, than control plants (Fig.
4A). The salt concentration in the soil increased gradually
with time, so that growth became increasingly inhibited
(not shown). As a consequence, the ratio of fresh weights in
salt-stressed plants over fresh weights in control plants
decreased steadily with time (Fig. 4B). There was no dif-
ference in this respect between FeSOD-overproducing and
control plants. In addition, there was no difference in dry
matter accumulation between FeSOD-overproducing and
control plants at the end of the experiment (Fig. 4C). At the
end of the experiment, the concentration of dissolved
so-
dium in the soil was 120 mM in the salt-treated pots,
compared with
5
mM in the controls (not shown), and the
sodium content of salt-stressed plants was approximately
twice that of the control plants (Fig. 4D).
Salt stress was accompanied by oxidative stress, as indi-
cated by increases in activities of a11 tested antioxidant
enzymes in nontransgenic plants. Specifically, the activities
of chloroplastic FeSOD, cytosolic Cu
/
ZnSOD, chloroplastic
Cu
/
ZnSOD, APx,
GR,
and DHAR were approximately two
to
three times higher, on a protein basis, in salt-stressed
nontransgenic plants than in unstressed nontransgenic
plants at the end of the experiment (Fig.
4E).
FeSOD-
overproducing plants exhibited a similar increase in over-
a11 activity of APx,
GR,
and DHAR during salt stress.
However the induction of cytosolic and chloroplastic Cu
/
ZnSOD, observed in control plants under salt stress, was
suppressed in FeSOD-overproducing plants (Fig.
4E).
To discriminate between chloroplastic and cytosolic
APx isozymes, we electrophoresed leaf extracts on non-
denaturing polyacrylamide gels, and stained the
gels
for
APx activity (Fig.
5).
Three bands of APx activity can be
distinguished after nondenaturing electrophoresis of leaf
extracts from bolting tobacco plants: one band corre-
sponding to cytosolic APx, and two bands corresponding
to chloroplastic APx
(L.
Slooten, K. Capiau,
S.
Kushnir, M.
Van Montagu, and D.
Inzé,
unpublished data). The latter
two will be denoted as chlAPx-1 and chlAPx-2 for ease of
reference. The plants used in the present experiments
were still in the rosette stage, and were grown at a rela-
tively low light intensity. Under those circumstances, only
the cytosolic APx and chlAPx-2 were observed in extracts
from unstressed, nontransgenic plants. Under salt stress,
nontransgenic plants usually exhibited an increase in ac-
tivity of chlAPx-2 in comparison with nonstressed plants.
Unstressed, FeSOD-overproducing plants exhibited a sim-
ilar isozyme pattern as unstressed nontransgenic plants.
However, during salt stress, the FeSOD-overproducing
plants exhibited an increase in activity of both chloroplas-
tic APx isozymes. Thus, the induction of chlAPx-1 was
accelerated specifically in FeSOD-overproducing plants
under salt stress.
4-(Chloromercuri)benzenesulphonic
acid, a specific inhibitor of ascorbate peroxidase (Chen
Table
VI. Comparison
of
the recoveries
of
overproduced
SODs
in
isolated chloroplasts
Washed chloroplasts were resuspended in 2.4 mL of the extraction medium used for determination
of GA3P-DH in whole leaf extracts (see "Materials and Methods"), except that polyvinylpolypyrroli-
done, Triton, and ascorbate were omitted. The chloroplasts were broken by sonication. Part of the
sonicate was used for chlorophyll determination. The remainder was centrifuged for 1 h at 45,OOOg. The
supernatants were used for assay of
SOD
(spectrophotometric, as well as after nondenaturing PACE) and
of GA3P-DH. All data are presented as mean
t
SD
from five independent experiments. The activities
of
trMnSOD and trFeSOD in leaf extracts from transgenic plants were 11.8
2
1.7 and 13.8
t
2.3 units/mg
chlorophyll, respectively. The activities of GA3P-DH in leaf extracts from
MnSOD-
and FeSOD-
overproducing plants were 10.3
t
0.9 and 10.4
t
2.4 units/mg chlorophyll, respectively. R
is
the
recovery in isolated chloroplasts
of
the enzyme indicated in the subscript. For other details, see text.
FeSOD-Overproducing- Plants
~
~
MnSOD-Overproducing Plants
RM",OV 39.9
t
i4.a~~
RkSOD
50.4
t
18.4%
RCA3PDH
38.8
t
11.4% RGA3P.DH 35.5
t
18.8%
R,,,,,$LSOD
1
.O3
t
0.41 0.69
t
0.25
RGA3P-DH/RFeSOV
RF~SOD
~- -
1.50
2
0.12
RMnSOD
1710
Van
Camp et
al.
Plant
Physiol.
Vol.
11
2,
1996
e!
Days
after
sowing
Days
after
sowing
0.4
E
%
03
E
D
02
a
-
6
0.1
SRI SR1-14 SRI-14
0.0
E
c7
co
C
.-
$2
n
31
E"
o
Fe
cytCu chlCu APx GR*10 DHAR
Figure
4.
Effects
of
salt
stress
on
FeSOD-overexpressing
(SR1-14)
and
control
plants.
NaCl
was
given
from
d
12
after
sowing (see
"Materials
and
Methods").
Data
are
presented
as
mean
i-
SE.
A,
Growth
measured
as
fresh
weight accumulation
in
leaves
and
stems
of
transgenic
(solid
symbols)
and
control
plants
(open
symbols)
(n
=
10).
B, Ratio
of
fresh weights between
unstressed
and
salt-stressed
plants.
C,
Dry
weights
of
the leaves
and
stems
of
unstressed
and
salt-stressed
plants
at
42
d
after
sowing
(n
=
5).
D,
Sodium
content
of
the
leaves
and
stems
of
transgenic
and
control
plants
at
42
d
after
sowing
(n
=
5).
E,
Antioxidant
enzyme activities
in
the
leaves
of
transgenic
and
control
plants
at
42
d
after
sowing
(n
=
5).
From
left
to
right,
FeSOD, cytosolic
Cu/ZnSOD,
chloroplastic CulZnSOD,
APx,
GR,
and
DHAR.
The activities of
GR
were multiplied
by
10.
and Asada, 1989), abolished all activity on the gels, as
shown in the bottom curve of Figure 5.
DISCUSSION
Severa1 reports have been published on the overproduc-
tion of Cu/ZnSOD in plants (Tepperman and Dunsmuir,
1990; Pitcher et al., 1991; Perl et al., 1993; Sen Gupta et al.,
1993a, 1993b) or MnSOD (Bowler et al., 1991; McKersie et
al., 1993; Foyer et al., 1994; Van Camp et al., 1994b; Slooten
et al., 1995).
To
our knowledge, the present report is the
first to describe overproduction of FeSOD in plants. The
level of overexpression of FeSOD in line SR1-14, which was
studied most extensively, was around 7.4-fold (Fig.
1;
plants grown at 135 pmol m-'
sK1)
to 16-fold
(Fig.
4E;
plants grown at 65 Fmol m-'
sK1).
These variations were
due mainly to differences in activity of the endogenous
FeSOD, depending on the light intensity received during
growth (Slooten et al., 1995). After nondenaturing electro-
phoresis and activity staining, trFeSOD showed up as a
double band in transgenic tobacco (Fig.
1,
bands
1
and
2).
This double band was also observed in extracts from
A.
tkaliana,
the source organism of the transgene (not shown).
The band splitting is presumably due to a posttranslational
modification. In transgenic tobacco, the fast-moving peak
was about three times lower in root extracts than in leaf
extracts (not shown), suggesting that posttranslational
modification occurs to different extents in different organs
of the plant. It may be added that the MnSOD also becomes
posttranslationally modified when it is overproduced in
the chloroplasts (C. Bowler,
W.
Van Camp, and D.
Inzé,
unpublished data).
Tolerance
to
MV
Transgenic plants with different levels
of
FeSOD over-
production did not differ significantly with respect to en-
hancement of MV tolerance (Fig. 3). Apparently, the lowest
level of
SOD
overproduction, around
11
units
/
mg chloro-
phyll (Table
11),
was already sufficient to relieve the rate
limitation of superoxide scavenging by endogenous chlo-
roplastic
SODs.
Judging by the enhanced MV tolerance,
SOD-
overproducing plants have an enhanced superoxide-
scavenging capacity, and therefore they produce more
H202
than control plants during illumination in the presence of
MV. This would be highly toxic if it were not removed by
ascorbate peroxidase (see the introduction section). How-
ever, in the MV experiments, the leaf discs were illuminated
with low-intensity light (30 pmol mK2
s-').
This was done
partly to avoid photoinhibition per se, but especially to
Figure
5.
APx
banding
pattern
of
leaf extracts
from
FeSOD-
overproducing
plants
(SR1-14)
and
control
plants
(SR1).
The
plants
were watered
with
(solid
curves) or without (dashed
curves)
60
mM
NaCl
during
growth.
Where indicated, the
gel
was
treated
with
0.2
mM
4-(chloromercuri)benzenesulphonic
acid
prior
to
staining.
Mi-
gration
is
from
left to
right.
Band
a,
Cytosolic
APx;
bands
b
and
c,
chloroplastic
APx
corresponding
with
chlAPx-2
and
chlAPx-1,
respectively.
Tobacco Overproducing Fe-Superoxide Dismutase
1711
avoid a situation in which the H,O,-scavenging capacity of
endogenous APx would be overwhelmed by a high rate of
superoxide production and scavenging. In addition, we
have evidence that in tobacco leaf discs, H,O, by itself does
not readily inactivate either the PSII reaction center or the
plasmalemma
(L.
Slooten,
K.
Capiau,
S.
Kushnir, M. Van
Montagu, and D. Inzé, unpublished data). Presumably, both
of these factors contributed to the clear-cut protection by
trFeSOD of the PSII reaction center against damage induced
by superoxide.
We reported previously that in var SR1, overproduction
of MnSOD provided protection against MV-induced ion
leakage (due to cell membrane deterioration), but not
against MV-induced inactivation of PSII (Slooten et al.,
1995). In contrast, overproduction
of
FeSOD resulted in
protection against both types of damage. This difference
cannot be attributed to differences in activity levels of
overproduced SOD, since plants overproducing MnSOD
contained on average 13 units/mg chlorophyll of the
overproduced activity (Slooten et al., 1995), which is sim-
ilar to that for FeSOD-overproducing plants. However,
trFeSOD is by origin a chloroplastic enzyme, whereas
trMnSOD is by origin a mitochondrial enzyme. During
illumination, the major site of electron donation to
MV
has been shown to be the Fe-S center
B
in PSI (Fujii et al.,
1990). From there the oxygen radicals (produced by reoxi-
dation of reduced MV) must propagate out of the chloro-
plasts and to the cell membrane, resulting in ion leakage,
and to the reaction center of PSII, resulting in the loss of
F,
/
F,,,.
To prevent a11 types
of
superoxide-induced dam-
age, the overproduced
SOD
should scavenge the super-
oxide at the site of its formation, i.e. at the acceptor side
of
PSI.
To
achieve this the SOD probably has to bind elec-
trostatically to a specific domain in the stromal membrane
in which PSI is embedded. The charge distribution on the
SODs may be such that trFeSOD is better able to recognize
these binding domains than trMnSOD. The results pre-
sented in Table VI are consistent with this hypothesis:
trFeSOD is at least partially membrane-bound, whereas
trMnSOD behaves like a stromal enzyme. The protection
provided by trFeSOD against inactivation of the PSII re-
action center may thus be due to trFeSOD binding in the
vicinity of the
PSI
reaction center, enabling it to scavenge
superoxide at the site of its formation. By contrast, trMn-
SOD, behaving like a stromal enzyme, may only be capa-
ble of providing protection against damage spreading
through the stroma toward the cell membrane and lead-
ing to ion leakage (Slooten et al., 1995).
It will certainly be necessary to confirm this hypothesis
by determining the suborganellar localization of trFeSOD
in the chloroplasts. But it may be noted that in spinach,
both chloroplastic (Ogawa et al., 1995b) and cytosolic Cu/
ZnSOD (Ogawa et al., 1995a) are preferentially membrane-
associated; the chloroplastic Cu/ ZnSOD is associated with
the stromal membranes and with stroma-facing thylakoid
membranes (Ogawa et al., 199513). Furthermore, functional
differences between FeSOD and MnSOD were also ob-
served in
E.
coli,
in which MnSOD
is
more effective than
FeSOD in protecting DNA and FeSOD is more effective
than MnSOD in protecting a cytosolic enzyme against
MV-
mediated oxygen radical damage. Here, too, the differences
were attributed to differences in charge and/or charge
distribution between MnSOD and FeSOD (Hopkin et al.,
1992).
Tolerance
to
Other
Types
of
Oxidative
Stress
The lack of enhancement of tolerance to systems gener-
ating eosin or H,O, (Table IV) is in accordance with the fact
that these toxic oxygen species are not utilized by SOD.
Overproduction of FeSOD did not enhance the tolerance to
photoinhibitory conditions either (Table
V).
Mainly from in
vitro studies with various
PSII
preparations, it was con-
cluded that the primary event in photoinhibition is inhibi-
tion of whole-chain electron transport, either at the accep-
tor side or at the donor side
of
the
PSII
reaction center (for
review, see Prasil et al., 1992; Aro et al., 1993; Barber, 1994).
During acceptor-side photoinhibition, active oxygen spe-
cies formed by the still-functional primary radical pair
initiate the degradation of the D1 protein of the reaction
center. Most of the attention has been focused on singlet
oxygen (Vass et al., 1992; Hideg et al., 1994), but superoxide
has been implicated as well (Miyao, 1994). During donor-
side photoinhibition, degradation of the D1 protein is not
strictly dependent on oxygen, yet the degradation is
strongly accelerated by superoxide (Chen et al., 1995),
which can be generated by the still-functional primary
radical pair. Superoxide production becomes manifest only
after complete removal of Mn, which is somehow involved
in a SOD-like activity displayed by the reaction center itself
(Ananyev et al., 1994; Chen et al., 1995). Donor-side pho-
toinhibition can occur under conditions that destabilize the
water-splítting complex, such as exposure
to
low temper-
ature (Wang et al., 1992). In contrast to acceptor-side pho-
toinhibition, donor-side photoinhibition can occur at low
light intensities (Eckert et al., 1991).
In the present study, overproduction of FeSOD did not
enhance the tolerance to chilling-induced photoinhibition,
either at low or high light intensities (Table
V).
This is in
agreement with the results obtained with MnSOD-
overproducing plants (Slooten et al., 1995). There are sev-
era1 possible explanations of this result: (a) photoinhibition
was not due to superoxide production in the reaction cen-
ter; (b) photoinhibition was due to superoxide production
in the reaction center, but the superoxide brought about
oxidative damage without becoming accessible to overex-
pressed
SOD;
and
(c)
the reaction centers did produce
superoxide accessible to transgenic SOD, but this was then
converted to H,O,, which can be equally damaging
(Miyao-Tokutomi et al., 1995). In light of the above discus-
sion, we assume that explanations b and c are more likely
than explanation a. In view of the results discussed in the
previous section (indicating that trFeSOD does protect the
PSII
reaction center against superoxide produced by PSI), it
would seem that explanation b is more likely than expla-
nation c.
Sen Gupta et al. (1993a, 199313) found that transgenic
tobacco
(N.
tabacum
cv Xanthi) overproducing pea Cu/
ZnSOD in the chloroplasts exhibited an enhanced tolerance
1712
Van
Camp
et al. Plant
Physiol.
Vol.
11
2,
1996
to inhibition of CO, fixation that was induced by high light
at low temperatures. This indicates that in the experiments
of Sen Gupta et al. (1993a, 1993b), inhibition of CO, fixation
was caused by the production of superoxide at a rate
exceeding the endogenous scavenging capacity. Sen Gupta
et al. (1993a, 1993b) used lower temperatures and higher
light intensities during the stress treatment than we did.
Hence, the rate of superoxide production may have been
higher in their experiments than in ours. However, in the
experiments of Sen Gupta al. (1993a), the recovery from
chilling-induced inhibition of CO, fixation was virtually
complete within about 20 min after return to 25°C. Recov-
ery from oxidative damage to the PSII reaction center
generally requires severa1 hours (Thiele et al., 1995; cf.
Greer et al., 1986). Therefore, it seems unlikely that in the
experiments of Sen Gupta et al. (1993a), the inhibition of
CO, fixation was due to oxidative damage to the PSII
reaction center.
Effect
of
trFeSOD on lnduction
of
Antioxidant
Enzymes during Salt Stress
We observed two chloroplastic APx isozymes on APx
activity gels (Fig. 5)
(L.
Slooten, K. Capiau,
S.
Kushnir, M.
Van Montagu, and D. Inzé, unpublished data). These are
water-soluble enzymes and are therefore different from the
intrinsically membrane-bound APx first found in spinach
(Miyake and Asada, 1992). We found a similar membrane-
bound APx activity in cuvette assays in membrane prepa-
rations from tobacco chloroplasts, but could not visualize
this activity on APx activity gels
(L.
Slooten,
K.
Capiau,
S.
Kushnir, M. Van Montagu, and D. Inzé, unpublished data).
In
nonstressed tobacco plants, overproduction
of
FeSOD
had no effect on activity levels of other antioxidant en-
zymes, including APx. This was also the case when the
plants were grown at a light intensity of 650 pmol m-'s-'
(see "Materials and Methods") (not shown). This agrees
with earlier data obtained with transgenic plants overpro-
ducing MnSOD in the chloroplasts (Slooten et al., 1995).
However, when we applied salt stress, only the transgenic,
FeSOD-overproducing plants exhibited at least a 3-fold
increase in activity of chlAPx-1. In contrast, chlAPx-2 was
enhanced in both the transgenic and the nontransgenic
plants in response to salt stress (Fig. 5). This suggests that
at least chlAPx-1 is induced by H,O,. During salt stress,
H,O, is probably generated at higher rates in transgenic,
FeSOD-overproducing plants than in nontransgenic plants.
The differential response of the two chloroplastic APx
isozymes to salt stress indicates some functional differen-
tiation of these isozymes. In var SR1, chlAPx-1 represents
only a minor fraction of the total chloroplastic APx. We
noted that leaf extracts from cv PBDG contained higher
activities of chlAPx-1 than leaf extracts from cv SR1 after
electrophoresis on nondenaturing gels
(L.
Slooten, K. Ca-
piau,
S.
Kushnir,
M.
Van Montagu, and D. Inzé, unpub-
lished data). Sen Gupta et al. (1993b) reported that trans-
genic tobacco
(N.
tabacum
cv Xanthi) overproducing pea
Cu
/
ZnSOD in the chloroplasts exhibited a 3-fold increase
in APx activity. The difference between the results of their
experiments and ours may be due to differences in the APx
isozyme distribution pattern between cv Xanthi and cv
SR1.
In accordance with data from other groups, we found
that salt stress leads to increases in the activity of many
antioxidant enzymes (chloroplastic FeSOD, cytosolic Cu
/
ZnSOD, chloroplastic Cu/ZnSOD, APx, GR, and DHAR) in
nontransgenic plants, indicating that oxidative stress is one
of the components of salt stress (Fig. 4E). In the absence of
stress, the activities of a11 of these enzymes except FeSOD
were the same in nontransgenic and FeSOD-overproducing
plants. However, the increase in activity
of
cytosolic and
chloroplastic Cu/ ZnSOD, observed in control plants under
salt stress, was suppressed in FeSOD-overproducing plants
(Fig. 4E). Cytosolic Cu/ZnSOD and trFeSOD are located in
different cellular compartments, indicating that trFeSOD
interferes with a signal pathway leading to induction of
cytosolic Cu/ ZnSOD, and probably also of chloroplastic
Cu/ZnSOD. This interference is probably due to the fact
that trFeSOD lowers the concentration of superoxide in the
chloroplasts. Similar results were obtained previously with
plants overproducing a mitochondrial MnSOD in the chlo-
roplasts (Slooten et al., 1995). It remains to be determined
whether superoxide itself or a molecule derived from a
reaction with superoxide constitutes the signal for induc-
tion of cytosolic Cu/ZnSOD.
Salt stress is complex, imposing a water deficit because of
osmotic effects and exerting ion-specific toxic effects on a
wide variety of metabolic activities (Greenway and Munns,
1980; Cheeseman, 1988). The water deficit might be expected
to cause oxidative stress, which is similar to drought stress
(Bowler et al., 1992). Indeed, Singha and Choudhuri (1990)
reported that superoxide and H,O, could be important in
the mechanism
of
salt injury. This notion was corroborated
by subsequent reports indicating an increase in the activities
of antioxidant enzymes in response to high salinity, and by
correlations of salt tolerance with antioxidant enzyme levels
(Gossett et al., 1994; Olmos et al., 1994; Hernandez et al.,
1995; Sehmer et al., 1995). In our experiments the chloro-
plasts of salt-stressed transgenic plants exhibited elevated
activities not only of
SOD
but also of APx in comparison
with salt-stressed control plants (see above). Nevertheless,
FeSOD-overproducing plants were no more tolerant to salt
stress (as indicated by biomass accumulation; Fig.
4,
A-C)
than were control plants. This suggests that in the present
experiments, growth of salt-stressed plants was not limited
by the capacity of the chloroplasts to scavenge superoxide
and
H,O,.
It cannot be excluded that the lack of induction of
cytosolic Cu
/
ZnSOD in transgenic plants overproducing
FeSOD contributed to the lack of enhancement of salt toler-
ante
in these plants.
CONCLUSIONS AND PROSPECTS
The results obtained with transgenic plants overproduc-
ing either FeSOD or MnSOD in the chloroplasts indicate that
FeSOD provides better protection against MV-dependent
oxidative stress than does MnSOD. We attribute this tenta-
tively to the higher membrane affinity of trFeSOD, allowing
this enzyme to scavenge superoxide radicals at the site of
their formation, i.e. near
PSI.
This difference between Mn-
Tobacco Overproducing Fe-Superoxide Dismutase
1713
SOD
and
FeSOD
is probably connected to the original
sub-
cellular localization of these enzymes. From a11 this it may
be
anticipated that FeSOD-overproducing plants
will
be
more
tolerant to physiological stresses entailing enhancement
of
light-induced superoxide formation than
MnSOD-
overproducing plants. FeSOD-overproducing plants
were
not more tolerant to salt stress than control plants, indicating
that, at least under the present assay conditions, the
superoxide-scavenging capacity was not a limiting factor for
growth
under
salt stress. However
in
salt-stressed plants the
overproduced
enzyme
interfered with signal pathways
for
induction
of
antioxidant
enzymes
in such a manner that
induction
of
one
chloroplastic APx isozyme
was
promoted
and induction
of
cytosolic
and
chloroplastic
Cu/
ZnSOD
was
inhibited. Thus, these plants may provide clues for the
elu-
cidation
of
signal pathways involved in induction
of
other
antioxidant enzymes. Furthermore, it
seems
likely that
FeSOD-overproducing plants can provide interesting mate-
rial to
assess
the importance
of
oxidative stress in various
physiological types
of
stress, and
in
addition, provide a
good
starting point
for
studying cooperative effects between
different overproduced antioxidant
enzymes
in
the scaveng-
ing
of
toxic oxygen species.
Received May
13,
1996; accepted September 9, 1996.
Copyright Clearance Center: 0032-0889/96/ 112/ 1703/12.
LITERATURE
CITED
Allen RD
(1995) Dissection
of
oxidative stress tolerance using
transgenic plants. Plant Physiol107 1049-1054
Ananyev G, Renger G, Wacker
U,
Klimov V
(1994) The produc-
tion of superoxide radicals and the superoxide dismutase activ-
ity of photosystem
11.
The possible involvement of cytochrome
b559. Photosynth Res 41: 327-338
Aro E-M, Virgin
I,
Andersson B
(1993) Photoinhibition of photo-
system
11:
inactivation, protein damage and turnover. Biochim
Biophys Acta 1143: 113-134
Asada K
(1994) Production and action of active oxygen species in
photosynthetic tissues.
In
CH Foyer, PM Mullineaux, eds,
Causes of Photooxidative Stress and Amelioration of Defense
Systems in Plants. CRC Press, Boca Raton, FL, pp 77-104
Asada
K,
Takahashi M
(1987) Production and scavenging of active
oxygen in photosynthesis.
In
DJ
Kyle, CB Osmond, CJ Arntzen,
eds, Photoinhibition (Topics in Photosynthesis,
Vol9).
Elsevier,
Amsterdam, pp 227-287
Badger MR
(1985) Photosynthetic oxygen exchange. Annu Rev
Plant Physiol 36: 27-53
Barber
J
(1994) Molecular basis of the vulnerability
of
photosystem
I1 to damage by light. Aust
J
Plant Physiol 22: 201-208
Bowler C, Slooten
L,
Vandenbranden
S,
De Rycke R, Botterman
J, Sybesma C, Van Montagu M, Inzé D
(1991) Manganese
superoxide dismutase can reduce cellular damage mediated by
oxygen radicals in transgenic plants. EMBO
J
10:
1723-1732
Bowler C, Van Montagu M, Inzé D
(1992) Superoxide dismutase
and stress tolerance. Annu Rev Plant Physiol Plant Mo1 Biol 43:
Bradford MM
(1976) A rapid and sensitive method for the quan-
tification of microgram quantities of protein utilizing the prin-
ciple of protein-dye binding. Ana1 Biochem
72:
248-254
Buchanan BB
(1980) Role
of
light in the regulation of chloroplast
enzymes. Annu Rev Plant Physiol31: 341-374
Buchanan BB
(1991) Regulation of CO, assimilation in oxygenic
photosynthesis: the ferredoxin/ thioredoxin system. Arch Bio-
chem Biophys 288:
1-9
83-116
Cerovic ZG, Plesnicar M
(1984) An improved method for the
isolation
of
intact chloroplasts of high photosynthetic capacity.
Biochem
J
223: 543-545
Cheeseman JM
(1988) Mechanism of salt tolerance in plants. Plant
Physiol87 547-550
Chen G-X, Asada K
(1989) Ascorbate peroxidase in tea leaves:
occurrence of two isozymes and the differences in their enzy-
matic and molecular properties. Plant Cell Physiol 30 987-998
Chen G-X, Blubaugh DJ, Homann PH, Golbeck JH, Cheniae GM
(1995) Superoxide contributes to the rapid inactivation of spe-
cific secondary donors of the photosystem I1 reaction center
during photodamage of manganese-depleted photosystem
I1
membranes. Biochemistry 34: 2317-2332
Chen
Z,
Silva H, Klessig DF
(1993) Active oxygen species in the
induction of plant systemic acquired resistance by salicylic acid.
Science 262 1883-1886
De Block
M,
Botterman
J,
Vandewiele M, Dockx
J,
Thoen C,
Gosselé V, Movva R, Thompson C, Van Montagu M, Leemans
J
(1987) Engineering herbicide resistance in plants by expression
of
a
detoxifying enzyme. EMBO
J
6: 2513-2518
Eckert H-J, Geiken B, Bernarding
J,
Napiwotzki A, Eichler H-J,
Renger G
(1991) Two sites of photoinhibition of the electron
transfer in oxygen evolving and Tris-treated PSII membranes
from spinach. Photosynth Res 27 97-108
Foyer CH, Descourvières P, Kunert KJ
(1994) Protection against
oxygen radicals: an important defense mechanism studied in
transgenic plants. Plant Cell Environ
17:
507-523
Foyer CH, Halliwell
B
(1976) The presence
of
glutathione and
glutathione reductase in chloroplasts: a proposed role in ascor-
bic acid metabolism. Planta 133: 21-25
Fujii T, Yokoyama
E-i,
Inoue
K,
Sakurai H
(1990) The sites of
electron donation of photosystem
1
to methyl viologen. Biochim
Biophys Acta 1015: 41-48
Furbank
RT,
Badger MA
(1982) Oxygen exchange associated with
electron transport and photophosphorylation in spinach chloro-
plasts. Biochim Biophys Acta 723: 400-409
Geller BL, Winge DR
(1984) Subcellular distribution of superox-
ide dismutases in rat liver. Methods Enzymol 105: 105-114
Genty B, Briantais J-M, Baker
NR
(1989) The relationship between
the quantum yield of photosynthetic electron transport and
quenching
of
chlorophyll fluorescence. Biochim Biophys Acta
Gossett DG, Millhollon EP, Lucas MC
(1994) Antioxidant re-
sponse to NaCl stress in salt-tolerant and salt-sensitive cultivars
of cotton. Crop Sci 34 706-714
Greenway H, Munns
R
(1980) Mechanism of salt tolerance in
non-halophytes. Annu Rev Plant Physiol 31: 149-190
Greer DH, Berry JA, Bjorkman
O
(1986) Photoinhibition
of
pho-
tosynthesis in intact bean leaves: role
of
light and temperature,
and requirement for chloroplast-protein synthesis during recov-
ery. Planta 168: 253-260
Halliwell B
(1984) Chloroplast Metabolism: The Structure and
Function of Chloroplasts in Green Leaf Cells. Clarendon Press,
Oxford, UK
Halliwell
B
(1987) Oxidative damage, lipid peroxidation and anti-
oxidant protection in chloroplasts. Chem Phys Lipids 44: 327-340
Havir EA
(1992) The
in
vivo
and
in
vitro
inhibition of catalase from
leaves of
Nicotiana sylvestris
by 3-amino-1,2,4-triazole. Plant
Physiol 99: 533-537
Hernandez JA, Olmos
E,
Corpas
FJ,
Sevilla
F,
De1 Rio LA
(1995)
Salt-induced oxidative stress in chloroplasts of pea plants. Plant
Sci
105:
151-167
Hideg
E,
Spetea C, Vass
I
(1994) Singlet oxygen production in
thylakoid membranes during photoinhibition as detected by
EPR spectroscopy. Photosynth Res 39: 191-199
Hopkin KA, Papazian MA, Steinman HM
(1992) Functional dif-
ferences between manganese and iron superoxide dismutases in
Escherichia
coli
K-12.
J
Biol Chem 267: 24253-24258
Hosein
B,
Palmer G
(1983) The kinetics and mechanism of oxida-
tion of reduced spinach ferredoxin by molecular oxygen and its
reduced products. Biochim Biophys Acta 723: 383-390
990: 87-92
1714
Van
Camp
et
al.
Plant Physiol.
Vol.
11
2,
1996
Kaiser WM (1979) Reversible inhibition of the Calvin cycle and
activation
of
oxidative pentose phosphate cycle in isolated intact
chloroplasts by hydrogen peroxide. Planta
145:
377-382
Knox JP, Dodge AD (1985) The photodynamic action
of
eosin, a
singlet-oxygen generator: some effects on leaf tissue of
Pisum
sativum
L. Planta
164:
22-29
Landgraf
P,
Doege M, Ohmann
E,
Tschiersch H (1995) Light
stress and reactive oxygen species: consequences for photosyn-
thesis in
Euglena
gradis.
In
P Mathis, ed, Photosynthesis: From
Light to Biosphere, Vol IV. Kluwer Academic Publishers, Dor-
drecht, The Netherlands, pp 465-468
Margoliash
E,
Novogrodsky A, Schejter A (1960) Irreversible
reaction of 3-amino-l:2:4-triazole and related inhibitors with the
protein of catalase. Biochem
J
74:
339-350
McKersie BD, Chen
Y,
de Beus M, Bowley SR, Bowler C, Inzé D,
DHalluin
K,
Botterman J (1993) Superoxide dismutase en-
hances tolerance of freezing stress in transgenic alalfa
(Medicago
sativa
L.).
Plant Physiol
103:
1155-1163
Mittler R, Zilinskas BA (1993) Detection of ascorbate peroxidase
activity in native gels by inhibition of ascorbate-dependent re-
duction of nitroblue tetrazolium. Ana1 Biochem
212:
540-546
Miyake
C,
Asada K (1992) Thylakoid-bound ascorbate peroxidase
in spinach chloroplasts and photoreduction of its primary oxi-
dation product monodehydroascorbate radicals in thylakoids.
Plant Cell Physiol
33:
541-553
Miyake C, Asada K (1994) Ferredoxin-dependent photoreducion
of the monodehydroascorbate radical in spinach thylakoids.
Plant Cell Physiol
35:
539-549
Miyao M (1994) Involvement
of
active oxygen species in degra-
dation
of
the D1 protein under strong illumination in isolated
subcomplexes of photosystem
11.
Biochemistry
33:
9722-9730
Miyao-Tokutomi M, Okada
K,
Yamamoto N (1995) Specific cleav-
age of the D1 protein of PSII by exogenous active oxygen spe-
cies.
In
P Mathis, ed, Photosynthesis: From Light to Biosphere,
Vol
IV.
Kluwer Academic Publishers, Dordrecht, The Nether-
lands,
pp
243-246
Nakano
Y,
Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell
Physiol
22:
867-880
Ogawa
K,
Kanematsu
S,
Asada
K
(1995a) Spinach chloroplastic
and cytosolic CuZnSODs are localized at the sites of superoxide
generation.
In
P Mathis, ed, Photosynthesis: From Light to Bio-
sphere, Vol IV. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp 339-342
Ogawa
K,
Kanematsu
S,
Takabe K, Asada K (199513) Attachment
of
CuZn-superoxide dismutase to thylakoid membranes at the
site
of
superoxide generation (PSI) in spinach chloroplasts: de-
tection by immunogold labeling after rapid freezing and substi-
tution method. Plant Cell Physiol
36
565-573
Okada
K,
Satoh K, Katoh
S
(1991) Chloramphenicol is an inhibitor
of photosynthesis. FEBS Lett
295:
155-158
Olmos
E,
Hernandez JA, Sevilla
F,
Hellin E (1994) Induction of
severa1 antioxidant enzymes in the selection of a salt-tolerant
cell line of
Pisum
sativum.
J
Plant Physiol
144
594-598
Perl A, Perl-Treves R, Galili
S,
Aviv
D,
Shalgi
E,
Malkin
S,
Galun
E
(1993) Enhanced oxidative-stress defense in transgenic
potato overexpressing tomato Cu,Zn superoxide dismutases.
Theor Appl Genet
85:
568-576
Pitcher LH, Brennan
E,
Hurley A, Dunsmuir P, Tepperman JM,
Zilinskas BA (1991) Overproduction of petunia chloroplastic
copper
/
zinc superoxide dismutase does not confer ozone toler-
ante
in transgenic tobacco. Plant Physiol97: 452455
Prasil
O,
Adir
N,
Ohad
I
(1992) Dynamics
of
photosystem
11:
mechanism
of
photoinhibition and recovery processes.
In
J
Bar-
ber, ed, Topics in Photosynthesis, Vol
11.
The Photosystems:
Structure, Function and Molecular Biology. Elsevier, Amster-
dam, pp 295-348
Sehmer
L,
Alaoui-Sosse
B,
Dizengremel
P
(1995) Effect of salt
stress on growth and on the detoxifying pathway of pedunculate
oak seedlings
(Quercus
robur
L.).
J
Plant Physiol 147: 144-151
Sen Gupta A, Heinen JL, Holaday AS, Burke JJ, Allen RD (1993a)
Increased resistance to oxidative stress in transgenic plants that
overexpress chloroplastic Cu
/
Zn superoxide dismutase. Proc
Natl Acad Sci USA 90: 1629-1633
Sen Gupta A, Webb RP, Holaday AS, Allen RD (1993b) Overex-
pression of superoxide dimutase protects plants from oxidative
stress. Plant Physiol
103:
1067-1073
Singha
S,
Choudhuri MA (1990) Effect of salinity (NaCl) on
H,02
metabolism in
Vigna
and
Oryza
seedlings. Biochem Physiol
Pflanz
186:
69-74
Slooten
L,
Capiau K, Van Camp W, Van Montagu M, Sybesma C,
Inzé
D (1995) Factors affecting the enhancement of oxidative
stress tolerance in transgenic tobacco overexpressing manganese
suyeroxide dismutase in the chloroplasts. Plant Physiol
107:
Stitt M, McC Lilley R, Gerhardt
R,
Heldt HW (1989) Metabolite
levels in specific cells and subcellular compartments of plant
leaves. Methods Enzymol
174:
518-552
Takahashi M, Asada
K
(1988) Superoxide production in the
aprotic interior of thylakoid membranes. Arch Biochem Biophys
Takeda T, Yokota A, Shigeoka
S
(1995) Resistance of photosyn-
thesis to hydrogen peroxide in algae. Plant Cell Physiol
36
Tepperman JM, Dunsmuir
P
(1990) Transformed plants with el-
evated levels of chloroplastic
SOD
are not more resistant to
superoxide toxicity. Plant Mo1 Biol
14
501-511
Thiele A, Winter K, Krause GH (1995) Photoinhibition of photo-
synthesis related to xanthophyll cycle and D1 protein turnover
in higher plants.
In
P Mathis, ed, Photosynthesis: From Light to
Biosphere, Vol
IV.
Kluwer Academic Publishers, Dordrecht,
The
Netherlands, pp 437-440
Van Camp W, Bowler C, Villarroel R, Tsang EWT, Van Montagu
M, Inzé D (1990) Characterization of iron superoxide dismutase
cDNAs from plants obtained by genetic complementation
in
Escherichia
coli.
Proc Natl Acad Sci USA
87:
9903-9907
Van Camp W, Van Montagu M,
Inzé
D (1994a) Superoxide dis-
mutases.
In
CH Foyer, PM Mullineaux, eds, Causes
of
Photooxi-
dative Stress in Plants and Amelioration
of
Defense Systems.
CRC Press, Boca Raton, FL, pp 317-341
Van Camp W, Willekens H, Bowler C, Van Montagu M, Inzé
D,
Reupold-Popp R, Sandermann
H
Jr, Langebartels C (199413)
Elevated levels of superoxide dismutase protect transgenic
plants against ozone damage. Bio/ Technology
12:
165-168
Vass
I,
Styring
S,
Hundal T, Koivuniemi A, Aro E-M, Andersson
B
(1992) Reversible and irreversible intermediates during pho-
toinhibition
of
photosystem 11: stable reduced
Q,
species pro-
mote chlorophyll triplet formation. Proc Natl Acad Sci USA
89:
Wang WS, Chapman DJ, Barber J (1992) Effect
of
cold treatments
on
the binding stability of photosystem two extrinsic proteins
and associated increase in susceptibility to photoinhibition.
Plant Physiol
99:
21-25
737-750
267:
714-722
1089-1095
1408-1412
