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Plant Physiol. (1998) 116: 1593-1602 A Cyanobacterium Lacking Iron Superoxide Dismutase Is Sensitized to Oxidative Stress Induced with Methyl Viologen but Is Not Sensitized to Oxidative Stress Induced with Norflurazon1
University of Idaho, Biological Sciences Department, Moscow, Idaho 83844-3051
A strain of
Synechococcus sp. strain PCC 7942 with no functional Fe
superoxide dismutase (SOD), designated
sodB
Oxygenic PET and aerobic respiration evolved during the
Precambrian period, improving the efficiency of C metabolism many-fold. The benefits of these new pathways were partially offset, however, by
their tendency to form reactive oxygen species that cause oxidative damage to biological molecules. The most significant reactive oxygen
species include excited singlet-state oxygen
(1O2*), the superoxide ion
(O2 Reactive oxygen species are detoxified in cells by a system of
antioxidants that co-evolved with PET and respiration. SODs catalyze
the conversion of O2 The SODs are metalloenzymes that may be separated into three classes,
depending on their metal cofactor. MnSODs are found in the cytosol
of eubacteria, in the cytosol and thylakoid membrane of cyanobacteria,
and in the mitochondrial lumen of eukaryotes. FeSODs are found in the
cytosol of eubacteria and cyanobacteria and in the chloroplast stroma
of photosynthetic plant cells. FeSODs are not usually found in
eukaryotes other than plants. Cu/ZnSODs are present only in eukaryotes
and may be found in the cytosol, chloroplast, and mitochondrial
intermembrane spaces (Okada et al., 1979 We have chosen cyanobacteria as models for the study of SODs. Like
eukaryotes, cyanobacteria have compartmentalized cells (cytosol and
thylakoid lumen) and multiple SODs (Okada et al., 1979 The cyanobacterium Synechococcus sp. strain PCC7942
(hereafter referred to as PCC7942) possesses two SODs. The MnSOD
encoded by the sodA gene is thylakoid associated, whereas
the FeSOD encoded by the sodB gene is cytosolic (Herbert et
al., 1992 Culture and Experimental Conditions
Growth Measurements Rapidly growing cells were centrifuged and resuspended in fresh BG-11 broth to an A750 of 0.1 to 0.3 and illuminated at 30 µmol photons m 2
s1. Aliquots of 3 mL were removed at
approximately 24-h intervals and A750 was
measured. Periodically, 10-mL samples were removed, vacuum filtered
onto 0.45-µm membrane filters, and dried for 24 h at 100°C to
determine dry mass.
Oxidative Stress Induction O2 generation was catalyzed by adding MV
(paraquat) to cultures to obtain a final concentration of 0.1 to 5.0 µm. MV catalyzes the formation of
O2 at a variety of electron-transport sites,
but in photosynthetic organisms in light the vast majority of
O2 is generated at the FA
and FB centers of PSI (Fujii et al.,
1990 ; Dodge, 1991). MV is a competitive inhibitor of the PSI cyclic pathway at concentrations of 100 µm (Yu et al., 1993).
However, the low concentrations of MV used in our experiments had no
effect on the PSI cyclic pathway (Herbert et al., 1995; Martin et al., 1997). 1O2* generation was catalyzed indirectly
by adding the carotenoid inhibitor NF to cultures to obtain a final
concentration of 0.1 to 5.0 µm. NF inhibits phytoene
desaturase and thus blocks synthesis of  -carotene and other
carotenoids (Ben-Aziz and Koren, 1974; Kümmel and Grimme, 1975).
Since carotenoids (-carotene in particular) normally quench
1O2* in the chlorophyll antenna, the addition
of NF promotes the formation of 1O2* within the
thylakoid membrane.
P700 Oxidation Reduction The photooxidation and dark-reduction kinetics of P700 were measured in intact cells using the broadband A820 change ( A820), as described elsewhere (Herbert
et al., 1995). The A820 was monitored by
reflectance using a modulated detection system (Walz, Effeltrich, Germany) consisting of a PAM 101 control unit and an ED 800T
emitter-detector unit. A branched fiber optic cable was used to deliver
modulated 820 nm and white actinic light to the sample and to collect
reflected 820 nm light. Actinic light (1000 µm photons
m2 s1) was provided by
a tungsten projector lamp (model EJV, General Electric) fitted with
three Calflex C heat filters (Balzers, Liechtenstein) and a mechanical
shutter (Uniblitz VS25, Vincent Associates, Rochester, NY). Output from
the PAM 101 control unit was collected and analyzed with a MacLab/2e
data acquisition system using Scope v3.3 software (AD Instruments,
Milford, MA) on a Macintosh computer. Samples for
A820 measurements were prepared at room
temperature by vacuum filtering 10 mL of culture onto 0.45-µm
membrane filters (type HA, Millipore). The filter and sample were then
placed under an acrylic light guide at the end of the trifurcated fiber
optic cable. Electron transport inhibitors (DCMU and/or DBMIB) were added to the samples prior to filtration.
Pigment Measurements
Catalase Assay Activity of the catalase in wild-type and sodB strains of PCC 7942 was determined
in intact cells by oxygen evolution in response to exogenous
H2O2. Cultures were washed
and resuspended to an A750 of 0.3 in fresh
medium. Aliquots (1.5 mL) of these samples were placed in a DW1
liquid-phase oxygen electrode cuvette (Hansatech, Norfolk, UK) and
stirred vigorously at 27°C. Oxygen in the cuvette was decreased to
30% of air-saturated values with a stream of N2
gas, and the cuvette was sealed and allowed to stabilize. A rate-saturating amount of
H2O2 was then injected (100 µL of a 30% solution), and the rate of oxygen evolution was measured
10 to 30 s after injection within the linear range of the
reaction. The background rate of spontaneous oxygen formation was
measured by injecting the same amount of
H2O2 into a sample of fresh
medium containing no cells. This background rate was subtracted from the rates obtained with cell samples and was typically less than 20%
of the rate obtained with cells. The assays were performed in darkness
to avoid contributions from photosynthetic oxygen evolution. Since
PCC7942 cells are small and a rate-saturating amount of
H2O2 was added for the
assay, breakage of the cells was not considered necessary to obtain
comparable catalase activities of the two strains. Both
H2O2 and oxygen diffuse
rapidly in and out of cells, and cell breakage introduces the risk of
catalase degradation during the assay.
Growth Data Figure 2 shows the growth rates of both strains with and without oxidative stresses. Without oxidative stress, the wild-type strain grew somewhat faster than the sodB strain. In the presence of 0.5 µm MV, the growth rate of the wild type was markedly
slower, but the sodB strain did not grow
at all. In the presence of 5 µm NF, both strains had
lower growth rates, but initially the
sodB strain had a higher growth rate than
the wild type. Eventually, both strains died in NF, but the wild type
was the first to succumb.
Photosynthetic Pigments The amount of phycocyanin changed little during all treatments (data not shown). Measurements of chlorophyll a in acetone extracts were consistently lower in the sodB strain than in the wild type during
all treatments, as shown in Figure 3.
Measurements of chlorophyll a in whole cells were very
similar to the acetone extracts (data not shown). The most marked
difference between the two strains was seen during the MV treatments,
when the wild type had approximately 3 times more chlorophyll
a than the sodB strain at the
end of 48 h. The ratio of total carotenoids to chlorophyll, shown
in Figure 4, followed a pattern similar
to that seen with total chlorophyll. In the control and NF groups, the
ratio remained similar in both strains, increasing slowly in the
control and decreasing slowly in the NF treatment. The MV treatment
resulted in a larger difference between the two strains at the end of
48 h, with the sodB strain losing
carotenoids. HPLC separation of the pigments, shown in Figure
5, coincides with the spectrophotometry
results. Before any treatments, the two strains had approximately the
same amount of total carotenoids per unit chlorophyll. Four carotenoids
were identified from the HPLC data by absorbance spectra and comparison with previous chromatograms (Masamoto and Furukawa, 1998):
nostoxanthin, caloxanthin, zeaxanthin, and -carotene. As has been
shown previously, zeaxanthin and -carotene were the most abundant
carotenoids in this cyanobacterium (Omata and Murata, 1983; Guillard et
al., 1985). No evidence of tocopherols was found (data not shown), confirming previous investigations (Powls and Redfearn, 1967).
P700 Oxidation Rate The rates of P700 oxidation, and thus the efficiency of excitation energy transfer from the photosynthetic antennae to P700, are shown in Figure 6. DBMIB (25 µm) was added to slow competing re-reduction of P700 and to ensure the maximum oxidation rate. In the wild-type control, the oxidation rate initially increased during the first 4 h and then decreased to a steady rate that was higher than the initial rate. The sodB control oxidation rate decreased
slightly during the 24-h period. During treatments with MV, the
wild-type oxidation rate remained constant throughout most of the
treatment, with a slight decrease by the end of 24 h. The
sodB oxidation rate with MV showed only a
slight decrease as well but was lower than the rate in the wild type. A
much more dramatic difference was seen in the NF treatments. The
wild-type oxidation rate decreased almost linearly to less than 20% of
the original rate after 48 h. During the same duration, the
sodB rate decreased to only about 70% of
the original rate.
P700 Activity The data of Figure 7 show that the extent of P700 oxidation remained approximately the same in both strains during the control and the 0.5 µm MV treatments. However, the sodB strain had slightly lower values and
was more inhibited by MV than the wild type. When treated with 5 µm NF, the wild-type strain showed a marked decrease in
P700 oxidation extent after 12 h, whereas
the sodB strain showed a steadier and
much slower decrease. To ensure that the P700 was
fully oxidized when measuring the extent of P700
oxidation, saturating actinic light was used and the samples were
treated with 25 µm each of DCMU and DBMIB.
PSI Electron Transport The rate of P700 re-reduction, shown in Figure 8, was measured in both strains in the presence of 25 µm DCMU. This rate is an indicator of PSI-driven cyclic electron transport (Herbert et al., 1995). The
wild-type control group initially showed an increase in cyclic electron
transport, as indicated by the increased rate of re-reduction. After
12 h, cyclic electron transport activity stabilized at a rate of
almost 160% of the initial rate. The
sodB control group had relatively
constant cyclic electron transport activity throughout the time course.
Both strains exhibited decreased cyclic activity when treated with MV,
but the sodB strain lost activity twice
as fast as the wild type. In the NF treatment the re-reduction rate in
the wild-type strain increased during the first 4 h and then
decreased drastically after 12 h. The re-reduction rate in the
sodB strain increased to a lesser degree
during the first 8 h and then remained fairly constant at a rate
of approximately 130% of the starting rate.
Catalase Activity Activity of the single catalase known in PCC7942 (Mutsuda et al., 1996) was measured by oxygen evolution in response to
H2O2 in whole cells. The
rates obtained from wild-type and sodB
cells were similar. Wild-type cultures diluted to an
A750 of 0.3 produced oxygen at a maximum
rate of 0.28 ± 0.09 µmol min1
mL1 of sample in response to exogenous
H2O2.
sodB cultures measured in the same way
produced oxygen at a maximum rate of 0.30 ± 0.10 µmol
min1 mL1 of sample
(values are means ± sample sd). The sample size for both numbers was seven. Each of these seven samples was a separate culture that was assayed two or three times to get an average value.
Values from any one culture were typically very repeatable.
Loss of cytosolic FeSOD activity in the
sodB MV The earliest target of MV inhibition in both strains was electron transport into P700 (Fig. 8). Without DCMU, PSII is the major source of these electrons. With DCMU added, the major sources of these electrons are the two cyclic paths of photosynthetic electron transport present in cyanobacteria (Herbert et al., 1992, 1995; Yu et al., 1993). The rate of this cyclic input to
P700 declines rapidly in the wild type when
treated with MV and much more rapidly in the
sodB strain. The
sodB strain of PCC7942 was shown
previously to be sensitive to MV, and the primary site of damage to PET
was observed to be in the cyclic electron transport path between the
FA/FB centers
of PSI and Cyt f (Herbert et al., 1992; Samson et al.,
1994). The present study confirms and extends those previous results.
Inhibition of the two cyclic paths in cyanobacteria is consistent with
oxidative damage to the
FA/FB centers
of PSI or to Fd. The
FA/FB centers of PSI are known to be an early site of oxidative damage to this photosystem (Fujii et al., 1990; Dodge, 1991). They and Fd also contain
Fe-S centers, which are known to be disrupted by
O2 (Liochev, 1996).
CO2 and Irradiance Side Effects When the cells were transferred from growth conditions (30 µm photons m2
s1, 3% CO2) to the
stress chambers (100 µm photons
m2 s1, ambient
CO2), the wild type exhibited an increase in both
the amount of photooxidizable P700 and in the
P700 oxidation rate. Under the same conditions,
the sodB strain was less variable for
both parameters. Previous research has shown that cyclic electron
transport increases in response to increased light intensity (Herbert
et al., 1995). In addition, the transfer from high to low
concentrations of CO2 would activate inorganic
carbon-concentrating mechanisms (for review, see Kaplan et al., 1991)
that require the cyclic pathway (Ogawa, 1991).
NF The most interesting result from this study was that the sodB strain is at least partially
resistant to the effects of NF. Unlike MV, NF caused a decrease in the
rate and extent of P700 oxidation. Both effects
would result from oxidation of chlorophyll in PSI by the formation of
1O2*
within the thylakoid membrane. When treated with NF, the wild type
shows a decreased rate of P700 photooxidation
(Fig. 6C), a loss of photooxidizable P700 (Fig.
7C), decreased cyclic PET (Fig. 8C), and a low growth rate (Fig. 2C).
The effects of NF on PSI activity in the wild type are consistent with
oxidative damage at or near the reaction center. Loss of
photooxidizable P700 represents damage to the
reaction center itself. Decreased oxidation rate suggests an
interruption of the delivery of excitation to the reaction center.
Figure 3C shows that oxidative loss of total chlorophyll did not
coincide with the decrease of the oxidation rate, so the interruption
must occur close to P700.
The Cause of NF Resistance in the sodB ), catalase (Mutsuda et al., 1996), and -carotene. The ascorbate
peroxidase and glutathione systems found in plants have not been found
in PCC7942 (Takeda et al., 1994). Also, unlike other cyanobacteria and
higher organisms, PCC7942 lacks the lipid-soluble antioxidant
 -tocopherol (Powls and Redfearn, 1967). Both the wild-type and
sodB strains have similar catalase
activities and carotenoid complements. Increased expression of the
MnSOD in the sodB strain has been
reported, however (Herbert et al., 1992). Presumably, the MnSOD
activity is increased to compensate for the lack of FeSOD. We propose
that the sodB strain resists the effects
of NF because of overexpression of MnSOD, which detoxifies the
O2 formed within the thylakoid as a product
of 1O2*. Work in progress
to obtain a sodA deletion mutant will
confirm this putative role of MnSOD in PCC4972 and further define the
different intracellular roles of antioxidants in this organism.
* Corresponding author; e-mail skherbe{at}uidaho.edu; fax 1- 208-885-7905. Received October 29, 1997;
accepted December 29, 1997.
Abbreviations:
DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.
MV, methyl
viologen.
NF, norflurazon.
PET, photosynthetic electron transport.
SOD, O2
This paper is dedicated to the late Prof. David E. Laudenbach, whose leadership of this work was cut short by his untimely death.
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Top
Abstract
Introduction
20°C in the
dark. Fifty microliters of the extract was injected into a 20-µL
sample loop. Carotenoid and chlorophyll peaks were identified at 420 nm
using known standards, absorbance spectra, and/or previously published
chromatograms. The presence or absence of tocopherols was investigated
using the same HPLC system at 290 nm in a 100% methanol carrier with
known standards (Lang et al., 1992
) and
sodB
) strains. Growth curves measured
at A750 are shown here for control (A), 0.5 µm MV-treated (B), and 5 µm NF-treated (C)
groups. Each point is the mean of three samples. Error bars are
sds.
