Plant Physiol.

(1986) 82, 1169-1171

0032-0889/86/82/1169/03/$0 1.00/0

Communication
Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and
Root Nodules of Medicago sativa'
Received for publication June 26, 1986 and in revised form September 29, 1986

MANUEL BECANA*, PEDRO APARICIO-TEJO, JUAN JOSE IRIGOYEN, AND MANUEL SANCHEZ-DIAZ

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Department of Plant Physiology, University ofNavarra, 31080 Pamplona, Spain

ABSTRACT tive inhibition or inactivation. Thus the Cu,Zn-SOD is both CN-

and H202-sensitive, the Fe-SOD is only H202-sensitive, and the
Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa Mn-SOD is resistant to either reagent; in addition, the Fe- and
L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 Mn-SOD are inactivated by organic solvents (chloroform plus
have been analyzed for the presence of the enzymes superoxide dismutase ethanol) whereas the Cu,Zn-SOD is not (2, 4, 6, 14, 15). Subcel-
(SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC lular location and distribution of SODs among organisms in an
1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular evolutionary context have been recently reviewed (3).
cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol During the last decade the enzymes of H202 metabolism in
of nodules possess CN-insensitive SOD activities. Studies of SOD leaves of higher plants have been studied thoroughly (see e.g. 3,
inactivation with H202 indicate that, very likely, a Mn-SOD is present 7, 10, 14) but much less is known about their occurrence in the
in the bacteroids, and suggest that the cytosol contain both Mn-SOD and root nodules of legumes. This is somewhat surprising since LHb2+
Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By is rapidly oxidized by O2 and H202, respectively, to the non-
contrast, the nodule cytosol exhibits an elevated peroxidase activity as functional forms LHb3+ and LHb4+ (17, 25, 26). We report here
compared with the foliar tissue; this activity was completely inhibited by the presence of substantial amounts of SOD, catalase and per-
50 to 100 micromolar KCN. The significantly lower contents of H202 oxidase in alfalfa nodules, in comparison with those found in
and malondialdehyde (a product of lipid peroxidation) in nodules with the leaves.
respect to those in leaves reveal that the above-mentioned bacteroid and
cytosol enzymes act in an efficient and combined manner to preserve MATERIALS AND METHODS
integrity of nodule cell membranes and to keep leghemoglobin active.
Plant and Bacterial Material: Growth Conditions. Seedlings of
alfalfa (Medicago sativa L. cv Aragon) were inoculated with
Rhizobium meliloti strain 102F51 (kindly provided by R. S.
Smith, The Nitragin Co., Milwaukee, WI). Plants were grown
for 3 months in a glasshouse and watered with Evans' N-free
nutrient solution (12). One week prior to the measurements, the
Activated forms of 02 produced by its incomplete reduction, plants were transferred to a controlled-environment chamber
such as superoxide anion radical (02 ) and H202, are very toxic with a 25/15°C and 60/80% RH day/night-regime; a fluorescent
to all forms of life. Even more reactive 02 species (singlet O2 (Sylvania F48T12-CW-WHO) tubes-incandescent lamps mixture
['02], hydroxyl radical [OH-]) are generated by the interaction provided a photon fluence rate of 200 ,uE.m-2-s-' (PAR) at the
between O2 and H202 (10, 15). A direct association between top plants for a 14-h photoperiod.
activated-02 metabolism and peroxidation of membrane lipids Enzyme Assays. All operations were conducted at 0 to 2°C.
(membrane deterioration) has been demonstrated (14, 19). Yet Fresh leaves (0.5 g) or nodules (1 g for bacteroids; 0.5 g for
plants, like other respiring organisms, have developed defensive cytosol) were immediately homogenized with 10% (w/w) poly-
mechanisms against the harmful effects of free radicals, either by vinylpolypyrrolidone and 10 ml of an extraction medium con-
directly scavenging them when already present or by preventing sisting of 50 mm K-phosphate (pH 7.8), and 0.1 mm Na2EDTA,
their formation (19). Thus SOD2 (EC 1.15.1.1) catalyzes the in an Omni-Mixer homogenizer (Sorvall, DuPont Instruments)
dismutation of 02 to H202 and 02, catalase (EC 1.11.1.6) at 50% speed for 3 min. The homogenate was filtered through
mediates the cleavage of H202 evolving O2, and peroxidase (EC four layers of cheesecloth, centrifuged at 500g for 2 min to
1.11.1.7) reduces H202 to H20 using several reductants available eliminate cell debris, and centrifuged again at 18,000g for 10
to the cells (15). min. The supematants, hereafter referred to as 'leaves' and
Three types of SOD are known at present: Cu plus Zn-, Mn-, 'nodule cytosol,' were used as enzyme sources. The pellet of the
and Fe-containing enzymes, which can be distinguished by selec- nodule extract containing the bacteroids was washed twice and
resuspended in 6.5 ml of the same buffer, then the bacteroids
' Partly financed by the United States-Spain Joint Committee for were disrupted by sonication in two 30-s pulses (M.S.E. Sonifier)
Scientific and Technological Cooperation in Basic Sciences (1985 Pro- in an ice-bath, and centrifuged at 35,000g for 20 min. The
gram). resulting supernatant, hereafter referred to as 'bacteriods,' was
2Abbreviations: SOD, superoxide dismutase (EC 1.15.1.1); Cu,Zn- used for enzymic assays.
SOD, copper plus zinc-containing superoxide dismutase; Fe-SOD, iron- SOD activity was assayed by the inhibition of the photochem-
containing superoxide dismutase; Mn-SOD, manganese-containing su- ical reduction of NBT, as described by Giannopolitis and Ries
peroxide dismutase; LHb2', LHb3+, ferrous, ferric leghemoglobin; NBT, (16) with minor modifications (29). The reaction medium com-
nitro blue tetrazolium; MDA, malondialdehyde. prised 0.37 ml 50 mm K-phosphate (pH 7.8), with 0.1 mm
1169
1170 BECANA ET AL.
Na2EDTA, 4 ml 02r-generating solution and 30 Ml enzyme. The
02--generating solution contained 2.2 AM riboflavin, 14.3 mM
methionine, and 82.5 AM NBT. Glass test tubes containing the
mixtures were placed in a cylindrical water-bath lined with
aluminum foil at 25°C and fitted with a 22-W circular fluorescent
lamp (Phillips). The reaction was initiated by turning the light
on and the reduction of NBT was followed by reading the A560
for 7 min; during this period the reaction was linear. Blanks and
controls were run the same way but without illumination and
enzyme, respectively. One unit of SOD was defined as the
amount of enzyme which produced a 50% inhibition of NBT
reduction under the assay conditions (5).
Catalase activity was determined by the procedure of Aebi (1),
which is suitable for crude extracts- even in the presence of
Plant Physiol. Vol. 82, 1986
maintained in ice until use. The decrease of A508 against distilled
H20 was followed and taken the minimal value. Blanks were
obtained by making 25 or 100 IAI 5% TCA to 2 ml with buffer.
Contents of H202 were determined from differences of A508
between samples and blanks, using H202 (30%, Merck) (5-50
Mm) as a standard.
Lipid peroxidation was estimated in terms of MDA content,
as indicated by Dhindsa et al. (8).
Enzymes were expressed as total activities (per unit dry weight
tissue). Contents of H202 and MDA were also expressed on a
dry weight basis. Dry weights of leaves and nodules were obtained
by drying at 80C for 48 h.
RESULTS AND DISCUSSION

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hemoglobin-like substances. One unit of activity was defined as
the amount catalyzing 1 Mmol perborate decomposed/min. Per-
oxidase activity was assessed by the guaiacol oxidation method
(27). One unit was defined as the enzymic amount which oxidizes
1 Mmol guaiacol/min.
Prior to determination of SOD and peroxidase activities in the
nodule cytosol, LHb was precipitated selectively by the Tsuchi-
hashi (chloroform-ethanol) (21) treatment to avoid interferences
(26).
H202 and Lipid Peroxidation. Content of H202 was deter-
mined based on the method of Patterson et al. (22). A 0.5 g
sample of leaves or nodules was homogenized in an Omni-Mixer
with 0.2 g activated charcoal and 12 ml 5% TCA. The homoge-
nate was filtered through four layers of cheesecloth and centri-
fuged at 18,000g for 10 min. The supernatant was filtered
through a Millipore filter (0.45 Mm) and used for the assay. A 25
Ml (leaves) or 100 Ml (nodules) aliquot was brought to 2 ml with
100 mm K-phosphate (pH 8.4) and 1 ml of colorimetric reagent
was added. This reagent was made daily by mixing 1:1 (v/v) 0.6
mM 4-(2-pyridylazo)resorcinol (disodium salt) (Sigma) and 0.6
mm potassium titanium oxalate (BDH Chemicals Ltd) and was
SOD activity from the soluble fraction of alfalfa foliar tissue
was entirely inhibited by 3 mM CN- (Table 1). Hence the enzyme
can be ascribed to the Cu,Zn-SOD type, in accordance with the
fact that Cu,Zn-containing SODs are characteristic ofthe cytosol
of eucaryotic cells (14, 15).
Both R. meliloti bacteroids and the nodule cytosol contain
considerable amounts of SOD. These results, however, are less
clear-cut than in the case of leaves, and deserve a larger comment.
Cyanide (1-3 mM) caused a 21 to 66% loss of bacteroidal SOD
activity (Table I). This observation was striking inasmuch as
procaryotes contain Mn- and/or Fe-SODs, both well-known to
be resistant to CN- (14, 15) even at 10 mM (2). Cyanide-sensitive
SOD activity of bacteroids can be attributed neither to contam-
ination by the cytosolic enzyme as bacteroids were washed up to
5 times with identical results, nor to SOD-like activity of perox-
idase (5) since only traces of this enzyme were found in the
bacteroids (Table I). Also other sources of error as metal chelates
which may mimic SOD activity were discarded because immer-
sion of bacteroidal and nodule cytosolic extracts in boiling water
for 2 min completely supressed SOD activity (11). Very likely a
Cu,Zn-SOD is present in nodule bacteroids. The occurrence of

Table I. Some Parameters of H202 Metabolism in Leaves, Bacteroids, and Nodule Cytosol ofAlfalfa Plants
The inhibitory effect of CN- on SOD activity was tested by including 1 or 3 mM KCN in the assay medium.
Inactivation of SOD with H202 was tested by preincubating the enzyme at 25°C for 15 min in 50 mm K-
phosphate (pH 7.8), containing 0.1 mM EDTA, the indicated concentrations of H202, and 3 mM KCN to
suppress peroxidase and catalase activities; then a small aliquot of the treated enzyme was withdrawn and
assayed for residual SOD activity in the presence of the same KCN concentration. Controls were performed
to ensure that the inhibitory compound added along with the enzyme did not interfere with the SOD assay.
Nodules
Parameter Leavesa
Bacteroidsa Cytosola
SOD (units/mg dry Wt)b
Noaddition 2.18±0.18 (100)a 1.02±0.08 (21)a 1.49±0.07 (33)a
+ I mM KCN 0.76±0.21 (65)b 0.80±0.06 (21)b 0.99±0.05 (33)b
+ 3 mm KCN 0 (100) c 0.35 ± 0.03 (66) c 0.49 ± 0.02 (67) c
+ 3 mM KCN + 5 mM
H202 0 (100)c 0.29 ±0.02 (72)c 0.21 ± 0.01 (86)d
+ 3 mM KCN + 10 mM
H202 0 (100) c 0.26 ± 0.01 (74) c 0.22 ± 0.01 (85) d
+ 3 mM KCN + 20 mM
H202 0 (100) c 0.30 ± 0.02 (71) c 0.16 ± 0.01 (89) d
Catalase (units/mg dry wt)b 37.7 ± 0.9 12.6 ± 1.1 10.1 ± 0.0
Peroxidase (units/g dry
wt)b 56.2 ± 7.2 Traces 222.8 ± 6.8
H202 (jsmol/g dry wt) 7.6 ± 2.0 4.3 0.6
±
Lipid peroxidation (nmol 26.2 ± 4.7
MDA/g dry wt) 119.6 ± 13.1 2 ±4.
a Data are the mean ± SE (n = 4-8 replicates corresponding to different extracts). Means denoted by the
same letter within each column do not differ at P < 0.05 (Duncan's multiple range test). Values in parentheses
are percent inhibition. b Units of enzymic activity are defined in "Materials and Methods."
 HYDROGEN PEROXIDE METABOLISM IN ALFALFA LEAVES AND NODULES
a Cu,Zn-containing enzyme has been so far reported in only one
other procaryote; curiously, in the also symbiotic bacterium
Photobacterium leiognathi (23). The possibility that this marine
bacterium has incorporated the enzyme through a gene transfer
from its ponyfish host has been speculated (20, 23). By analogy,
a similar proposal may be raised for Rhizobium bacteroids, which
are also involved in a long-standing symbiosis. The 34%-residual
SOD activity of bacteroids upon treatment with 3 mm CN- may
be assigned to a Mn-containing enzyme because of its resistance
to H202 inactivation, even at 20 mm (Table I). A point of interest
is, however, that treatment with chloroform plus ethanol did not
result in any reduction of this activity (data not shown), in sharp
contrast with reports on the denaturation of Mn-SOD (6, 15, 16)
and Fe-SOD (4) by organic solvents.
On grounds of inactivation studies with H202 and NaN3,
Stowers and Elkan (30) concluded that a single, inducible Fe-
containing SOD is present in free-living R. japonicum. Our
results are closer to those of Dimitrijevic et al. (9), who found
both Mn-SOD (largely predominant under normal conditions)
and Fe-SOD in R. phaseoli, but only Mn-SOD in bean nodule
bacteroids. None of these latter authors detected a Cu,Zn-SOD
in the bacteroids. Further work with polyacrylamide gels is clearly
needed to solve definitively this point, comparing SOD isozymes
from free-living and symbiotic rhizobia.
To avoid serious interferences between LHb and determina-
tions of SOD and peroxidase activities, the chloroform-ethanol
treatment was applied to the cytosolic extract. Nonetheless, a
33% of cytosolic SOD activity remained after treatment with 3
mM) KCN (Table I). Judging from data with the inhibitors H202
(5-20 mM) and CN- (3 mM), we suggest that both Fe- and Mn-
containing SODs are present in the cytosolic fraction. Again this
finding was unexpected since these activities should have been
destroyed during extraction. Since Salin and Bridges (28) first
reported the presence of a Fe-SOD in the eucaryote Brassica
campestris, the enzyme has been found in a number of higher
plants (1). In a survey of 43 families, the Fe-SOD was present in
the leaves of species belonging to only 3 of them (7). Although
these authors did not observe Fe-SOD in the Leguminosae
members Glycine max and Phaseolus vulgaris, the enzyme has
been subsequently detected in bean leaves ( 18).
The nodule cytosol and bacteroids exhibit substantial catalase
activities, albeit they were inferior to those of the foliar tissue if
expressed per dry weight tissue (Table I). A relationship has been
established between elevated catalase levels in the bacteroids or
in the cytosol of soybean nodules and a superior effectiveness of
the symbiosis (13).
The alfalfa nodule cytosol possesses much greater total perox-
idase activity than the leaf tissue but the bacteroids, interestingly,
devoid of it (Table I). Studies by Puppo et al. (24) on purified
soybean peroxidase have indicated that the pseudoperoxidatic
acitvity of LHb is probably not important in nodules; neverthe-
less, we cannot totally exclude the presence of small quantities
of LHb in the cytosol even after its selective precipitation by
chloroform-ethanol. Exposition to 50 to 100 LM CN- led to
complete suppression of peroxidase, in agreement with the high
CN-sensitivity of 'true' peroxidase activity (27).
Levels of free H202 and MDA, produced during the oxidation
of polyunsaturated fatty acids of membrane lipids, are consist-
ently lower in nodules than in leaves (Table I). This observation
indicates that the bacteroid and cytosol enzymes SOD, catalase
and peroxidase act jointly to scavenge the deleterious species
02-, H202 and their derivatives, thus limiting: (a) the deteriora-
tion of cellular membranes (8) and (b) the functional inactivation
1 171
of LHb2+/ LHb2+ *02 (25, 26).
Acknowledgments-We thank Miss A. Eguilaz and Miss A. Castillo for assistance
in the work reported here. Dr. Marvin Salin's (Mississippi State University) valuable
help in reviewing this manuscript is greatly appreciated.
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