Soil Biology & Biochemistry 36 (2004) 15531558

www.elsevier.com/locate/soilbio

Influence of selenium on oxidoreductive enzymes

activity in soil and in plants
Janina Nowaka,*, Krzysztof Kaklewskia, Marek Ligockib
a
Department of Biochemistry, University of Agriculture, Ul. Sowackiego 17, 71-434 Szczecin, Poland
b
Department of Poultry Breeding, University of Agriculture, Ul. Doktora Judyma 22, 71-434 Szczecin, Poland
Received in revised form 14 March 2004

Abstract
The aim of this greenhouse experiment was the assessment of the influence of H2SeO3 at soil concentrations of 0.05, 0.15 and
0.45 mmol kgK1, on the activity of selected oxidoreductive enzymes in wheat (Triticum aestivum). The wheat plants were grown in 2 dm3
pots filled with dust-silt black soil of pH 7.7. Applied H2SeO3 caused activation of plant nitrate reductase at all concentrations, but activation
of plant polyphenol oxidase at only two lower concentrations. The highest concentration caused inhibition of polyphenol oxidase and
peroxidase. Plant catalase activity decreased under the influence of 0.15 and 0.45 mmol kgK1 concentration. After the final analysis Se was
quantified in plants and soil. The amounts in plants were: control (unamended soil) 1.95 mg kgK1; I dose (0.05 mmol kgK1) 18.27 mg kgK1;
II dose (0.15 mmol kgK1) 33.20 mg kgK1 and III dose (0.45 mmol kgK1) 38.37 mg kgK1, in soil: 0.265 mg kgK1; 3.61 mg kgK1;
10.53 mg kgK1; 30.53 mg kgK1; respectively. Simultaneously, a laboratory experiment was performed, where the activity of soil catalase
and peroxidase were tested after 1, 3, 7, 14, 28, 56, and 112 days after Se treatment. Peroxidase activity in soil decreased with increasing Se
content, over the whole experiment. The lowest dose of Se caused activation a significant 10% increase in catalase activity, but the influence
of others doses was unclear.
q 2004 Elsevier Ltd. All rights reserved.

Keywords: Selenium; Peroxidase; Catalase; Nitrate reductase; Polyphenol oxidase; Wheat (Triticum aestivum)

1. Introduction properties (Shrift and Ulrich, 1969; Harbone, 1997).

Selenium is an essential element for human and animal
metabolism. Its antioxidant properties, comparable to those
of vitamin E, are widely known (Kabata-Pendias and
Pendias, 1999). However, when absorbed in higher concen-
trations, it can be harmful and catalyse the oxidation of thiols
and simultaneously generate superoxide O$K 2 ; which means
it acts as a prooxidant (Zayed et al, 1998; Stewart et al.,
1999). Due to its chemical similarity to S, Se can replace it in
compounds containing sulfhydryl groups, such as sulphur
amino acids, glutathione and coenzyme A. When replacing
sulphur in proteins, Se can modify their structure and
* Corresponding author. Tel.: C48-91-4250-370; fax: C48-91-4871-
962.
E-mail address: biochem@agro.ar.szczecin.pl (J. Nowak).
0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2004.07.002
Enzymes of the oxidoreductase class can be the most Se
sensitive, owing to the antioxidant properties of the element.
The literature provides abundant information on the role
Se plays in animals. Many authors have indicated a strong
influence of Se on the activities of oxidoreductase enzymes,
such as catalase, glutathione peroxidase and superoxide
dismutase. Howewer, there have been relatively few reports
on the contribution of Se to biochemical processes in soil
and plants.
Worldwide research on the Se content of soils has shown
that it varies from deficit quantities of 0.01 mg kgK1 at the
Russian Plane to heavily toxic values of 1200 mg kgK1 in
organic soils at Meath, Ireland. Selenium content in soils
originating from light and medium clays in Poland falls into
the middle range of concentrations and varies between 0.20
and 0.34 mg kgK1 of soil (Piotrowska, 1984).
1554 J. Nowak et al. / Soil Biology & Biochemistry 36 (2004) 15531558
Selenium in soils occurs in inorganic forms as selenite IV
SeO2K 2K
3 and selenate VI SeO4 ; as well as in organic
forms. Selenate is not adsorbed in soil and is easily leached.
In contrast, selenite is rapidly adsorbed and is resistant to
leaching, hence it can provide a long-lasting Se source for
plants (Fio et al., 1991).
Selenium content in plants depends on several factors
that determine the availability of the Se present in the
soil and in the plants. Cruciferae plants contain four
times more Se than Papilionaceae plants, whose Se
content is twice that of grasses and cereals. Most cereal
crops and fodder plants show relatively weak ability to
absorb Se, even when grown on soils with higher Se
content. Selenium in plants is found in the form of
elemental Se, as selenite, selenate, or as organic
compounds (Zabocki, 1990).
Our research on the effect of Se on soil enzyme activities
(Nowak et al., 2002), has proven that addition of Se to soil
slightly affects the activities of hydrolytic enzymes, such as
acid and alkaline phosphatases or b-glucosidase, but
substantially alters activities of oxidoreductases, e.g.
dehydrogenase or nitrate reductase.
In this study, effects on selected enzyme activities
of 0.05, 0.15 and 0.45 mmol kgK1 selenium introduced
into soil, in the form of H2SeO3, were investigated.
The selected enzymes included soil peroxidase
and catalase, as well as plant enzymes of the oxydor-
eductase family, such as nitrate reductase, peroxidase,
catalase and polyphenol oxidase in wheat of the Kobra
cultivar.
2. Materials and methods
2.1. Laboratory experiment
Enzyme activities in soil were studied under optimal
laboratory conditions at 20 8C and 60% mwc (maximum
water capacity). The samples were collected from upper soil
layer (030 cm) at Pyrzycka Plain. The soil texture was silty
clay and the Corg. content was 1.92%. The soil was dried at
room temperature (about 20 8C) and sieved (!2 mm). An
aqueous solution of H2SeO3 was added, to give the following
four concentrations: zero, 0.05, 0.5, 5.0 mmol kgK1 (control,
I, II and III). The prepared samples were then analysed for
catalase and peroxidase activity at frequent intervals at the
start of the experiment and at longer intervals at the later
stages.
Activity of soil catalase was determined according to
Ladd (1978), using H2O2 as substrate, in units of H2O2
decompositon gK1 dry soil over 30 min (mg H 2O 2
(30 minK1) gK1 d.m. soil).
Activity of soil peroxidase was determined according
to Ladd (1978), using pyrogallol as substrate; the amount
of purpurogaline produced was adopted as the activity
unit (mmol of purpurogaline gK1 d.m.soil hK1).
2.2. Greenhouse experiment
A pot experiment was carried out under greenhouse
conditions, using Kobra cultivar wheat as the test plant.
Plants were grown in pots of 2 dm3 volume filled with the
soil described above, to which various amounts of H2SeO3
solution were added. Four combinations, control, I, II, and
III, that contained no selenium, 0.05, 0.15 and
0.45 mmol kgK1 of soil, respectively, were used, each in
four pots. The soil humidity was balanced at 60% of the
maximum water-holding capacity. The experiment was set
on 7 May 2002 and maintained until 20 August 2002.
Activity measurements were performed over four plant
developmental stages, namely a 3 leaves stage, shooting
stage, an ear formation stage, and early maturity stage, each
replicated three-fold. The respective stages corresponded to
the following days of the experiment: 42, 58, 70 and 112,
respectively.
In the greenhouse experiment activities of the following
plant enzymes were investigated: nitrate reductase (EC
1.7.99.4), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6),
and polyphenol oxidase (EC 1.10.3.2).
Activity of nitrate reductase was determined according to
Tweedy and Ries (1966), with KNO3 as the substrate, and
the amount of NOK 2 in mg produced by 1 g of fresh plant
tissue over 1 h of incubation adopted as the activity unit
(mg NOK 2 g
K1
f.m. plant hK1).
Plant peroxidase activity was determined according to
Chance and Maehly (1955), using pyrogallol as the
substrate, with the amount of purpurogaline produced over
1 min by 1 g of fresh tissue (mmol purpurogaline gK1 f.m.
plant minK1) adopted as the activity unit.
Polyphenol oxidase activity was determined according to
Chance and Maehly (1955), using pyrogallol as substrate,
and activity was expressed as mmol purpurogaline gK1 f.m.
plant minK1.
Catalase activity was determined according to Beers and
Sizer (1952), with H2O2 as the substrate, and activity
expressed as mg H2O2 gK1 f.m. plant minK1.
The results were presented as linear plots together with
LSD values calculated with Students t-test, at PZ0.05.
Additionally, the total effect of each Se dose on soil and
plant enzyme activities was calculated, as the mean of all
measurements during the whole experiment.
Upon completion of the final measurement, the plants
were harvested and dried. Subsequently, both soil and plant
samples were analysed for Se content in compliance with
branch standard Fodderdetermination of selenium con-
tent in fodder and fodder premixes No BN-91/9160-44.
Prior to analysis samples were digested in a microwave
oven in 65% HNO3. Inductively coupled plasma optical
emission spectroscopy (ICP OES) with an Optima 2000
DV apparatus manufactured by Perkin Elmer was used to
determine Se content in the studied material; a multielement
Merck. ICP Multielement Standard VI was used. The
measurements were recorded at a wavelength of 196.26 nm,
 J. Nowak et al. / Soil Biology & Biochemistry 36 (2004) 15531558
with axial optical route option (along plasma flame). The
mean recovery of Se from soil was 98.97G0.21%, and from
plants was 97.21G0.27%
3. Results
3.1. Laboratory experiment
Soil peroxidase activity was inhibited significantly at all
H2SeO3 concentrations (Fig. 1a). Activity of the enzyme in
the control soil and in the soil exposed to Se concentration I,
after a small increase observed on day 3, declined for the
rest of the experiment. Application of Se at concentrations II
and III resulted in a small activity increase initially, but a
decline later. Enzyme activity declined with increased Se
content in soil, to as low as 50% of the control sample
activity for concentration III (Fig. 3a).
Catalase activity in soil was observed to diminish on day
3 of the experiment both in the control and in all treated
soils, regardless of the applied Se concentration. It later rose
steadily until day 14, but in the following days the
differences in catalase activity were found to be immaterial,
apart from its rise in the soil with concentrations I and II on
days 56 and 112, respectively (Fig. 1b). Despite the
inconsistent behaviour of catalase activity in soils treated
with various Se doses, a slight activation from H2SeO3
concentrations I and II, and an inhibition by concentration
III, can be seen (Fig. 3b).
Fig. 1. Changes in enzymes activities in soil: (a) peroxidase, (b) catalase, caused by various doses of selenie acid.
1555
3.2. Greenhouse experiment
In the greenhouse research large impacts of the applied
Se on activity of the selected oxidoreductase enzymes were
found in the wheat cultivated on the treated soils.
For plant peroxidase activity, only the highest dose
(0.45 mmol kgK1) caused a significant change. Initially
peroxidase activities were the same irrespective of dose.
Subsequently, until the third measurement (70 d)
activities increased and only for the highest dose, was
there a difference in the rate of increase (Fig. 2a). For
concentrations I and II, added Se did not affect
peroxidase activities significantly over the entire duration
of the experiment, whereas concentration III caused a
significant inhibition (O20%) compared with the activity
in the control plants (Fig. 4a).
No marked changes were noted for catalase activity in
plants over the entire experiment. In plants grown on the
soil with Se concentration I, the enzyme was found to be
significantly activated when compared to the control
plants. In plants grown on substrate treated with concen-
trations II and III, an inhibition of the enzyme was observed
that was significant for all the times, apart from the first
(Figs. 2b and 4b).
During the experiment an increase in polyphenol
oxidase activity was observed as a result of Se addition
at 0.05 and 0.15 mmol kgK1, while Se concentration III
resulted in reduced activity. Such differences, though
varying slightly, remained stable over the entire
1556 J. Nowak et al. / Soil Biology & Biochemistry 36 (2004) 15531558

Fig. 2. Changes in enzymes activities in wheat: (a) peroxidase, (b) catalase, (c) polyphenol oxidase, (d) nitrate reductase, caused by various doses of selenium.

experiment (Fig. 2c). Activation caused by Se concen-
tration I, although exceeding 180%, remained smaller than
that caused by concentration II, which reached 210% of the
activity in the control plants. Concentration III caused a
60% inhibition (Fig. 4c).
Particularly pronounced positive influence of all con-
centrations of Se were observed for nitrate reductase
(Fig. 2d). Activity in the plants grown on the control soil
did not vary significantly over the experiment. Activity of
this enzyme increased in the plants grown on all soils treated
Se. The nitrate reductase activity was very high in relation to
the control plants and for the highest Se concentration
reached over 6500% (Fig. 4d).
During the development of plants, the increased Se
concentration in soils was accompanied by a rise in the
amount of selenium in plants (Table 1). The plants grown on
the control substrate contained 1.95 mg kgK1 d.m. of Se,
whereas those grown on the soils treated by concentrations

Fig. 3. Influence of various concentrations of selenic acid on the activity of soil enzymes: (a) peroxidase, (b) catalase.
 J. Nowak et al. / Soil Biology & Biochemistry 36 (2004) 15531558 1557

Fig. 4. Influence of various concentrations of selenic acid added to soil on the activity of plant enzymes: (a) peroxidase, (b) catalase, (c) poliphenol oxidase, (d)
nitrate reductase.

I, II and III were characterised with contents of 18.27, 33.20 release peroxidase from membrane structures. Similar
and 38.73 mg kgK1 d.m. Se, respectively. activity changes of peroxidase and catalase were observed
During the experiment the pH of soil was not in plants under O2, Fe, and Al influence (Cakmak and Horst,
significantly affected by Se amendment. 1991).
Applied Se at 0.05 and 0.15 mmol kgK1 activated of
polyphenol oxidase, while the highest concentration caused
4. Discussion inhibition.
Polyphenol oxidase and peroxidase catalyse the oxi-
Our results revealed significant changes in the activities dation of the same substrates, therefore plants usually
of oxidoreductase enzymes in response to added Se, both manifest activity of only one of these enzymes. It seems
soil and wheat plants. The nature of the changes can not be possible also, that the affinity of various izoenzymes differs
clearly determined, however, because they were found to (Kar and Mishra, 1976).
depend on both the concentration of Se and the enzyme. Comparing our results to those of other authors, it can be
The greenhouse experiment revealed an increase in generalised that the lowest Se concentration positively
activity of both catalase and peroxidase in plants, induced affected the antioxidant defence in wheat plants, but higher
by Se at 0.05 mmol kgK1, whereas these activities were concentrations provoked stress responses. Hence, higher
respectively, reduced and increased at 0.15 mmol kgK1. concentrations can be regarded as a prooxidant.
The highest concentration (0.45 mmol kgK1 caused a
reduction in both enzyme activities. Table 1
Increased plant peroxidase activity is usually correlated Amount of selenium added to soil and amount of selenium in soil and in
with environmental stress (i.e. temperature extremes, plants after completing experiment
drought, mineral deficiency), increased ozone concentration Combination Amount of Se Amount of Se Amount of Se
or ageing. According to Zhang and Kirkham (1994), wheat added to soil in soil in plant
plants subjected to water stress exhibited increased (mg kgK1) (mg kgK1) (mg kgK1)
peroxidase activity and simultaneously a small reduction Control 0.00 0.27G0.02 1.95G0.10
in catalase activity. As suggested by the authors, I concentration 3.95 3.61G0.02 18.27G0.44
II concentration 11.85 10.53G0.12 33.20G0.43
increased peroxidase activity may indicate production of
III concentration 35.50 30.53G0.19 38.73G0.33
large amounts of H2O2. Elevated H2O2 concentrations could
1558 J. Nowak et al. / Soil Biology & Biochemistry 36 (2004) 15531558
Catalase and peroxidase activities in soil and in plants
compared against each other, allow the observation that
adding Se to soil causes activation of catalase both in soil
and plant, although the activity in soil is much more
variable. Peroxidase activity in soil was inhibited by all of
the Se concentrations, but inhibition of this enzyme in plants
occurred only in those grown on the soil treated with the
highest Se concentration, and only from plants which were
shooting into stalk.
In this experiment, a significant increase in nitrate
reductase activity was observed in the later development
stages of wheat plants grown on Se treated soil, probably
due to incorporation of selenocysteine to one of the active
sites, where NAD(P)H is bound. Enzymes with selenocys-
teine in the active site acquire a greater, more powerful
redox potential towards their substrates, because selenocys-
teine has a greater nucleophilic power and a lower pK than
cysteine (Eshdat et al., 1997; Campbell, 2001).
Plant nitrate reductase reacts to Se treatment of soil quite
differently from soil nitrate reductase. Research conducted
previously reported the soil enzyme to be inhibited even by
the lowest applied concentration. The highest concentration,
i.e. 5.0 mmol kgK1 caused 50% inhibition (Nowak et al.,
2002).
In our experiment nitrate reductase activity decreased
slightly in plants grown on the control substrate, but did not
vary significantly over the experiment. This confirmed the
results of Wojcieska et al. (1992) who reported a drop in
nitrate reductase for triticale, as the plants grew.
The quantity of Se in plants increased as Se concentration
in soil increased. Plants grown on the soil with no Se added
contained 1.95 mg kgK1 d.m., exceeding the average value
range for Se content in plants (0.2 mg kgK1 d.m.) according
to Kabata-Pendias and Pendias (1999). Plants grown on the
soil treated with Se at the lowest concentration contained
18.27 mg kgK1 d.m. of the element, which, although the Se
content was significantly higher, was still below the toxic
limit (30 mg kgK1 d.m.). However, the plants from the
higher Se treatments absorbed Se to concentrations of 33.20
and 38.73 mg kgK1 d.m., respectively. These results show
that Se can be absorbed by arable crops from soils of higher
Se content. This disproves the premise of weak selenium
absorption capacity for crops grown on soils with higher
selenium content. Apart from other factors, the form
of selenium present in the substrate mainly determines
the degree of selenium availability. Plants absorb mainly
selenite and selenate, and to a smaller extent, organic
selenium.
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