Plant and Soil 237: 5561, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

55

Antioxidative and growth-promoting effect of selenium on senescing

lettuce

Tailin Xue1 , Helin Hartikainen2,3 & Vieno Piironen2

1 Institute
of Geography, Chinese Academy of Sciences, Beijing 100101 China. 2 Department of Applied Chemistry
and Microbiology, P.O. Box 27, SF-00014 University of Helsinki, Finland. 3 Corresponding author

Received 2 February 2001. Accepted in revised form 30 July 2001

Key words: antioxidant, GSH-Px, lettuce, selenium, senescence, SOD, tocopherol

Abstract
In human and animal cells, Se plays an essential role in antioxidation and exerts an antiaging function but it is toxic
at high dietary intake. To increase its intake in forage and foodstuffs, Se fertilization is adopted in some countries
where soils are low in bioavailable Se, even though higher plants are regarded not to require Se. To test its ability
to counteract senescence-related oxidative stress in higher plants, a pot experiment was carried out with lettuce
(Lactuca sativa) cultivated with increasing amounts of H2 SeO4 . The yields harvested 7 or 14 weeks after sowing
revealed that a low Se dosage (0.1 mg kg1 soil) stimulated the growth of senescing seedlings (dry weight yield
by 14%) despite a decreased chlorophyll concentration. The growth-promoting function was related to diminished
lipid peroxidation. In young and senescing plants, the antioxidative effect of Se was associated with the increased
activity of glutathione peroxidase (GSH-Px). In the senescing plants, the added Se strengthened the antioxidative
capacity also by preventing the reduction of tocopherol concentration and by enhancing superoxide dismutase
(SOD) activity. When no Se was added, tocopherols and SOD activity diminished during plant senescence. The
higher Se dosage (1.0 mg kg1 soil) was toxic and reduced the yield of young plants. In the senescing plants, it
diminished the dry weight yield but not the fresh weight yield.

Abbreviations: GSH reduced glutathione; GSH-Px glutathione peroxidase; MDA malondialdehyde; SOD
superoxide dismutase; TBARS thiobarbituric acid- reactive substances; TR thioredoxin reductase

Introduction and animal cells for Type I, II and III iodothyronine

Selenium (Se) was first recognized as an essential nu-
trient by Schwartz and Foltz (1957), who found that
it is the critical element in factor 3 that prevents liver
necrosis in rats. Later, Hogue et al. (1962) and Muth
(1963) found that Se and vitamin E prevented muscu-
lar lesions in young ruminants. Before these findings,
Se was considered to be a toxicant causing diseases
in livestock grazing selenoferous pastures (reviewed
by OToole and Raisbeck, 1998). However, Rotruck
et al. (1973) identified Se to be an essential compon-
ent of the enzyme glutathione peroxidase (GSH-Px).
Subsequently it was identified as essential in human
FAX No: +358-9-191-58475.
E-mail: helina.hartikainen@helsinki.fi
deiodinases (Berry et al., 1991; Pallud et al., 1997;
Ramauge et al., 1996), thioredoxin reductase (Tamura
and Stadtman, 1996) and for a number of selenopro-
teins (Behne et al., 1997; Gladyshev et al., 1998;
Whanger et al., 1997).
In Finland, where soils are very low in bioavail-
able Se, the daily Se intake in humans was below the
recommended level (Ekholm et al., 1995). Therefore,
since 1984 multinutrient fertilizers have been supple-
mented with Se in order to improve the nutritional
value of forage and foodstuffs. Even though higher
plants are thought not to require Se and to have a
low tolerance to it, there are increasing indications
that Se may also have beneficial biological functions
in higher plants. Recently, Hartikainen et al. (2000)
56
demonstrated that, depending on the dosage, Se ex-
erts a dual effect on ryegrass: At low concentrations,
it acts as an antioxidant and can stimulate the plant
growth, whereas at higher concentrations it acts as a
pro-oxidant reducing the yields.
In plants, numerous oxygen-containing radicals
are produced in various metabolic processes involving
oxygen, e.g. photosynthesis (Elstner, 1991) and res-
piration (Werf et al., 1991). An abnormal accumu-
lation of these species and their derivatives triggers
radical chain reactions that may cause damage to bio-
molecules such as chlorophyll, proteins, lipids and
nucleic acids, thus promoting cell senescence and cell
death. Senescence, an integral part of plant develop-
ment, may coincide with the production of free oxygen
radicals and be regulated by a variety of environmental
and autonomous factors. On the basis of the antioxid-
ative role of Se, it is theoretically possible that Se is
able to delay plant senescence and to diminish post-
harvest losses of agricultural plants. In this study, the
effect of Se on the quantity and quality of plant yield
of lettuce was investigated in a glasshouse experiment.
The objectives were (1) to investigate the ability of Se
to increase the antioxidative capacity of plants and to
counteract senescence-related oxidative stress and (2)
to improve the growth of senescing plants.
Materials and methods
Pot experiment
Lettuce (Lactuca sativa L. Australischer gelber) was
grown in a glasshouse in winter under low light con-
ditions. The daylight in December was supplemented
with Osram Viatox Nav-T 400 W lamps to give a
photoperiod of 17 h and quantum flux density of
160 mol m2 s1 at the top of the pots. The tem- Briefly, for determination of chlorophyll, the homo-
perature in the greenhouse varied between 10 C (by
night) and 20 C (by day). The plants were cultivated
in plastic pots (22 cm dia.) containing coarse-textured
soil (Inceptisol, pH 6.3 in 0.01 m CaCl2 and 2.8% of
organic C) taken from a field never amended with Se-
containing fertilizers. Prior to the experiment, the soil
was air-dried, sieved (< 35 mm) and homogenized.
Two sets of 4-kg soil samples (12 samples in each
set) were then weighted for the pot experiment. In
both sets, the soil units were treated in quadruplic-
ate with Se (supplied as H2 SeO4 , Fluka Chemie AG
product, analytical grade) at the rate of 0.1 and 1.0 mg
kg1 soil, or left without Se addition. As the range of
total Se in Finnish mineral soils is 0.040.7 mg kg1
(Ylranta, 1985), Se addition markedly increased the
Se reserves in soil. After Se treatment, a portion of
3.5 kg of soil in each unit was taken apart and 4.5 g
of ground NPK fertilizer (16-16-16, Se-background
0.1 mg kg1 , Kemira Agro Oy) was mixed into it.
This fertilized portion was put into the pot and covered
with the remaining unfertilized 0.5 kg of soil. Seeds
(15 per pot) were sown onto the upper layer of soil
and covered with 100 g of soil unamended with Se
and NPK fertilizer. Nitrogen, P and K added in the
fertilizer amounted to 180 mg per kg of soil. No mi-
cronutrients were added. During the growth period
plants were watered from below with deionized water.
The experiment was designed to contain two ran-
domized complete blocks with four replications of
three Se addition levels: 0, 0.1 and 1.0 mg kg1 . The
blocks represented the ages of the plants: (1) young
seedlings (YS), harvested 7 weeks after sowing, and
(2) senescing seedlings (SS), harvested 14 weeks after
sowing when chlorosis in the shoots, especially in the
oldest leaves, became evident.
Plant analyses
The fresh weight (f.wt) of the plant material from each
pot was determined at harvest. The dry weight (d.wt)
of the yields was determined by drying sub-samples
at 60 C for 48 h. The fresh material was stored at
70 C. For chemical analysis, three subsamples were
randomly taken from the fresh plant material repres-
enting the whole leaf biomass and their mean was
taken to represent the result of each replicate. Three
separate subsamples for tocopherols were stored at
90 C in a vacuum.
The analytical methods are described in more de-
tail in a previous paper (Hartikainen et al., 2000).
genized fresh leaf material was extracted with pre-
chilled 80% acetone solution and concentrations were
measured with a Shimadzu UV-120-02 spectropho-
tometer (Arnon, 1949). Selenium in the dry plant
material was analyzed by the method of Kumpulainen
et al. (1983), and an in-house reference sample was
included in every analytical round. Lipid peroxidation
was assayed by measuring thiobarbituric acid-reactive
substances (TBARS) according to a modified method
of Yagi (1982).
With respect to antioxidants, attention was fo-
cused on Se-induced changes in glutathione perox-
idase (GSH-Px), superoxide dismutase (SOD) and

tocopherols (vitamin E). SOD scavenges superox-
ide anion radicals, which are regarded as initiators
in free radical chain reactions and are produced in
all oxygen-requiring processes. Tocopherols scavenge
lipid peroxide radicals and singlet oxygen, and they
act cooperatively with Se in inhibiting lipid peroxid-
ation (Douillet et al., 1998). GSH-Px (EC 1.11.1.9)
activity was measured by a modification of the method
of Flohe and Gunzler (1984) by using H2 O2 (Riedel-
de Han product, GA) as substrate. The enzyme was
extracted by a pre-chilled KNaHPO4 (Merck product,
analytical grade) buffer, pH 7.0, with a homogenizer.
The supernatant obtained by centrifugation (10 min
at 3000 rpm) was used as an enzyme coarse extract.
For the enzyme reaction, 0.2 ml of the supernatant
was placed into a tube and mixed with 0.4 ml GSH
(reduced glutathione, Sigma product, analytical grade)
(0.1 mM) and 0.2 ml KNaHPO4 (0.067 M). The above
reagents without supernatant extract were used for
the non-enzyme reaction. After pre-heating the mix-
tures on water bath at 25 C for 5 min, 0.2 ml H2 O2
(1.3 mM) was added to initiate the reaction. The re-
action lasted 10 min and was terminated by adding 1
ml 1% trichloric acetic acid (Merck product, analytical
grade), and the mixture was put into an ice bath for 30
min. Then the mixture was centrifuged for 10 min at
3000 rpm, 0.48 ml of the supernatant was placed into
a cuvette, and 2.2 ml of 0.32 M Na2 HPO4 (Riedel-
de Han product, analytical grade) and 0.32 ml of
1.0 mM 5,5 -dithio-bis (2-nitrobenzoic acid) (DTNB,
Sigma product, analytical grade) were added for color
development. The absorbance at wavelength 412 nm
was measured with a Shimadzu UV-120-02 spectro-
photometer within 5 min. The enzyme activity was
calculated as a decrease in GSH within the reaction
time as compared to that in the non-enzyme reaction.
SOD (EC 1.15.1.1) activity was analysed by a
modified method of Giannopolitis and Ries (1977).
Concentrations of - and -tocopherol were measured
by a small-scale HPLC method described by Xue et al.
(1997). The protein concentration in the enzyme ex-
tract was determined spectrophotometrically (Murphy
and Kies, 1960).
Data analysis
The responses of each harvest to Se applications were
tested separately using Duncan test. The senescence-
induced changes in plant characteristics at each Se
level (expressed in percentages) were tested by con-
ventional t-test.
57
Results
The lower Se addition did not affect either fresh weight
or dry weight of the younger plants, but did signi-
ficantly stimulate (p<0.05) growth of the senescing
plants (d.wt yield by 14%) (Table 1). The higher Se
dosage was toxic to the young lettuce seedlings and
reduced their shoot yields. In the senescing plants,
however, the detrimental effect was not seen in fresh
weight even though dry weight was markedly di-
minished. This response indicates that the higher Se
addition raised the water content of the leaves. Fur-
thermore, when no Se was added, the senescence of
the older plants appeared as a reduction in the con-
centration of total chlorophyll (Table 1). The added
Se increased (p<0.05) chlorophylls in the young seed-
lings but was not able to counteract their decrease in
the senescing plants (Table 1).
Without Se addition, the Se concentration in the
young plants was very low but increased markedly
(66%, p<0.01) during plant development (Table 2).
On the other hand, in the plants supplied with Se the
concentration was high and diminished significantly
(p<0.05) during senescence. In fact, the Se uptake
(yield Se concentration) at the addition level of
1.0 mg kg1 soil was 38% lower in the senescing seed-
lings than in the young seedlings, indicating that Se
losses had taken place during senescence.
In the control plants cultivated without Se, lipid
peroxidation increased significantly with senescence
(Table 2). The added Se reduced lipid peroxidation
irrespective of plant age. Consequently, during senes-
cence the accumulation of TBARS remained lower in
the Se-treated plants. It is noteworthy, however, that
at the higher Se addition the decrease in lipid perox-
idation was smaller, indicating that the antioxidative
effect of Se was diminished.
In lettuce cultivated without Se addition, GHS-Px
increased (p<0.05) during plant senescence to coun-
teract oxidative reactions, whereas the SOD activity
diminished (Table 3). In the plants supplied with a low
Se dosage, GSH-Px activity increased irrespective of
the plant age. On the other hand, added Se diminished
SOD in the younger seedlings and increased it in the
senesced ones. The net result was that SOD activity
increased in the Se-treated lettuce during senescence
(Table 3).
In the young seedlings, Se added at a low level
decreased the concentration of tocopherols, while a
high Se dosage increased them (Figure 1). In the sen-
escing seedlings, on the other hand, both Se additions
58
Table 1. Fresh and dry weight of shoot yields, the concentration of total chlorophylls in lettuce at various Se addition levels (means and standard
errors, n=4), and their changes during senescence. Each column was tested separately. Values followed by the same letter do not differ at P
0.05 (Duncan test). The changes during senescence (+/) were tested by t-test: = P 0.05, = P 0.01. YS = young seedlings, SS =
senescing seedlings

Se added Fresh weight Dry weight Total chlorophyll content

mg kg1 g pot1 g pot1 mg g1 DW
YS SS Change% YS SS Change% YS SS Change%

0 177a 5 256b 5 +44 20.7a 1.9 46.9b 2.2 +126 2.41c 0.13 1.14b 0.09 53
0.1 160a 12 286a 9 +78 20.3a 1.6 53.5a 2.8 +163 2.60b 0.05 1.17b 0.07 55
1.0 97b 28 256b 22 +164 6.9b 1.6 27.9c 1.7 +304 4.78a 0.12 2.09a 0.10 56

Table 2. Se uptake and lipid peroxidation (TBARS) in lettuce shoots at various Se addition levels, and their changes during senescence.
Values are means of four replicates standard errors. Each column was tested separately. The means followed by the same letter do not
differ at P 0.05 (Duncan test). The changes during senescence (+/) were tested by t-test: = P 0.05, = P 0.01. YS = young
seedlings, SS = senescing seedlings

Se added Se concentration in shoot material Total Se uptake TBARS

mg kg1 mg kg1 DW g pot1 nmol MDA g1 DM
YS SS Change% YS SS Change% YS SS Change%

0 0.080c 0.011 0.133c 0.040 +66 1.66c 0.22 6.22c 1.88 +276 128a 23 268a 28 +109
0.1 4.8b 0.3 1.9b 0.2 60 98b 6 103b 9 +5 74c 4 159c 18 +115
1.0 270a 54 41a 4 85 1860a 370 1150a 100 38 110b 7 199b 14 +81

to promote the synthesis of -tocopherol (p<0.01)

(Figure 1). In addition, there was a significant differ-
ence in the balance of / tocopherols: irrespective
of the Se treatment -tocopherol dominated over -
tocopherol in the young seedlings (the range in /
was 0.510.73), whereas the senescing plants exhib-
ited an opposite dominance (the range in / was
1.581.70).

Discussion

The effect of Se on plant growth was dependent on

Figure 1. - and -tocopherols in lettuce at various Se addi-
tion levels. In young and senescing seedlings, the differences in
both tocopherol species between various Se levels were significant
(Duncan test, P0.05). At a given Se level, the senescence-induced
changes in both tocopherols were significant (t-test, P0.01).
significantly (p<0.05) increased the total tocopherol
concentration. During senescence, total tocopherols
diminished when no Se was added, but the Se ad-
ditions counteracted their decrease. It is noteworthy
that in senescing lettuce a high Se dosage appeared
dosage and plant age: at a lower addition level it stim-
ulated the growth of senescing lettuce, but at a high
level it exerted a toxic effect irrespective of plant age.
These responses are similar to those obtained in a pre-
vious study by Hartikainen et al. (2000) with ryegrass.
A significant increase in total chlorophylls and an el-
evated water content in the senescing seedlings at the
higher Se level (Table 1) indicate that excess Se caused
metabolic disturbances. In fact, Se can indiscrimin-
antly replace S and incorporate Se-amino acids into
proteins (Brown and Shrift, 1982; Eustice et al., 1981;
Stadtman, 1974). The formation of Se-amino acids, in
 59
Table 3. The activity of gluthatione peroxidase (GSH-Px) and superoxide dismutase (SOD) in lettuce seed-
lings at various Se addition levels, and their changes during senescence. Values are means of four replicates
standard errors. Each column was tested separately. The means followed by the same letter do not differ
at P 0.05 (Duncan test). The changes during senescence (+/) were tested by t-test: = P 0.05, = P
0.01. YS = young seedlings, SS = senescing seedlings

Se added GSH-Px SOD

mg kg1 mol GSH mg1 protein min1 Unit mg protein min1
YS SS Change% YS SS Change%

0 5.58c 1.21 5.94b 0.94 +6 0.78a 0.15 0.67b 0.10 14

0.1 6.95a 0.57 6.86a 0.99 1 0.68b 0.02 0.75a 0.12 +10
1.0 6.55b 2.18 6.78a 1.09 +4 0.46c 0.11 0.74a 0.23 +61

turn, is found to enhance ethylene production (Konze
et al., 1978), which can modify membrane lipid com-
position (Thompson et al., 1982), increase membrane
permeability (Hanson and Kende, 1975; Vangronsveld
et al., 1993) and result e.g. in an increased K+ leakage
(Vangronsveld et al., 1993). Thus, it can be hypothes-
ized that high Se addition enhanced K+ leakage, and
more water was retained in the intercellular space to
counterbalance an increased osmotic pressure.
Similarly as in a previous study (Hartikainen et al.,
2000), the positive response of plant growth to low Se
addition was delayed appearing when the plants be-
came older. The growth-stimulating effect of low Se
addition was not related to total chlorophyll, which
diminished during senescence, but rather to its antiox-
idative function as demonstrated by diminished lipid
peroxidation in senescing plants. At the high addi-
tion level, however, the antioxidative function of Se
seemed to be diminished or reversed irrespective of
plant age, lipid peroxidation being higher than at the
low Se level (Tables 2 and 3). This response supports
the finding that at a high level Se can even act as
a pro-oxidant, and this mechanism can, in addition
to metabolic disturbances, contribute to Se toxicity
(Hartikainen et al., 2000).
No growth-promoting effect of Se was seen in the
young seedlings despite an increase in total chloro-
phylls. Lipid peroxidation was reduced concomitantly
with an increase in GSH-Px activity. This suggests
that in the young plants the antioxidative capacity was
sufficient for normal growth under the low-light con-
ditions prevailing in the experiment. However, greater
antioxidative capacity is needed to counteract the
enhanced lipid peroxidation during senescence. The
results reveal that in the senescing plants cultivated
without Se, antioxidative capacity was strengthened
by increased GSH-Px (Table 3) in accordance with
rising Se concentration (Table 2). In the Se-fertilized
seedlings, on the other hand, GSH-Px was not able
to increase further during senescence, which was in
agreement with the decrease in the Se concentration
(Table 2). The lower Se concentration could be attrib-
utable either to reduced Se absorption by the senescing
seedlings or to Se loss as volatile compounds. In fact,
a distinct reduction in the Se taken up by the senescing
seedlings (Table 2) supports the latter hypothesis pre-
viously shown to occur in lettuce (Terry et al., 1992;
Terry and Zayed, 1994).
The reduction in SOD (Table 3) and tocopher-
ols (Figure 1) found in the young seedlings at low
Se level agrees with the results reported earlier for
young ryegrass and lettuce seedlings (Hartikainen et
al., 1997, 2000; Xue and Hartikainen, 2000). In
addition, the young seedlings were dominated by
-tocopherol, whereas the senescing seedlings were
dominated by -tocopherol, which is a more active
species in antioxidation and is synthetized from -
tocopherol (Schultz et al., 1991). In other words, as
lettuce became older, more -tocopherol was syn-
thesized from -tocopherol to meet the increased
antioxidant requirement.
When no Se was added, tocopherols and SOD di-
minished during plant senescence. The added Se con-
tributed to the maintenance of antioxidative capacity
by diminishing the reduction in tocopherols and by
enhancing SOD. Thus, it provided extra antioxidative
capacity which might indirectly diminish the demand
for SOD. Superoxide radicals can be used in spon-
taneous disproportion reactions producing H2 O2 and
singlet oxygen (Thompson et al., 1987). Hartikainen
et al. (2000) hypothesized that at the same time that
Se promotes scavenging of produced H2 O2 through
increased GSH-Px it enhances the spontaneous dis-
proportion of superoxide radicals and, consequently,
60
reduces the need for their scavenger, SOD. Further-
more, at the non-toxic Se level the decrease in su-
peroxide radicals through spontaneous disproportion
diminished the production of lipid peroxide radicals
(LOO) and, consequently, the need for scavenging
tocopherols.
These results call for further studies of the role
of Se in senescing plants. However, they clearly in-
dicate that Se may exert a beneficial role in plants
under stress and, thus, they support the finding of
Hartikainen and Xue (1999) that Se defends plants
subjected to UV(B) episodes. Even if the present study
provides some support for the hypothesis of the exist-
ence of a Se-dependent GSH-Px in higher plants, the
Se-induced increase in this enzyme alone may not be
marked enough to explain the enhanced antioxidative
capacity and stimulated growth of senescing plants.
Acknowledgements
We thank Ms. Marjatta Koivisto, M. Sc. Satu
Pekkarinen and Ms. Xiuping Wang for skillful tech-
nical assistance. We are grateful to Docent Arja
Pennanen for constructive criticism on the manu-
script. Funding for this research was provided by the
Academy of Finland and Kemira Oyj.
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