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
demonstrated that, depending on the dosage, Se ex- total Se in Finnish mineral soils is 0.040.7 mg kg1
erts a dual effect on ryegrass: At low concentrations, (Ylranta, 1985), Se addition markedly increased the
it acts as an antioxidant and can stimulate the plant Se reserves in soil. After Se treatment, a portion of
growth, whereas at higher concentrations it acts as a 3.5 kg of soil in each unit was taken apart and 4.5 g
pro-oxidant reducing the yields. of ground NPK fertilizer (16-16-16, Se-background
In plants, numerous oxygen-containing radicals 0.1 mg kg1 , Kemira Agro Oy) was mixed into it.
are produced in various metabolic processes involving This fertilized portion was put into the pot and covered
oxygen, e.g. photosynthesis (Elstner, 1991) and res- with the remaining unfertilized 0.5 kg of soil. Seeds
piration (Werf et al., 1991). An abnormal accumu- (15 per pot) were sown onto the upper layer of soil
lation of these species and their derivatives triggers and covered with 100 g of soil unamended with Se
radical chain reactions that may cause damage to bio- and NPK fertilizer. Nitrogen, P and K added in the
molecules such as chlorophyll, proteins, lipids and fertilizer amounted to 180 mg per kg of soil. No mi-
nucleic acids, thus promoting cell senescence and cell cronutrients were added. During the growth period
death. Senescence, an integral part of plant develop- plants were watered from below with deionized water.
ment, may coincide with the production of free oxygen The experiment was designed to contain two ran-
radicals and be regulated by a variety of environmental domized complete blocks with four replications of
and autonomous factors. On the basis of the antioxid- three Se addition levels: 0, 0.1 and 1.0 mg kg1 . The
ative role of Se, it is theoretically possible that Se is blocks represented the ages of the plants: (1) young
able to delay plant senescence and to diminish post- seedlings (YS), harvested 7 weeks after sowing, and
harvest losses of agricultural plants. In this study, the (2) senescing seedlings (SS), harvested 14 weeks after
effect of Se on the quantity and quality of plant yield sowing when chlorosis in the shoots, especially in the
of lettuce was investigated in a glasshouse experiment. oldest leaves, became evident.
The objectives were (1) to investigate the ability of Se
to increase the antioxidative capacity of plants and to Plant analyses
counteract senescence-related oxidative stress and (2)
to improve the growth of senescing plants. 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
Materials and methods at 60 C for 48 h. The fresh material was stored at
70 C. For chemical analysis, three subsamples were
Pot experiment randomly taken from the fresh plant material repres-
enting the whole leaf biomass and their mean was
Lettuce (Lactuca sativa L. Australischer gelber) was taken to represent the result of each replicate. Three
grown in a glasshouse in winter under low light con- separate subsamples for tocopherols were stored at
ditions. The daylight in December was supplemented 90 C in a vacuum.
with Osram Viatox Nav-T 400 W lamps to give a The analytical methods are described in more de-
photoperiod of 17 h and quantum flux density of tail in a previous paper (Hartikainen et al., 2000).
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 genized fresh leaf material was extracted with pre-
night) and 20 C (by day). The plants were cultivated chilled 80% acetone solution and concentrations were
in plastic pots (22 cm dia.) containing coarse-textured measured with a Shimadzu UV-120-02 spectropho-
soil (Inceptisol, pH 6.3 in 0.01 m CaCl2 and 2.8% of tometer (Arnon, 1949). Selenium in the dry plant
organic C) taken from a field never amended with Se- material was analyzed by the method of Kumpulainen
containing fertilizers. Prior to the experiment, the soil et al. (1983), and an in-house reference sample was
was air-dried, sieved (< 35 mm) and homogenized. included in every analytical round. Lipid peroxidation
Two sets of 4-kg soil samples (12 samples in each was assayed by measuring thiobarbituric acid-reactive
set) were then weighted for the pot experiment. In substances (TBARS) according to a modified method
both sets, the soil units were treated in quadruplic- of Yagi (1982).
ate with Se (supplied as H2 SeO4 , Fluka Chemie AG With respect to antioxidants, attention was fo-
product, analytical grade) at the rate of 0.1 and 1.0 mg cused on Se-induced changes in glutathione perox-
kg1 soil, or left without Se addition. As the range of idase (GSH-Px), superoxide dismutase (SOD) and
57
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
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
Discussion
turn, is found to enhance ethylene production (Konze rising Se concentration (Table 2). In the Se-fertilized
et al., 1978), which can modify membrane lipid com- seedlings, on the other hand, GSH-Px was not able
position (Thompson et al., 1982), increase membrane to increase further during senescence, which was in
permeability (Hanson and Kende, 1975; Vangronsveld agreement with the decrease in the Se concentration
et al., 1993) and result e.g. in an increased K+ leakage (Table 2). The lower Se concentration could be attrib-
(Vangronsveld et al., 1993). Thus, it can be hypothes- utable either to reduced Se absorption by the senescing
ized that high Se addition enhanced K+ leakage, and seedlings or to Se loss as volatile compounds. In fact,
more water was retained in the intercellular space to a distinct reduction in the Se taken up by the senescing
counterbalance an increased osmotic pressure. seedlings (Table 2) supports the latter hypothesis pre-
Similarly as in a previous study (Hartikainen et al., viously shown to occur in lettuce (Terry et al., 1992;
2000), the positive response of plant growth to low Se Terry and Zayed, 1994).
addition was delayed appearing when the plants be- The reduction in SOD (Table 3) and tocopher-
came older. The growth-stimulating effect of low Se ols (Figure 1) found in the young seedlings at low
addition was not related to total chlorophyll, which Se level agrees with the results reported earlier for
diminished during senescence, but rather to its antiox- young ryegrass and lettuce seedlings (Hartikainen et
idative function as demonstrated by diminished lipid al., 1997, 2000; Xue and Hartikainen, 2000). In
peroxidation in senescing plants. At the high addi- addition, the young seedlings were dominated by
tion level, however, the antioxidative function of Se -tocopherol, whereas the senescing seedlings were
seemed to be diminished or reversed irrespective of dominated by -tocopherol, which is a more active
plant age, lipid peroxidation being higher than at the species in antioxidation and is synthetized from -
low Se level (Tables 2 and 3). This response supports tocopherol (Schultz et al., 1991). In other words, as
the finding that at a high level Se can even act as lettuce became older, more -tocopherol was syn-
a pro-oxidant, and this mechanism can, in addition thesized from -tocopherol to meet the increased
to metabolic disturbances, contribute to Se toxicity antioxidant requirement.
(Hartikainen et al., 2000). When no Se was added, tocopherols and SOD di-
No growth-promoting effect of Se was seen in the minished during plant senescence. The added Se con-
young seedlings despite an increase in total chloro- tributed to the maintenance of antioxidative capacity
phylls. Lipid peroxidation was reduced concomitantly by diminishing the reduction in tocopherols and by
with an increase in GSH-Px activity. This suggests enhancing SOD. Thus, it provided extra antioxidative
that in the young plants the antioxidative capacity was capacity which might indirectly diminish the demand
sufficient for normal growth under the low-light con- for SOD. Superoxide radicals can be used in spon-
ditions prevailing in the experiment. However, greater taneous disproportion reactions producing H2 O2 and
antioxidative capacity is needed to counteract the singlet oxygen (Thompson et al., 1987). Hartikainen
enhanced lipid peroxidation during senescence. The et al. (2000) hypothesized that at the same time that
results reveal that in the senescing plants cultivated Se promotes scavenging of produced H2 O2 through
without Se, antioxidative capacity was strengthened increased GSH-Px it enhances the spontaneous dis-
by increased GSH-Px (Table 3) in accordance with proportion of superoxide radicals and, consequently,
60
reduces the need for their scavenger, SOD. Further- Eustice D C, Kull F J and Schrift A 1981 Selenium toxicity:
more, at the non-toxic Se level the decrease in su- Aminoacylation and peptide bond formation with selenome-
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These results call for further studies of the role Gladyshev V N, Jeang K T, Wootton J C and Hatfield D L 1998 A
of Se in senescing plants. However, they clearly in- new human selenium-containing protein: Purification, character-
dicate that Se may exert a beneficial role in plants ization, and cDNA sequence. J. Biol. Chem. 273, 89108915.
under stress and, thus, they support the finding of Hanson A D and Kende H 1975 Ethylene enhanced ion and sucrose
efflux in morning glory flower tissue. Plant Physiol. 55, 663669.
Hartikainen and Xue (1999) that Se defends plants Hartikainen H, Ekholm P, Piironen V, Xue T, Koivu T and Yli-Halla
subjected to UV(B) episodes. Even if the present study M 1997 Quality of the ryegrass and lettuce yields as affected by
provides some support for the hypothesis of the exist- selenium fertilization. Agr. Food Sci. Finland 6, 381387.
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plant growth as triggered by ultraviolet irradiation. J. Environm.
Se-induced increase in this enzyme alone may not be Qual. 28, 13721375.
marked enough to explain the enhanced antioxidative Hartikainen H, Xue T and Piironen V 2000 Selenium as an anti-
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Acknowledgements Konze J R, Schilling N and Kende H 1978 Enhancement of ethylene
formation by selenoamino acids. Plant Physiol. 62, 397401.
We thank Ms. Marjatta Koivisto, M. Sc. Satu Kumpulainen J, Raittila A-M, Lehto J and Koivistoinen P 1983
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Pennanen for constructive criticism on the manu- Murphy J B and Kies M W 1960 Note on spectrophotometer de-
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