Quality of the ryegrass and lettuce yields as affected by selenium fertilization

Seleeni on ihmisille ja elaimille valttamaton alkuaine, jonka puute yhdessa E-vitamiinin puutteen kanssa lisaa hapettumisvaurioiden riskia soluissa. Suurina annoksina se on kuitenkin myrkyllinen. Suomessa maaperassa on luontaisesti vahan kasveille kayttokelpoista seleenia, minka vuoksi sita on vuodesta 1984 lisatty moniravinteisiin lannoitteisiin, jotta elintarvikkeiden seleenipitoisuus olisi ravitsemuksen kannalta riittava. Seleenin merkitys kasveissa on kuitenkin viela epaselva. Taman vuoksi tehtiin astiakoe, jossa alustavasti selvitettiin, miten nousevina maarina annettu lannoiteseleeni vaikutti kasvien kemialliseen koostumukseen ja antioksidatiivisiin ominaisuuksiin. Koekasveina kaytettiin raiheinaa ja salaattia. Seleenilisayksen noustessa kasvoi liukoisiin ja liukenemattomiin valkuaisaineisiin sitoutuneen seleenin suhteellinen osuus samalla kun vapaissa aminohapoissa olevan osuus pieneni. Tama osoittaa, etta kasvin ottamaaseleenia kaytettiin tehokkaasti proteiinisynteesissa. Seleenilannoitus vaikutti myos kasvien antioksidatiivisiin ominaisuuksiin. Molemmissa kasveissa havaittiin glutationi-peroksidaasientsyymin (GSH-Px) aktiivisuuden lisaantyvan seleenilisayksen noustessa, mika viittaa siihen, etta korkeammissa kasveissa toimii samantapainen seleenista riippuva GSH-Px kuin elaimissa. GSH-Px:n aktiivisuuden kasvaessa vaheni kuitenkin toisen antioksidatiivisen entsyymin, superoksididismutaasin (SOD) aktiivisuus ja E-vitamiinin pitoisuus pieneni. Tulos viittaa siihen, etta samalla kun seleeni nosti GSH-Px:n aktiivisuutta se vahensi muiden antioksidanttien tarvetta.

ntroduction Selenium is an essential element to human and animals. Low Se-intake connected with vitamin E deficiency increases oxidative stress and contributes to the development of oxidative damages (for references see e.g. May land 1994). However, at high intake Se is toxic. Typical symp-toms of selenosis are reported for humans in China and animals in USA (Yang et al. 1983, Oldfield 1987). Because of these dual effects it is important to control the concentration of Se in plant products. In global scale, Se-deficient areas are far larger than seleniferous ones (Kubota et al. 1967, Zhen et al. 1982. In Finland the soils are poor in bioavailable Se (Yläranta 1983) and the Se-concentration of crops used to be below the adequate level (e.g. Sippola 1979). Therefore, since 1984 all multinutrient fertilizers produced in Finland have been supplemented with sodium selenate, resulting in a substantial increase in Se in plants and food (Ekholm et al. 1995).
Nevertheless, the role of Se in plants is still unclear. Plant species capable of accumulating Se can assimilate it in different forms into their tissues (Peterson and Burler 1962). Selenium absorbed by the plants can be metabolized partly following the pathway of its chemical analogue sulfur, and it can be synthesized to Se-containing substituents (Shrift and Virupaksha 1965). However, Se in various chemical forms may be dissimilarly utilized by humans or animals, and its effectiveness in increasing the activity of glutathione peroxidase (GSH-Px), an important anti-oxidative enzyme, and Se-concentrations in tissues may vary (Mahan and Moxon 1978, Sankari 1985,Aspila 1991. The aim of this preliminary pot experiment was to investigate how Se added in increasing amounts is allocated into various compounds in different plant species. To study the effect of Sefertilization on the anti-oxidative properties of plant products and enzyme activities, the yields were also analyzed for the concentration of vitamin E and for GSH-Px and superoxide dismutase (SOD) activities. The study was carried out with ryegrass (monocotydelon), an important forage crop, and with lettuce (dicotydelon), a common vegetable in the human diet.

Pot experiment
The soil used in the pot experiment had a pH(CaCI,) of 6.3 and contained 2.8% of organic carbon. Its particle size composition was: 8% of clay (< 2 pm), 19% of silt (2-20 pm) and 64% of fine sand  pm) and 9% of coarser material. Each pot was filled with 7.43 kg of moist soil, equivalent to 6.43 kg of dry matter. The different Se-levels (0, 8, 16 and 33 pg Se kg ' of dry soil, four replicates) were created with two commercial multinutrient compound fertilizers, one virtually devoid of Se (18.0% N, 6.4% P, 13.1% K and 0.1 ug Se kg ') and one supplemented with sodium selenate during the manufacturing process (16.6% N, 7.1% P, 13.3% K and 19 mg Se kg '). Small amounts of Ca(H,P0 4 ) 2 • H 2 O and KCI were also applied, when needed, to end up with the same quantities of N (1.83 g), P (0.78 g) and K (1.47 g) in each pot. The pots were sown with 5 seeds of lettuce or 2.5 g of Italian ryegrass. Lettuce was thinned to three plants in each pot. Deionized water was used for watering.

Plant analyses
The ryegrass was harvested 21 days and the lettuce 46 days after sowing. To get enough plant material for vitamin and Se-fractionation analyses the fresh yields of all replicates in each treatment were combined and then weighed (FW). After careful mixing, about a quarter of the plant material was immediatedly packed in vacuum bags and stored in a freezer at -70°C for vitamin and enzyme analyses. Then rest of the yield was used for the determination of the dry weight (DW) and Se-analyses. The air-dried plant material was ground and a subsample of about 4 g was stored in a freezer for the analysis of total Se. The rest of the ground plant material was used to study the allocation of added Se into various compounds.
The Se in the inorganic fraction, in free amino acids and in the insoluble residue was fractionated in two replicates according to Gissel-Nielsen (1987). In addition, the Se in the soluble proteins was also determined. The supernatants obtained when a 10-g sample was extracted twice for 20 min with 100 ml of water and once with 50 ml of water were combined, and the soluble proteins were precipitated with 60 ml of 30% trichloroacetic acid. The solution was centrifuged in tared tubes for 40 min at 11 000 rpm, and the proteins in the precipitate were dried at 40-50°C overnight, cooled in a desiccator, weighed and stored in a freezer. The total Se-concentration in plant material (measured in dublicate) and Se in each fraction was determined by the electrothermal atomic absorption spectrometric method of Kumpulainen et al. (1983) Tocopherols were determined at least in duplicate according to Piironen et al. (1986). SOD activity was determined according to Giannopolitis and Ries (1977) and GSH-Px activity by a modified method of Flohe and Gunzler (1984) In the fractionation procedure the ion exchange in the amino acid analyses was tested by using a standard sample containing 16 amino acids. Their retention in and elution from the column were checked by the ninhydrin reaction. To control the reproducibility of the Se-analyses, in-house reference samples were included in every determination round. The mean and standard deviation in the reference lettuce sample was 4.609±0.278 (n=7). The corresponding figures obtained for the wheat flour sample within the study period 0.257±0.024 (n=7) were close to the long-term value of 0.246±0.008 (n= 332). In tocopherol analyses the recovery of «-tocopherol varied from 83 to 115%, and for the wheat germ oil used to control the level of tocopherol analyses the coefficient of variation was 5.1% (n=!4). In the enzyme analyses, the means of the coefficients of variation in the five replicates of each sample were 8.3% and 15.1% for GSH-Px and 14.1% and 20.4% for SOD in ryegrass and lettuce, respectively.

Results and discussion
The shoot yields and Se-concentrations The dry matter yield of the ryegrass tended to decrease slightly with increasing Se applications, whereas that of the lettuce increased (Table 1). At the highest Se-level, however, this positive response diminished. Nevertheless, the beneficial effect of Se found for lettuce in the present study cannot be taken for granted, because the treatments were not equal in the quantities of the commercial fertilizers added and a possible contribution by some trace element cannot be excluded.
In the unsupplemented plants, the Se-concentration (Table 1) was of the magnitude reported for agricultural plants in Finland before Se-supplementation of fertilizers (see Sippola 1979,  Yläranta 1983). In the Se-fertilized plants it was higher than normally found in field crops (cf. Varo 1983, Ekholm et al. 1995. Because lettuce was harvested at a later stage, the uptake of fertilizer Se by the yields was slightly greater than that by ryegrass. In both plants, the utilization of added Se diminished at the highest fertilization level.

Allocation of Se into different fractions
The figures in Table 2 show that in all treatments the Se concentration in the various fractions followed the order: soluble proteins > insoluble residue > inorganic > amino acids. The Se in the insoluble residue is mainly protein-bound. The relative allocation of Se between various forms was calculated by taking into account the weight ofeach fraction. Figure 1 reveals that, except for lettuce not amended with Se, the major part of Se in plant material was incorporated into proteins (soluble proteins plus residue). The Se-fertilization further increased the relative portion of protein-bound Se-fraction but decreased that bound by free amino acids. This indicates that the Se taken up was effectively utilized in the protein synthesis. In ryegrass, the relative portion of insoluble Se-proteins (the residual fraction) seemed to in-crease at the expense of soluble proteins (Fig. 1). This may to some extent be attributable e.g. to the formation of Se-methionine, because it is less water-soluble than its S-containing analogue methionine (Shepherd and Huber 1969). In lettuce the Se-fertilization also seemed to promote the synthesis of Se-containing proteins at the expense of the inorganic fraction, which was high in the control plants. This response was clear at the lowest Se-fertilization level. The result suggests that the nutritional value of the plants can be increased by a reasonable Se-addition, because organic Se is known to be more efficient than inorganic Se in increasing the Se-concentration and GSH-Px activity in plasma and muscle tissue (Mahan and Moxon 1978, Sankari 1985, Aspila 1991.

Vitamin E and enzyme activities
The two plant species differed in tocopherol compounds (Vit E ) detected, a-tocopherol dominated in ryegrass and only traces of (i-tocopherol were found (Table 3). Lettuce, on the contrary, contained about equal quantities aand y-tocopherols in addition to traces of p-tocopherol. The Se-fertilization tended to decrease their concentrations. The increase in both tocopherols in lettuce at the highest-Se level was the only exception and its reason remained un clear.
GSH-Px and SOD activities were higher in lettuce than in ryegrass (Table 4). In both species, the GSH-Px activity seemed to increase with increasing Se-fertilization, whereas an opposite trend was found for SOD. The increasing trend in GSH-Px possibly indicates the presence of Se-dependent GSH-Px in plants. However, further evidence is needed to prove the occurrence of this enzyme in higher plants. Anderson and Scarf (1983) refer to results which showed that in crude extracts of pea shoots catalyzing H 20 2 -dependent oxidation of GSH, a flavonoid was needed to express this activity. On the other hand, the opposite trends in GSH-Px and SOD as well as the Se-induced depletion in tocophe- 19.5 11 The proportion of y-tocopherol from the total amount of tocopherols is estimated by comparing its peak area with that of ce-tocopherol. rols are probably due to the fact that Se increased the anti-oxidative capacity of the plants and diminished the substrate for SOD (superoxide anion radical 0 2 ) and Vit E (lipid peroxide radical LOO). Therefore, the demand for the synthesis of SOD and Vit E could be reduced (Xue et al. 1993).