Research Note Quality of the ryegrass and lettuce yields as affected by selenium fertilization Helinä Hartikainen Department ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki, Finland, e-mail: helina.hartikainen@helsinki.fi Päivi Ekholm, Vieno Piironen Department ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki, Finland Tailin Xue Institute of Geography ofChinese Academy ofSciences, Beijing 100101, China Terhi Koivu, Markku Yli-Halla Department ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki, Finland The effect of Se-fertilization on the chemical composition and anti-oxidative properties of ryegrass and lettuce was studied in a pot experiment. The addition of Se enhanced its relative incorporation in soluble and insoluble proteins and diminished it in free amino acids. It also affected the anti-oxida- tive systems of the plants. The glutathione peroxidase (GSH-Px) activity found in both plant species increased with increasing Se-fertilization, whereas the superoxide dismutase (SOD) activity as well as the concentration of vitamin E decreased. This may indicate that the synthesis of SOD and vitamin E was reduced because the requirement of these anti-oxidants was diminished by antioxidative func- tion of Se. Key words: anti-oxidants, glutathione peroxidase, Se-fractions, superoxide dismutase, vitamin E ntroduction Selenium is an essential element to human and animals. Low Se-intake connected with vitamin E deficiency increases oxidative stress and con- tributes to the development of oxidative damag- es (for references see e.g. May land 1994). How- ever, 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 Fin- land the soils are poor in bioavailable Se (Yläran- ta 1983) and the Se-concentration of crops used © Agricultural and Food Science inFinland Manuscript received October 1997 381 Vol. 6 (1997): 381-387. AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=C7baIAR8JmOZJTHy.nlH4NV_4iwdtgZsrSUWwdQ.6zv1vz4RBz-GbvpDDpIO97u_FszKy5Vc1l-aLYRyOFUtgUQ1NRbjw7eynHblJvef46LvPZIByodLcdppuOFW7Z53IsKUke4ryUj0_jUsnQhPmiXEWPxERiagGdPhI-6P0vAiqLX3aJiZDTQgU054cvH5Nzitg_W8uSwDhhaRdiJomtCICBB9fmEPYk87KmHth9slh7kK8xsG_AIFn9JFRJvvUGcA7AvLSDl2rvRfQ-YakuUbT_Qk3a4YpnVuvygSUpRvqXezF36nM-xl7q6bYGzwD04chvW9n14U2nGp5Nah-odHHfs4vSnHSfnzGfcI_9WbSOHSGelm6fearIEjsB-6ynawDDeGXiIU8kzMBGdVkuP9ggM8WhAwHrA Hartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilization to be below the adequate level (e.g. Sippola 1979). Therefore, since 1984 all multinutrient fertilizers produced in Finland have been sup- plemented 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 part- ly following the pathway of its chemical ana- logue sulfur, and it can be synthesized to Se-con- taining substituents (Shrift and Virupaksha 1965). However, Se in various chemical forms may be dissimilarly utilized by humans or ani- mals, and its effectiveness in increasing the ac- tivity of glutathione peroxidase (GSH-Px), an importantanti-oxidative enzyme, and Se-concen- trations 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 Se- fertilization on the anti-oxidative properties of plant products and enzyme activities, the yields were also analyzed for the concentration of vi- tamin E and for GSH-Px and superoxide dis- mutase (SOD) activities. The study was carried out with ryegrass (monocotydelon), an impor- tant forage crop, and with lettuce (dicotydelon), a common vegetable in the human diet. Material and methods 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 (20-200 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 ' ofdry soil, four replicates) were created with two commercial multinutrient compound ferti- lizers, one virtually devoid of Se (18.0% N, 6.4% P, 13.1% K and 0.1 ug Se kg ‘) and one supple- mented with sodium selenate during the manu- facturing process (16.6% N, 7.1% P, 13.3% K and 19mg Se kg '). Small amounts of Ca(H,P04 ) 2 • H2 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 let- tuce 46 days after sowing. To get enough plant material for vitamin and Se-fractionation analy- ses the fresh yields ofall replicates in each treat- ment 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 ma- terial 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 ami- no acids and in the insoluble residue was frac- tionated in two replicates according to Gissel- Nielsen (1987). In addition, the Se in the solu- ble proteins was also determined. The superna- tants obtained when a 10-g sample was extract- ed 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 382 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table I. The fresh (FW) and dry weight (DW) of the shoot yields, their Se-concentration and Se-uptake at various Se-addition levels. Se added FW DW Se-conc. Se-uptake Hg kg'soil g g Hgg'DW (lg % of added Ryegrass 0 330 36.40.023 0.8 8 320 35.62.15 76.5 36 16 291 34.33.45 118.3 38 33 301 32.35.05 162.9 19 Lettuce 0 574 35.40.022 0.8 8 640 53.61.52 81.5 38 16 700 62.72.90 181.9 43 33 600 51.44.90 252.0 30 11 000 rpm, and the proteins in the precipitate were dried at 40-50°C overnight, cooled in a des- iccator, weighed and stored in a freezer. The to- tal Se-concentration in plant material (measured in dublicate) and Se in each fraction was deter- mined 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 Gi- annopolitis and Ries (1977) and GSH-Px activi- ty by a modified method of Flohe and Gunzler (1984) In the fractionation procedure the ion ex- change 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-analy- ses, in-house reference samples were included in every determination round. The mean and standard deviation in the reference lettuce sam- ple was 4.609±0.278 (n=7). The corresponding figures obtainedfor the wheat flour sample with- in the study period 0.257±0.024 (n=7) were close to the long-term value of0.246±0.008 (n= 332). In tocopherol analyses the recovery of «-toco- pherol 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 benefi- cial 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 con- tribution by some trace element cannot be ex- cluded. In the unsupplemented plants, the Se-concen- tration (Table 1) was of the magnitude reported for agricultural plants in Finland before Se-sup- plementation of fertilizers (see Sippola 1979, 383 Vol. 6 (1997): 381-387. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Hartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilization Table 2. Se-concentration in various fractions of the plant material. Se added Inorganic" Amino acid" Sol. proteins Residue Hg kg ' soil ng ml" 1 ng g ' DW of the fraction Ryegrass 0 4 5 78 37 8 143 15 3780 2640 16 291 38 6900 3200 33 535 67 9200 4200 Lettuce 0 9 4 54 35 8 119 13 2710 1860 16 230 16 4200 3650 33 471 67 7900 5600 1) = volume of the fraction was 20 ml 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 fer- tilizer Se by the yields was slightly greater than that by ryegrass. In both plants, the utilization of added Se diminished at the highest fertiliza- tion level. Allocation of Se into different fractions The figures in Table 2 show that in all treatments the Se concentration in the various fractions fol- lowed 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 pro- teins (soluble proteins plus residue). The Se-fer- tilization 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 ana- logue methionine(Shepherd and Huber 1969). In lettuce the Se-fertilizationalso seemed to promote the synthesis of Se-containing proteins at the ex- pense of the inorganic fraction, which was high in the control plants. This response was clear at the lowest Se-fertilization level. The result sug- gests 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 in- organic 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 dom- inated in ryegrass and only traces of (i-tocophe- rol were found (Table 3). Lettuce, on the con- trary, contained about equal quantities a- and y-tocopherols in addition to traces of p-toco- pherol. The Se-fertilization tended to decrease their concentrations. The increase in both to- copherols in lettuce at the highest-Se level was 384 AGRICULTURAL AND FOOD SCIENCE IN FINLAND 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 spe- cies, the GSH-Px activity seemed to increase with increasing Se-fertilization, whereas an op- posite 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 occur- rence of this enzyme in higher plants. Anderson and Scarf (1983) refer to results which showed that in crude extracts of pea shoots catalyzing H 202-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- Table 3. Tocopherol concentrations in the experimental plant species at various Se-addition levels I *. Se added a y Z pig kg’ 1 soil mg kg ' FW Ryegrass 0 13.8 13.8 8 12.0 12.0 16 11.6 11.6 33 11.3 11.3 Lettuce 0 5.9 5.8 11.7 8 6.8 6.1 12.9 16 2.1 5.1 7.2 33 9.5 10,0 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. Table 4. GSH-Px and SOD activities (mg' 1 protein •min) in the experimental plants at various Se-addition levels' Ryegrass Lettuce Se added GSH-Px SOD GSH-Px SOD Hg kg soil"' umol unit umol unit 0 34.82.8 nd nd 8 37.92.1 119.85.6 16 40.62.0 129.82.8 33 54.42.0 160.03.9 11 GSH-Px = glutathione peroxidase SOD = superoxide dismutase nd = no data Fig. 1.The relative distribution of various Se-fractions. 385 Vol. 6 (1997): 381-387. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Hartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilization rols are probably due to the fact that Se increased the anti-oxidative capacity of the plants and di- minished the substrate for SOD (superoxide an- ion radical 02 ) and VitE (lipid peroxide radical LOO). Therefore, the demand for the synthesis of SOD and VitE could be reduced (Xue et al. 1993). Acknowledgements.The financial support from the Acade- my ofFinland is gratefully acknowledged. References Anderson, J.W. & Scarf, A.R. 1983. Selenium and plant metabolism. In: Robb, D.A. & Pierpoint, W.S. (eds.). Metals and micronutrients: Uptake and utilization by plants. Academic Press. Orlando, p. 241-275. Aspila P. 1991. Metabolism of selenite, selenomethionine and feed-incorporatedselenium in lactating goats and dairy cows. Journal of Agricultural Science in Fin- land 63: 1-73. Ekholm, P., Ylinen, M., Koivistoinen, P. & Varo, P. 1995. Selenium concentration of Finnish foods: Effect of reducing the amount of selenate in fertilizers. Agri- cultural Science in Finland 4: 377-384. Flohe, L. & Gunzler, W.A, 1984. Assays of glutathione peroxidase. In: Packer, L. (ed.). Methods in Enzy- mology. Vol. 105. Academic Press, NewYork. p. 114- 121. Giannopolitis, C.N. & Ries, S.K. 1977. Superoxide dis- mutases: I Occurrence in higher plants. Plant Phys- iology 59: 309-314. Gissel-Nielsen G. 1987. Fractionation of selenium in bar- ley and ryegrass. Journal of Plant Nutrition 10: 2147- 2152. Kubota, J., Allaway, W.H., Carter, D.L., Cary, E.E. & La- zar, V.A. 1967. Selenium in crops in the United States in relation to selenium-responsive diseases of ani- mals. JournalofAgriculturaland Food Chemistry 15: 448-453. Kumpulainen, J., Raittila A.-M., Lehto, J. & Koivistoinen, P. 1983. Electrothermal atomic absorption spectro- metric determination of selenium in foods and diets. Journal of the Association ofOfficial Analytical Chem- ists 66: 1129-1135. Mahan, D.C. & Moxon A.L. 1978. Effects of adding inor- ganic and organic selenium sources to the diets of young swine. Journal ofAnimalScience 47: 456-466. Mayland, H.F. 1994. Selenium in plant and animal nutri- tion. In: Frankenberger, W.T. Jr. & Benson, S, (eds.). Selenium in the environment. Marcel Dekker, Inc. New York. p. 29-45. Oldfield, J.E. 1987. Two faces of selenium. Journal of Nutrition 117: 2002-2008. Peterson, P.J. & Burler, G.W. 1962. The uptake and as- similation of selenite by higher plants. Australian Jour- nal of Biological Sciences 15:126-146. Piironen, V., Syväoja, E.-L., Varo, P., Salminen, K. & Koivistoinen, P. 1986. Tocopherols and tocotrienols in Finnish foods: vegetables, fruits and berries. Jour- nal ofAgricultural and Food Chemistry34: 742-746. Sankari, S. 1985. Plasma glutathioneperoxidase and tis- sue selenium response to selenium supplementation in swine. Acta Veterinaria Scandinavica 81: 1-127. Shrift, A. & Virupaksha, T.K. 1965. Seleno-amino acids in selenium-accumulating plants. Biochimica et Bio- physica Acta 100: 65-75. Sippola, J. 1979. Selenium content of soils and timothy (Phleum pratense L.) in Finland. Annates Agricultur- ae Fenniae 18: 182-187. Shepherd, L. & Huber, R.E. 1969. Some chemical and biochemical properties of selenomethionine. Cana- dian Journal of Biochemistry 47; 877- 881. Varo, P. 1983. Selenium fertilization in Finland: selenium content in feed and foods. Norwegian Journal ofAg- ricultural Science Supplement 11: 151-158. Yang, G., Wang , W., Zhou, R. & Sun, S. 1983. Endemic selenium intoxication of humans in China, American Journal of Clinical Nutrition 37: 872-881. Yläranta, T. 1983. Effect of added selenite and selenate on the selenium content of rye grass (Lolium multi- florum ) in different soils. Annates Agriculturae Fen- niae 22: 139-151. Xue, T., Hou, S. & Tan, J. 1993. The antioxidative func- tion of selenium in higher plants. I The inhibitive ef- fect of selenium in lipid peroxidation and its enzy- matic mechanism approaching. Chinese Science Bulletin 38; 274-277. (In Chinese). Zhen, D., Li, R. & Wang, W. 1982. The world Se deficien- cy belt. Acta Scientiae Circumstantiae 2: 241-249. 386 AGRICULTURAL AND FOOD SCIENCE IN FINLAND SELOSTUS Seleenilannoituksen vaikutus raiheinän ja salaatin laatuun Helinä Hartikainen, Päivi Ekholm, Vieno Piironen, Tailin Xue, Terhi Koivu ja Markku Yli-Halla Helsingin yliopisto ja Kiinan tiedeakatemia Seleeni on ihmisille ja eläimille välttämätön alkuai- ne, jonka puute yhdessä E-vitamiinin puutteen kans- sa lisää hapettumisvaurioiden riskiä soluissa. Suuri- na annoksina se on kuitenkin myrkyllinen. Suomes- sa maaperässä on luontaisesti vähän kasveille käyt- tökelpoista seleeniä, minkä vuoksi sitä on vuodesta 1984 lisätty moniravinteisiin lannoitteisiin, jotta elin- tarvikkeiden seleenipitoisuus olisi ravitsemuksen kannalta riittävä. Seleenin merkitys kasveissa on kui- tenkin vielä epäselvä. Tämän vuoksi tehtiin astiakoe, jossa alustavasti selvitettiin, miten nousevina määri- nä annettu lannoiteseleeni vaikutti kasvien kemialli- seen koostumukseen ja antioksidatiivisiin ominai- suuksiin. Koekasveina käytettiin raiheinää ja salaat- tia. Seleenilisäyksen noustessa kasvoi liukoisiin ja liukenemattomiin valkuaisaineisiin sitoutuneen selee- nin suhteellinen osuus samalla kun vapaissa amino- hapoissa olevan osuus pieneni. Tämä osoittaa, että kasvin ottamaa seleeniä käytettiin tehokkaasti pro- teiinisynteesissä. Seleenilannoitus vaikutti myös kas- vien antioksidatiivisiin ominaisuuksiin. Molemmis- sa kasveissa havaittiin glutationi-peroksidaasientsyy- min (GSH-Px) aktiivisuuden lisääntyvän seleenili- säyksen noustessa, mikä viittaa siihen, että korkeam- missa kasveissa toimii samantapainen seleenistä riip- puva GSH-Px kuin eläimissä. GSH-Px:n aktiivisuu- den kasvaessa väheni kuitenkin toisen antioksidatii- visen entsyymin, superoksididismutaasin (SOD) ak- tiivisuus jaE-vitamiinin pitoisuus pieneni. Tulos viit- taa siihen, että samalla kun seleeni nosti GSH-Px:n aktiivisuutta se vähensi muiden antioksidanttien tar- vetta. 387 Vol. 6 (1997): 381-387. AGRICULTURAL AND FOOD SCIENCE IN FINLAND