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-oxidative 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 function of Se

Helinä Hartikainen

Markku Yli-Halla

Päivi Ekholm

Tailin Xue

Terhi Koivu

Vieno Piironen

Agricultural and Food Science

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Research NoteQuality of the ryegrass and lettuce yieldsas affected by selenium fertilizationHelinä HartikainenDepartment ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki,Finland, e-mail: helina.hartikainen@helsinki.fiPäivi Ekholm, Vieno PiironenDepartment ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki, FinlandTailin XueInstitute of Geography ofChinese Academy ofSciences, Beijing 100101, ChinaTerhi Koivu, Markku Yli-HallaDepartment ofApplied Chemistry and Microbiology, PO Box 27, FIN-00014 University ofHelsinki, FinlandThe effect of Se-fertilization on the chemical composition and anti-oxidative properties of ryegrassand lettuce was studied in a pot experiment. The addition of Se enhanced its relative incorporation insoluble 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 speciesincreased with increasing Se-fertilization, whereas the superoxide dismutase (SOD) activity as wellas the concentration of vitamin E decreased. This may indicate that the synthesis of SOD and vitaminE 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 EntroductionSelenium is an essential element to human andanimals. Low Se-intake connected with vitaminE 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 inChina and animals in USA (Yang et al. 1983,Oldfield 1987). Because of these dual effects itis important to control the concentration of Sein plant products. In global scale, Se-deficientareas 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 inFinlandManuscript received October 1997381Vol. 6 (1997): 381-387.AGRICULTURAL AND FOOD SCIENCE IN FINLANDHartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilizationto be below the adequate level (e.g. Sippola1979). Therefore, since 1984 all multinutrientfertilizers produced in Finland have been sup-plemented with sodium selenate, resulting in asubstantial increase in Se in plants and food(Ekholm et al. 1995).Nevertheless, the role of Se in plants is stillunclear. Plant species capable of accumulatingSe can assimilate it in different forms into theirtissues (Peterson and Burler 1962). Seleniumabsorbed 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 Virupaksha1965). However, Se in various chemical formsmay be dissimilarly utilized by humans or ani-mals, and its effectiveness in increasing the ac-tivity of glutathione peroxidase (GSH-Px), animportantanti-oxidative enzyme, and Se-concen-trations in tissues may vary (Mahan and Moxon1978, Sankari 1985,Aspila 1991).The aim of this preliminary pot experimentwas to investigate how Se added in increasingamounts is allocated into various compounds indifferent plant species. To study the effect of Se-fertilization on the anti-oxidative properties ofplant products and enzyme activities, the yieldswere also analyzed for the concentration of vi-tamin E and for GSH-Px and superoxide dis-mutase (SOD) activities. The study was carriedout with ryegrass (monocotydelon), an impor-tant forage crop, and with lettuce (dicotydelon),a common vegetable in the human diet.Material and methodsPot experimentThe soil used in the pot experiment had apH(CaCI,) of 6.3 and contained 2.8% of organiccarbon. Its particle size composition was: 8% ofclay (< 2 pm), 19% of silt (2-20 pm) and 64%of fine sand (20-200 pm) and 9% of coarsermaterial. Each pot was filled with 7.43 kg ofmoist soil, equivalent to 6.43 kg of dry matter.The different Se-levels (0, 8, 16 and 33 pg Sekg ' ofdry soil, four replicates) were created withtwo 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% Kand 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 potswere sown with 5 seeds of lettuce or 2.5 g ofItalian ryegrass. Lettuce was thinned to threeplants in each pot. Deionized water was used forwatering.Plant analysesThe ryegrass was harvested 21 days and the let-tuce 46 days after sowing. To get enough plantmaterial 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 plantmaterial was immediatedly packed in vacuumbags and stored in a freezer at -70°C for vitaminand enzyme analyses. Then rest of the yield wasused 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 gwas stored in a freezer for the analysis of totalSe. The rest of the ground plant material wasused to study the allocation of added Se intovarious 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 andonce with 50 ml of water were combined, andthe soluble proteins were precipitated with60 ml of 30% trichloroacetic acid. The solutionwas centrifuged in tared tubes for 40 min at382AGRICULTURAL AND FOOD SCIENCE IN FINLANDTable I. The fresh (FW) and dry weight (DW) of the shoot yields, their Se-concentration and Se-uptake atvarious Se-addition levels.Se added FW DW Se-conc. Se-uptakeHg kg'soil g g Hgg'DW (lg % of addedRyegrass0 330 36.40.023 0.88 320 35.62.15 76.5 3616 291 34.33.45 118.3 3833 301 32.35.05 162.9 19Lettuce0 574 35.40.022 0.88 640 53.61.52 81.5 3816 700 62.72.90 181.9 4333 600 51.44.90 252.0 3011 000 rpm, and the proteins in the precipitatewere 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 (measuredin dublicate) and Se in each fraction was deter-mined by the electrothermal atomic absorptionspectrometric method of Kumpulainen et al.(1983) Tocopherols were determined at least induplicate 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 byusing a standard sample containing 16 aminoacids. Their retention in and elution from thecolumn were checked by the ninhydrin reaction.To control the reproducibility of the Se-analy-ses, in-house reference samples were includedin every determination round. The mean andstandard deviation in the reference lettuce sam-ple was 4.609±0.278 (n=7). The correspondingfigures obtainedfor the wheat flour sample with-in the study period 0.257±0.024 (n=7) were closeto 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 wheatgerm oil used to control the level of tocopherolanalyses the coefficient of variation was 5.1%(n=!4). In the enzyme analyses, the means ofthe coefficients of variation in the five replicatesof each sample were 8.3% and 15.1% for GSH-Px and 14.1% and 20.4% for SOD in ryegrassand lettuce, respectively.Results and discussionThe shoot yields and Se-concentrationsThe dry matter yield of the ryegrass tended todecrease slightly with increasing Se applications,whereas that of the lettuce increased (Table 1).At the highest Se-level, however, this positiveresponse diminished. Nevertheless, the benefi-cial effect of Se found for lettuce in the presentstudy cannot be taken for granted, because thetreatments were not equal in the quantities of thecommercial 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 reportedfor agricultural plants in Finland before Se-sup-plementation of fertilizers (see Sippola 1979,383Vol. 6 (1997): 381-387.AGRICULTURAL AND FOOD SCIENCE IN FINLANDHartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilizationTable 2. Se-concentration in various fractions of the plant material.Se added Inorganic" Amino acid" Sol. proteins ResidueHg kg ' soil ng ml" 1 ng g ' DW of the fractionRyegrass0 4 5 78 378 143 15 3780 264016 291 38 6900 320033 535 67 9200 4200Lettuce0 9 4 54 358 119 13 2710 186016 230 16 4200 365033 471 67 7900 56001) = volume of the fraction was 20 mlYläranta 1983). In the Se-fertilized plants it washigher than normally found in field crops (cf.Varo 1983,Ekholm et al. 1995). Because lettucewas harvested at a later stage, the uptake of fer-tilizer Se by the yields was slightly greater thanthat by ryegrass. In both plants, the utilizationof added Se diminished at the highest fertiliza-tion level.Allocation of Se into different fractionsThe figures in Table 2 show that in all treatmentsthe Se concentration in the various fractions fol-lowed the order: soluble proteins > insolubleresidue > inorganic > amino acids. The Se in theinsoluble residue is mainly protein-bound. Therelative allocation of Se between various formswas calculated by taking into account the weightofeach fraction. Figure 1 reveals that, except forlettuce not amended with Se, the major part ofSe in plant material was incorporated into pro-teins (soluble proteins plus residue). The Se-fer-tilization further increased the relative portionof protein-bound Se-fraction but decreased thatbound by free amino acids. This indicates thatthe Se taken up was effectively utilized in theprotein synthesis.In ryegrass, the relative portion of insolubleSe-proteins (the residual fraction) seemed to in-crease at the expense of soluble proteins(Fig. 1). This may to some extent be attributablee.g. to the formation of Se-methionine, becauseit is less water-soluble than its S-containing ana-logue methionine(Shepherd and Huber 1969). Inlettuce the Se-fertilizationalso seemed to promotethe synthesis of Se-containing proteins at the ex-pense of the inorganic fraction, which was highin the control plants. This response was clear atthe lowest Se-fertilization level. The result sug-gests that the nutritional value of the plants canbe increased by a reasonable Se-addition, becauseorganic Se is known to be more efficient than in-organic Se in increasing the Se-concentration andGSH-Px activity in plasma and muscle tissue(Mahan and Moxon 1978, Sankari 1985, Aspila1991).Vitamin E and enzyme activitiesThe two plant species differed in tocopherolcompounds (VitE) 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- andy-tocopherols in addition to traces of p-toco-pherol. The Se-fertilization tended to decreasetheir concentrations. The increase in both to-copherols in lettuce at the highest-Se level was384AGRICULTURAL AND FOOD SCIENCE IN FINLANDthe only exception and its reason remained unclear.GSH-Px and SOD activities were higher inlettuce than in ryegrass (Table 4). In both spe-cies, the GSH-Px activity seemed to increasewith increasing Se-fertilization, whereas an op-posite trend was found for SOD. The increasingtrend in GSH-Px possibly indicates the presenceof Se-dependent GSH-Px in plants. However,further evidence is needed to prove the occur-rence of this enzyme in higher plants. Andersonand Scarf (1983) refer to results which showedthat in crude extracts of pea shoots catalyzingH 202-dependent oxidation of GSH, a flavonoidwas needed to express this activity. On the otherhand, the opposite trends in GSH-Px and SODas well as the Se-induced depletion in tocophe-Table 3. Tocopherol concentrations in the experimental plantspecies at various Se-addition levels I *.Se added a y Zpig kg’ 1 soil mg kg ' FWRyegrass0 13.8 13.88 12.0 12.016 11.6 11.633 11.3 11.3Lettuce0 5.9 5.8 11.78 6.8 6.1 12.916 2.1 5.1 7.233 9.5 10,0 19.511 The proportion of y-tocopherol from the total amount oftocopherols is estimated by comparing its peak area withthat of ce-tocopherol.Table 4. GSH-Px and SOD activities (mg' 1 protein •min) in the experimental plants at various Se-addition levels'Ryegrass LettuceSe added GSH-Px SOD GSH-Px SODHg kg soil"' umol unit umol unit0 34.82.8 nd nd8 37.92.1 119.85.616 40.62.0 129.82.833 54.42.0 160.03.911 GSH-Px = glutathione peroxidase SOD = superoxide dismutase nd = no dataFig. 1.The relative distribution ofvarious Se-fractions.385Vol. 6 (1997): 381-387.AGRICULTURAL AND FOOD SCIENCE IN FINLANDHartikainen, H. et al. Ryegrass and lettuce yields as affected by seleniumfertilizationrols are probably due to the fact that Se increasedthe anti-oxidative capacity of the plants and di-minished the substrate for SOD (superoxide an-ion radical 02 ) and VitE (lipid peroxide radicalLOO). Therefore, the demand for the synthesisof SOD and VitE could be reduced (Xue et al.1993).Acknowledgements.The financial support from the Acade-my ofFinland is gratefully acknowledged.ReferencesAnderson, J.W. & Scarf, A.R. 1983. Selenium and plantmetabolism. In: Robb, D.A. & Pierpoint, W.S. (eds.).Metals and micronutrients: Uptake and utilization byplants. Academic Press. Orlando, p. 241-275.Aspila P. 1991. Metabolism of selenite, selenomethionineand feed-incorporatedselenium in lactating goats anddairy 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 ofreducing the amount of selenate in fertilizers. Agri-cultural Science in Finland 4: 377-384.Flohe, L. & Gunzler, W.A, 1984. Assays of glutathioneperoxidase. 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 Statesin 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 ofyoung 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 ofNutrition 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 tocotrienolsin 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 supplementationin swine. Acta Veterinaria Scandinavica 81: 1-127.Shrift, A. & Virupaksha, T.K. 1965. Seleno-amino acidsin 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 andbiochemical properties of selenomethionine. Cana-dian Journal of Biochemistry 47; 877- 881.Varo, P. 1983. Selenium fertilization in Finland: seleniumcontent in feed and foods. Norwegian Journal ofAg-ricultural Science Supplement 11: 151-158.Yang, G., Wang , W., Zhou, R. & Sun, S. 1983. Endemicselenium intoxication of humans in China, AmericanJournal of Clinical Nutrition 37: 872-881.Yläranta, T. 1983. Effect of added selenite and selenateon 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 ScienceBulletin 38; 274-277. (In Chinese).Zhen, D., Li, R. & Wang, W. 1982. The world Se deficien-cy belt. Acta Scientiae Circumstantiae 2: 241-249.386AGRICULTURAL AND FOOD SCIENCE IN FINLANDSELOSTUSSeleenilannoituksen vaikutus raiheinän ja salaatin laatuunHelinä Hartikainen, Päivi Ekholm, Vieno Piironen, Tailin Xue, Terhi Koivu ja Markku Yli-HallaHelsingin yliopisto ja Kiinan tiedeakatemiaSeleeni 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 vuodesta1984 lisätty moniravinteisiin lannoitteisiin, jotta elin-tarvikkeiden seleenipitoisuus olisi ravitsemuksenkannalta 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 jaliukenemattomiin valkuaisaineisiin sitoutuneen selee-nin suhteellinen osuus samalla kun vapaissa amino-hapoissa olevan osuus pieneni. Tämä osoittaa, ettäkasvin 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:naktiivisuutta se vähensi muiden antioksidanttien tar-vetta.387Vol. 6 (1997): 381-387.AGRICULTURAL AND FOOD SCIENCE IN FINLAND

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

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

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