Enhanced glutathione peroxidases (GPx) activity in young barley seedlings enriched with selenium

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1 Africn Journl of Biotechnology Vol. 10(55), pp , 21 Septemer, 2011 Aville online t DOI: /AJB ISSN Acdemic Journls Full Length Reserch Pper Enhnced glutthione peroxidses (GPx) ctivity in young rley seedlings enriched with selenium Kunlun Liu 1, Yn Zho 1, Fusheng Chen 1, Zhenxin Gu 2 * nd Gunho Bu 1 1 College of Food Science nd Technology, Henn University of Technology, Zhengzhou , Chin. 2 College of Food Science nd Technology, Nnjing Agriculturl University, Nnjing , Chin. Accepted 27 July, 2011 To ssess whether young rley seedlings could e used s selenium-enriched food mteril, field experiment ws crried out. Brley seeds were soked in queous solutions t 20 C for 24 h. The solutions contined selenium in the form of sodium selenite nd were t 0, 10, 20 nd 30 µg/ml, respectively. Then the seeds were sowed in non-selenium-fertilized field. Seedlings were hrvested 2, 3, 4, nd 5 weeks fter sowing. Results show tht selenium tretment did not ffect (P>0.05) the iomss yield, height nd stem dimeter of young rley seedlings ut significntly incresed (P<0.05) selenium content nd enhnced chlorophyll ccumultion nd glutthione peroxidses (GPx) ctivity in young rley seedlings. This study indictes tht the method of soking seeds in selenium solutions ws fesile in enhncing selenium content in young rley seedlings which could e used s seleniumenriched food mteril. Key words: Selenium, young rley seedlings, growth, chlorophyll, glutthione peroxidses. INTRODUCTION Selenium (Se) is n essentil trce element for humn eings. Se deficiency is ssocited with some diseses nd generlly impirs the immune system. The recommended dietry llownce of Se is 55 µg per dy for helthy humn dults. However, Se content in foods is reltively low nd fils to meet the dily dietry requirement. It is estimted tht there re 500 to 1000 million Se-deficient people in the world (Coms, 2001). In Chin, Keshn disese nd Kshin-Beck disese in the popultion hve een ssocited with reltively low Se sttus cused y low-se-food diets. It is necessry to try to increse people s Se intke, nd this prolem is effectively solved y incresing Se in food. Therefore, Seenriched plnt products hve een investigted (Fng et l., 2008; Hu et l., 2002; Smrkolj et l., 2007; Stiilj et l., 2004), ut no studies hve een done on Se-enriched young rley seedlings to our knowledge. Brley (Hordeum vulgre L.) is one of the widely consumed cerel crops. Trditionlly, lmost 80 to 90% of rley productions hve een used for niml feeds nd eer mlts. Recently, young rley seedlings hve een used s food mteril for people in Asin countries such s Chin, Jpn, nd Kore. Young rley seedlings re rich in dietry fier, chlorophyll, crotene, vitmins, nd such minerl elements s clcium, potssium, iron, nd zinc. They lso contin some physiologiclly eneficil enzymes, such s superoxide dismutse, cytochrome oxidse, lipse, protese, ctlse, etc. If the Se content in young rley seedlings could e enriched, it will e helpful in solving the Se deficiency prolem mentioned erlier. Therefore, this study ws to investigte the effect of Se tretment on growth of young rley seedlings, Se nd chlorophyll contents nd GPx ctivity in young rley seedlings, which would determine whether young rley seedlings could e used s type of Se-enriched food mteril. MATERIALS AND METHODS Preprtion of Se-enriched young rley seedlings * Corresponding uthor. E-mil: guzx@nju.edu.cn. Tel/Fx: Brley seeds (vriety: gng-2) were soked in solutions t 20 C for 24 h. The solutions contined Se in the form of sodium selenite t concentrtions of 0 (s control), 10, 20, nd 30 µg/ml, respectively.

2 11484 Afr. J. Biotechnol. Tle 1. Effect of Se concentrtion on iomss yield, height nd stem dimeter of young rley seedlings. Week fter sowing N 2SeO 3 Biomss yield (g/100 plnts DW) Height of seedling (cm) Stem dimeter (mm) (µg/ml) (0.36) 8.55 (0.65) (1.34) (1.98) (1.32) (2.09) (2.04) (3.51) 2.08 (0.12) 2.96 (0.15) 3.97 (0.21) 4.70 (0.23) (0.41) 7.64(080.) (0.98) (2.08) (1.45) (1.97) (1.99) (4.12) 2.13 (0.09) 2.88 (0.15) 3.92 (0.22) 4.87 (0.35) (0.29) 9.05 (0.77) (1.28) (2.61) (1.29) (2.13) (2.01) (3.19) 2.09 (0.16) 2.79 (0.19) 3.85 (0.30) 4.66 (0.41) (0.32) 8.63 (0.81) (1.01) (2.29) (1.17) (1.53) (2.18) (4.06) 2.14 (0.11) 3.06 (0.21) 4.01 (0.26) 4.78 (0.31) The results re expressed s men ± SD (in prentheses) with three replictions. Brley seeds were soked in solutions t 20 C for 24 h with different concentrtions of selenite. The seeds were then sown in field never mended with Se-contining fertilizers. Field experiment ws designed y rndomized complete locks with three replictions, 3 2 m of ech plot. The totl Se content of the top soil (30 cm) ws 0.17 µg/g, of which the wter-solule Se ws µg/g. The experiment ws crried out in Tizhou City (119 54' N, 32 29' E), Jingsu Province, Chin. Young rley seedlings were hrvested 2, 3, 4, nd 5 weeks fter sowing. Biomss mesurement Biomss production ws represented y the dry weight yield of seedlings. 100 young rley seedlings (including stems nd leves, ut not the under-ground prts which were not consumed) were hrvested from ech plot. After drying t 55 ± 1 C for 48 h, the dry weight of smples ws mesured. Totl Se nd protein-ound Se determintion The totl Se (T-Se) nd protein-ound Se (PB-Se) contents of Se-enriched young rley seedlings were determined ccording to the method reported y Liu nd Gu (2009). Briefly, the fresh leves were cut into 2 to 3 cm in length nd dried for 24 h y freeze-drying system (Lconco, USA). 1 g of the smple ws digested with 5 ml of mixture of HNO 3 nd HClO 4 (v/v, 4:1) t 130 C for 1 h. After cooling, 5 ml of concentrted HCl ws dded nd incuted t 115 C for 20 min. A reflux condenstion equipment ws employed during digestion to void the voltiliztion loss of Se. After digesting, the solution ws cooled down to room temperture nd trnsferred into volumetric flsk nd mde up to 50 ml with ultrpure wter. Then, the digested solution ws used for totl Se determintion y hydride genertion of tomic fluorescence spectrometer (HG-AFS) method. For the PB-Se determintion, the semipermele memrne device (SPMD) technique nd the enzymtic hydrolysis (protese XIV, Sigm) were used (Liu nd Gu, 2009). Chlorophyll determintion 1 g of the fresh leves ws dded with 4 ml of pre-cooled 80% cetone solution nd ground. The homogenized smple ws centrifuged (4 C) t rpm for 10 min. The superntnt ws diluted to 100 ml with 80% cetone to extrct chlorophyll. The chlorophyll content ws mesured using UV/VIS Spectrometer System (Unico, UV-2800, USA) ccording to Arnon (1949). GPx ctivity determintion The GPx (EC ) ctivity ws exmined with the method descried y Hrtikinen et l. (2000).The totl solule proteins of the enzyme extrct were determined spectrophotometriclly y the method descried y Brdford (1976) using ovine serum lumin s stndrd. Sttisticl nlysis All trils were crried out in triplicte nd ll the dt were given s mens ± stndrd devition (SD). The sttisticl significnce ws evluted using Duncn s multiple-rnge test nd P<0.05 ws tken s significnt. RESULTS AND DISCUSSION Growth of young rley seedlings The dt in Tle 1 showed tht Se tretments hd no significnt effect (P>0.05) on iomss yield, height nd stem dimeter of young rley seedlings from 2 to 5 weeks fter sowing. These results indicte tht the concentrtions of selenite rnging from 0 to 30 µg/ml did not inhiit the growth of young rley seedlings. Similr results were oserved nd previously reported in lettuce (Xue et l., 2001). Se content in young rley seedlings Se is pprently not required for growth y mjority of higher plnts. In fct, high content of Se in the tissues of plnts my result in toxic effect. However, smll numer of plnts which re clled Se hyper-ccumultors re le to ccumulte extrordinrily high levels of Se. This study shows tht young rley seedlings hd reltively high cpcity to ccumulte Se. As shown in Tle 2, Se contents in young rley

3 Chlorophyll content (mg/g FW) Afr. J. Biotechnol. Tle 2. Accumultion of T-Se nd PB-Se contents in young rley seedlings. Week fter sowing N 2SeO 3 T-Se (µg DW) PB-Se (%) (µg/ml) (0.02) 0.05 (0.02) 0.04 (0.02) 0.07 (0.03) 96.0 (2.9) 93.6 (6.6) 94.1 (7.1) 94.3 (5.9) (0.04) 0.23 (0.06) 0.27 (0.06) 0.20 (0.05) 81.1 (7.0) 86.9 (6.0) 91.1 (6.1) 93.5 (5.0) c (0.04) 0.42 c (0.06) 0.43 c (0.05) 0.31 c (0.04) 81.7 (9.2) 89.6 (5.4) 82.1 (5.4) 88.4 (7.9) d (0.09) 0.59 d (0.09) 0.61 d (0.11) 0.48 d (0.08) 68.4 c (6.1) 79.2 (8.6) 84.4 (8.6) 86.5 (7.7) The results re expressed s men ± SD (in prentheses) with three replictions. Within the sme column, mens followed y the sme superscript letter re not significntly different (P>0.05). Brley seeds were soked in solutions t 20 C for 24 h with different concentrtions of selenite µg/ml 10 µg/ml 20 µg/ml 30 µg/ml c c Weeks fter sowing Figure 1. Effect of Se tretment on chlorophyll content (mg/g FW) in young rley seedlings. The results re expressed s men ± SD with three replictions. Columns mrked with sme letter re not significnt t P>0.05. Brley seeds were soked in solutions t 20 C for 24 h with different concentrtions of selenite. seedlings incresed significntly (P<0.05) s the selenite concentrtion supplied in the soking solutions incresed from 0 to 30 µg/ml. The results were in consistence with those otined in Se-enriched rice (Hu et l., 2002). In ddition, the rtio of PB-Se to T-Se in young rley seedlings incresed s the seedlings grew longer (Tle 2). 5 weeks fter sowing, the proportion of PB-Se to T-Se ws rnged from 83 to 93% when treted with vrious concentrtions of selenite. This indicted tht the trnsformtion of selenite to PB-Se species for young rley seedlings ws rpid nd effective. Chlorophyll content in young rley seedlings Figure 1 shows chlorophyll contents of young rley seedlings t vrious Se ddition levels nd in different growth stges. The effect of Se tretment on chlorophyll content ws relted to the Se concentrtion nd the growth period of young rley seedlings. A lower Se level of 10 µg/ml did not ffect chlorophyll content (P>0.05) t ll the growth stges. However, t higher Se dose of 30 µg/ml, significnt increse (P<0.05) of chlorophyll content in young rley seedlings ws detected from 2 to 4 weeks fter sowing. Bsed on the Se content in young rley seedlings (Tle 2), the results suggest tht Se promoting influence on chlorophyll content ws dependent on its content in seedlings. This my e ecuse Se could stimulte the respirtion rtes nd the flow of electrons in respirtory chin nd ccelerted the chlorophyll iosynthesis (Germ et l., 2005). GPx ctivity in young rley seedlings The ctivity of GPx in different growth periods is shown in

4 GPx ctivity (U) Afr. J. Biotechnol µg/ml 10 µg/ml 20 µg/ml 30 µg/ml c Weeks fter sowing Figure 2. Effect of Se tretment on chlorophyll content (mg/g FW) in young rley seedlings. The results re expressed s men ± SD with three replictions. Columns mrked with sme letter re not significnt t P>0.05. Brley seeds were soked in solutions t 20 C for 24 h with different concentrtions of selenite. One unit (U) of the enzyme ctivity ws clculted s the mount of enzyme ctlyzing the conversion of 1 µmol of sustrte per min s compred to tht in the nonenzyme rection. Figure 2. At ll the four hrvest time points, the GPx ctivity ws found to e incresing (P<0.05) with higher Se concentrtion supplied in the soking solutions, which suggested tht Se tretment hd positive effect on the enzyme ctivity. GPx is one of the min hydrogen peroxide scvenging enzymes in higher plnts. Though, Se ws not thought to e n essentil element for higher plnts, incresing findings reveled tht Se could increse the tolernce of plnts to oxidtive stress (Hrtikinen et l., 2000), s well s enhnced the growth of lettuce (Xue et l., 2001) nd ryegrss (Hrtikinen et l., 2000). In fct, ll the forementioned growthpromoting functions of Se were relted to the improved Se-dependent GPx ctivity. Conclusions This study investigtes the effects of Se tretment on growth, Se nd chlorophyll contents nd GPx ctivity in young rley seedlings. The results show tht Se tretment did not ffect the growth of young rley seedlings, ut it significntly incresed the chlorophyll content nd enhnced GPx ctivity. Young rley seedlings possessed reltively high cpcity of Se ccumultion nd trnsformtion. Se tretment might help young rley seedlings to ecome Se-enriched food mteril. ACKNOWLEDGEMENTS This work ws supported y the Doctor Reserch Fund of Henn University of Technology (No. 2010BS051). REFERENCES Arnon DI (1949). Copper enzyme in isolted chloroplsts, polyphenol oxidse in Bet vulgris. Plnt Physiol., 24: Brdford MM (1976). A rpid nd sensitive method for the quntifiction of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl. Biochem., 72: Coms GF (2001). Selenium in glol food systems. Brit. J. Nutr., 85(5): Fng Y, Wng L, Xin Z, Zho LY, An XX, Hu QH (2008). Effect of folir ppliction of zinc, selenium, nd iron fertilizers on nutrients concentrtion nd yield of rice grin in Chin. J. Agric. Food Chem., 56(6): Germ M, Kreft I, Osvld J (2005). Influence of UV-B exclusion nd selenium tretment on photochemicl efficiency of photosystem II, yield nd respirtory potentil in pumpkins (Cucurit pepo L.). Plnt Physiol. Bioch., 43(5): Hrtikinen H, Xue T, Piironen V (2000). Selenium s n nti-oxidnt nd pro-oxidnt in ryegrss. Plnt Soil, 225(1): Hu QH, Chen LC, Xu J, Zhng YL, Pn GX (2002). Determintion of selenium concentrtion in rice nd the effect of folir ppliction of Se-enriched fertiliser or sodium selenite on the selenium content of rice. J. Sci. Food.Agri., 82(8): Liu KL, Gu ZX (2009). Selenium ccumultion in different rown rice cultivrs nd its distriution in frctions. J. Agri. Food Chem., 57(2): Smrkolj P, Osvld M, Osvld J, Stiilj V (2007). Selenium uptke nd

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