EFFECTS OF CADMIUM-ADDED ON ASCORBATE-GLUTATHIONE CYCLE OF YOUNG CITRUS SEEDLINGS UNDER SELENITE OR SELENOMETHIONINE-ENRICHED SOIL

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1 Pk. J. Bot., 5(4): , 218. EFFECTS OF CADMIUM-ADDED ON ASCORBATE-GLUTATHIONE CYCLE OF YOUNG CITRUS SEEDLINGS UNDER SELENITE OR SELENOMETHIONINE-ENRICHED SOIL XIEPING SUN 1,2, GUOQIANG HAN 1, YOUJIN LUO 2*, GUANGWEN ZHOU 2 AND YU WANG 1 1 Life Siene nd Tehnology Institute, Yngtze Norml University, P.R. Chin 2 Fruit Reserh Institute, Chongqing Ademy of Agriulturl Sienes, Chongqing, P.R. Chin * Corresponding uthor s emils: luoyoujin1984@163.om Astrt In order to study the effet of dmium (Cd)-dded on itrus growth under selenite or selenomethionine ()- enrihed soil, the onentrtion of lef pigments, H2O2, ntioxidnts nd ntioxidses in sorte glutthione (AsA-GSH) yles were deteted in Dy itrus seedlings (Citrus sinensis L.). The results showed tht hlorophyll,, nd + were signifintly enhned y dmium-dded under -enrihed tretment, while lef pigments showed little influene dmium under selenite-enrihed tretment. Menwhile, dmium-dded signifintly inresed H2O2 onentrtion under oth selenite nd -enrihed soils. In AsA-GSH yle, the onentrtion of AsA, dehydrosorte (DHA), nd AsA/(AsA+DHA) rtio were inresed, while the levels of GSH, oxidized glutthione (GSSG), nd GSH/(GSH+GSSG) were deresed y dmium-dded under selenite or -enrihed soil. Menwhile, the tivities of sorte peroxidse (APX), monodehydrosorte redutse (DHAR), nd glutthione redutse (GR) inresed, nd the tivities of glutthione peroxidse (GPX) deresed under selenite or -enrihed soil with dmium-dded. Our results indited tht despite dmium-dded hd positive effets on hlorophyll onentrtions under Se-enrihed soil, lef ws injured y inresed H2O2 onentrtion. Furthermore, the AsA-GSH yle effiieny ws inresed to defend dmium stress under two selenium types-enrihed soils. Key words: Dy ornge, Selenium, Cdmium, AsA-GSH yle. Introdution Hevy metls known in nture re divided into two min groups: 1-the essentil hevy metls, 2-non-essentil hevy metls for plnts growth (Emmverdin, & Ding, 218). Cdmium (Cd) is highly toxi non-essentil hevy metl, whih redues plnt growth nd lter metoli proesses (Hssn & Mnsoor, 217). Citrus plnts possess remrkle tolerne to dmium. For instne, itrus ould exhiit no lef dmge or plnt deth under 3 µm dmium tretment (Aggrwl et l., 211). The plnts typilly umulted dmium re le to mintin the intrellulr redox lne nd defend the oxidtive stress. Plnts employ ntioxidnt enzymes nd the smll moleulr ntioxidnts to ope with the oxidtive stress. Asorte-glutthione (AsA-GSH) yle system involved in svenging of H 2O 2 ontins GSH, glutthione redutse (GR), oxidized glutthione (GSSG), sori id (AsA), sorte peroxidse (APX), dehydrosorte (DHA), nd monodehydrosorte redutse (MDHAR) (Ríos et l., 29; Cho et l., 21; López-Climent et l., 214). Plnt mintining high rtios of AsA/(AsA+DHA) s well s GSH/(GSH+GSSG) inresed the toxi retive oxygen speies defense (Wng et l., 212). Selenium (Se) is termed to e enefiil element for numerous plnts. Low-dose selenium enhned plnt growth, while high-dose hindered plnt growth (Jing et l., 215; Sun et l., 218). Low dose selenium indued ntioxidnt prodution, stimulting plnt growth (Ríos et l., 29). In ddition, selenium plys protetive funtion ginst dmium stress (Elguer et l., 214). For exmple, low-dose selenium hd enefiil effet on rie growth, together with mitigting the toxiity of dmium (Ding et l., 214). Adverse effet of dmium stress ws mitigted y regulting the osmoprotetnts, the ntioxidnt enzymes, nd the seondry metolites with pplition of selenium (Ahmd et l., 216). Selenium onsiderly indued the iosynthesis of meltonin, nd meltonin n lso redue dmium uptke together with mitigting dmium toxiity (Li et l., 216). Plnts growing on the seleniferous soil produe the selenium-enrihed foods (Ysin et l., 215). Some Seenrihed soil is inevitle to e polluted y dmium, or hs risk of dmium pollution. In our work, AsA-GSH yle systems of Dy ornge were ompred, whih grown under dmium-polluted or not polluted Se 4+ (from selenite) nd selenomethionine ()-enrihed soil. Mterils nd Methods The experiment ws rried out in greenhouse t Bishiyi Town (E 16 o 21 44, N29 o ), Jiulongpo Distrit, Chongqing ity of Chin. Uniform grfted seedlings of Dy ornge (Citrus sinensis L.) were plnted in 1. L plsti pots filled with grden soil, turf soil, nd puerile (1:1:1) mixture. Plnts were llowed to limte for one month under well plnted onditions. On Aug 24 th 215, helthy seedlings of uniform size were seleted s mterils. Four tretments were pplied, vi (1) pots ontined 3. mg/l selenium from selenite; (2) pots ontined 3. mg/l selenium from selenomethionine (); (3) pots ontined 3. mg/l selenium (from selenite) +.6 mg/l dmium (from dmium hloride); (4) pots ontined 3. mg/l selenium (from selenomethionine ()) +.6 mg/l dmium (from dmium hloride). The experiments ended on April 18 th, 216. Leves were olleted nd ground t temperture of -8 o C prior to the detetion.

2 1292 XIEPING SUN ET AL., Lef pigments were extrted using 95% ethyl lohol in drk, determined y spetrophotometry in ordne with the method of Lihtenhler & Wellurn (1983). The onentrtion of H 2O 2 ws determined s H 2O 2-titnium omplex rised from the retion of tissue- H 2O 2 with titnium tetrhloride (Zhng et l., 212). The tivity of APX, DHAR, GPX, GR, nd the ontent of AsA, DHA, GSH, GSSG were mesured y enzyme linked immunosorent ssy (ELISA)-sndwih pproh y Ryto RT-61 nd ELISA detetion kits. And then, the sum of AsA+DHA nd GSH+GSSG, nd the rtios of AsA/(AsA+DHA) nd GSH/(GSH+GSSG) were lulted. All dt were sttistilly nlyzed with Origin 8. softwre. The dt ws displyed s men ± stndrd devition (SD) performing of four replitions. One-wy nlysis of vrine (ANOVA) ws used to ssess the tretment effet for ompring signifine mong tretments. Signifine levels were expressed t p<.5 level. Results Pigment ontents: Dy pigment ontents were not signifintly influened y dmium-dded under selenite-enrihed soil. While hlorophyll, hlorophyll, rotenoid nd hlorophyll + inresed 18.3%, 14.8%, 15.3% nd 17.2% y dmium-dded under enrihed soil, respetively(fig. 1). Furthermore, higher onentrtions of hlorophyll (8.9%), hlorophyll (3.%), rotenoid (8.6%), nd hlorophyll + (7.1%) were found under selenite-enrihed soil ompred to those under -enrihed tretment. The ontents of the hlorophyll, hlorophyll, rotenoid, nd hlorophyll + were 11.5%, 15.5%, 1.2%, 12.4% deresed y dmium-dded under -enrihed tretment, respetively. H2O2 ontents: There ws no signifint differene in lef H 2O 2 onentrtion of Dy ornge etween selenite nd -enrihed soil. Leves H 2O 2 onentrtions were inresed y 125.8% nd 45.3% with dmium dditions of under selenite nd -enrihed soil (Fig. 2). AsA-GSH yle effiieny: GSH nd AsA re low moleulr ntioxidnts, menwhile GSSG nd DHA re their oxidtion sttes, respetively. APX nd GPX re mjor H 2O 2-svenging enzymes. GR nd DHAR re two key enzymes to mintin high onentrtion of GSH nd AsA. AsA ws inresed 31.7% (selenite-enrihed) nd 8.6% ( enrihed soil) y dmium ddition. Menwhile, DHA onentrtion ws lso inresed out 23.% (selenite-enrihed) nd 1.% ( enrihed soil). GSH onentrtion ws deresed 12.3% nd 14.3% y dmium ddition under selenite-enrihed nd enrihed soil, respetively. GSSG onentrtion ws lso redued to 6.1% nd 7.2% y pplition of dmium under selenite-enrihed nd -enrihed soil, respetively. A slight inrese of AsA/(AsA+DHA) rtio nd slight derese of GSH/(GSH+GSSG) rtio were found with dmium ddition under two types enrihed soil APX, GR nd DHAR tivities in lef were deresed y 28.%, 17.4% nd 64.2% y ddition of dmium under selenite-enrihed soil, respetively. While APX, GR nd DHAR tivities in lef were inresed y 23.1%, 13.1% nd 59.7% y dmium ddition under enrihed soil, respetively. GPX tivities were deresed 1.% nd 13.4% y dmium ddition under seleniteenrihed nd -enrihed soil, respetively. Disussion Retive oxygen speies re inresed in the plnt under ioti stress, dmging the struture s well s funtion of the ells. Chlorophyll pigments is one of the key ftors of dmium injury in plnts, wheres, different selenium type lso ffets plnt pigments (Murdoglu et l., 215, Grous et l., 216). In our study, Dy ornge hd higher pigments under seleniteenrihed soil thn tht under -enrihed soil, inditing tht plnt pigments were ffeted with selenium type. Dy pigment onentrtion hd no signifint hnge with dmium ddition under seleniteenrihed soil, while it ws signifintly inresed y dmium ddition under -enrihed soil (Fig. 1). The results were onsistent with report y Aggrwl et l., (211) tht itrus is resistnt to dmium. Cdmiumdded inresed H 2O 2 onentrtion under seleniumenrihed soils (Fig. 2). High level of H 2O 2 dmged ell strutures, using severe onsequenes (Ozyigit et l., 216). Dy ornge showed more sensitive to dmium stress under selenite-enrihed soil thn tht under enrihed soil. It my e due to muh more selenium gined y seedlings leves under ondition thn under selenium-enrihed soil (dt not shown). Selenium, espeilly relted with GPX, hs very speil iologil funtion, inluding protetion ginst oxidtive dmge, defenses ginst infetion, nd modultion of growth nd development (EI-Rmdy, et l., 214). A lrge numer of studies indited tht exogenous selenium mitigted dmium toxiity through the regultion of ntioxidtive system (Ahmd et l., 216; Luo et l., 217). Wu et l., (217) reported tht pplition of selenium inresed the ontrtion of GSH nd AsA, tivities of GR nd DHAR, improving the AsA-GSH yle effiieny. The tivities of GR nd GPX were inresed, the oxidtive stress ws redued, nd the plnt growth ws improved y selenium ddition under dmium stress (Khn et l., 215). Studies on physiologil nd iohemil retion of plnts under dmium-ontminted selenite-enrihed soil were few. In our study, AsA-GSH yle effiieny ws nlyzed with dmium ddition under selenium-enrihed soil (Figs. 2, 3 nd 4). The higher rtios of AsA/(AsA+DHA) nd GSH/(GSH+GSSG) indited the stronger ntioxidnt pity (Fotopoulos, et l., 21; Wng et l., 212). But in our study, higher AsA/(AsA+DHA) rtio nd lower GSH/(GSH+GSSG) rtio were found y ddition of dmium under selenium-enrihed soil (Figs. 3 nd 4). Inonsistent hnges of the two rtios my e due to GSH redox yle effiieny (GPX ws redued y dmium-dded) whih ws redued y dmium-dded. In AsA-GSH yles, GPX nd DHAR onsumed sme enzyme sustrte GSH. GSH is moleulr ntioxidnt tht diretly svenging retive oxygen speies. Moreover, GSH ws omined with dmium to llevite the toxiity of dmium (Brouwer et l., 1993; Ydv, 21). Thus, it explins why dmium-dded tretments redue GSH/(GSH+GSSG) rtio.

3 EFFECT OF CADMIUM-ADDED ON CITRUS GROWTH UNDER SELENITE OR SELENOMETHIONINE 1293 Pigment onentrtion (mgg -1 ) Chlorophyll Chlorophyll Crotenoid Chlorophyll + +dmium +dmium Fig. 1. Effet of dmium-dded tretment on the onentrtion of hlorophyll, hlorophyll, rotenoid, nd hlorophyll + of Dy ornge under selenite or soil. The dt shown re the verges of four replites, with the stndrd errors indited y the vertil rs. The mens demoted y the sme letter do not signifintly differ t p<.5 (Fisher LSD test) Fig.2. Effet of dmium-dded tretment on the onentrtion of H2O2 of Dy itrus under selenite or soil. The dt shown re the verges of four replites, with the stndrd errors indited y the vertil rs. The mens demoted y the sme letter do not signifintly differ t p<.5 (Fisher LSD test) AsA (μmol g 1 FW) d GSH ( mol g 1 FW). DHA ( mol g 1 FW) d GSSG ( mol g 1 FW).. AsA/(AsA+DHA) GSH/(GSH+GSSG). +dmium +dmium +dmium. +dmium Fig. 3. Effet of dmium-dded tretment on the onentrtion of AsA, DHA, GSH, GSSG, nd rdios of AsA/(AsA+ DHA), GSH/(GSH+ GSSG) of Dy itrus under selenite or soil. The dt shown re the verges of four replites, with the stndrd errors indited y the vertil rs. The mens demoted y the sme letter do not signifintly differ t p<.5 (Fisher LSD test).

4 1294 XIEPING SUN ET AL., APX tivity (U g 1 FW) GPX tivity (U g 1 FW) DHAR tivity (U g 1 FW) GR tivity (U g 1 FW) +dmium +dmium. +dmium +dmium Fig. 4. Effet of dmium-dded tretment on the tivtes of APX, DHAR, GPX, GR of Dy itrus under selenite or soil. The dt shown re the verges of four replites, with the stndrd errors indited y the vertil rs. The mens demoted y the sme letter do not signifintly differ t p<.5 (Fisher LSD test) Conlusion In our study, Cd-ddition showed positive effets on Dy ornge lef pigments under selenium-enrihed soil, espeilly under -enrihed soil. However, dding dmium signifintly inresed lef H 2O 2 under selenium-enrihed soil. The AsA-GSH yle effiieny ws inresed to defend dmium stress. Aknowledgments This work ws supported y the Bsi Sientifi Reserh Business Expenses in Chongqing Chin (215st-jky-514; 216st-jky-514); The Initition Projet of Introdution Tlents of Yngtze Norml University (217KYQD65); nd The Ntionl Nturl Siene Foundtion of Chin (316146). Referenes Aggrwl, M., S. Shrm nd N. Kur Exogenous proline pplition redues phytotoxi effets of selenium y minimising oxidtive stress nd improves growth in en (Phseolus vulgris L.) seedlings. Biol. Tre Elem. Res., 14(3): Ahmd, P., E.F.A. Allh nd A. Hshen Exogenous Applition of selenium mitigtes dmium toxiity in Brssi june L. (Czern & Cross) y up-regulting ntioxidtive system nd seondry metolites. J. Plnt Growth Regul., 35(4): Brouwer, M., T. Hoexum-Brouwer nd R.E. Cshon A puttive glutthione-inding site in Cd/Zn-metllothionein identified y equilirium inding nd moleulr-modeling studies. Biohem. J., 294(Pt1)(1): Cho, Y., C. Hong nd C. Ko. 21. The deline in sori id onentrtion is ssoited with dmium toxiity of rie seedlings. Plnt Physiol. Biohem., 48(5): Ding, Y., R. Feng, R. Wng, J. Guo nd X. Zheng A dul effet of Se on Cd toxiity evidene from plnt growth, root morphology nd responses of the ntioxidtive systems of pddy rie. Plnt Soil, 375(1-2): Elguer, J.C.T., E.Y. Brrientos nd K. Wroel Effet of dmium (Cd(II)), selenium (Se(IV)) nd their mixtures on phenoli ompounds nd ntioxidnt pity in Lepidium stivum. At Physiol. Plnt., 35(2): El-Rmdy, H.R., Év Domokos-Szolsy, N.A. Adll, T.A. Shly, A. Sztrik, J. Prokish, M. Fári Selenium nd nno-selenium in groeosystems. Enviro. Chem. Lett., 12(4): Emmverdin, A., nd Y. Ding Effets of inhiition threshold in hevy metls on the growth nd development of rundinri fortunei. Pk. J. Botny., 5(3): Fotopoulos, V., V. Ziogs, G.Tnou, A. Molssiotis. 21. Involvement of AsA/DHA nd GSH/GSSG rtios in gene nd protein expression nd in the tivtion of defene mehnisms under ioti stress onditions. In Asorte- Glutthione Pthwy nd Stress Tolerne in Plnts; Springer: Dordreht, The Netherlnds, Grousi, F., B. Kovás, D. Andrási nd S. Veres Selenium phytoumultion y sunflower plnts under hydroponi onditions. Wter Air Soil Pollut., 227(1): 382. Hssn M. nd S. Mnsoor Priming seeds with phytohormones llevites dmium toxiity in mung en (Vign rdit L. Wilzek) seedlings. Pk. J. Bot., 49(6): Jing, C., C. Zu, J. Shen, F. Sho nd T. Li Effets of selenium on the growth nd photosyntheti hrteristis of flue-ured too (Niotin tum L.). At So.Bot.Pol., 84(1):

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