Lead Absorption by Tea Leaves and its Distribution in Tea Plants.

Transcription

1 Led Asorption y Te Leves nd its Distriution in Te Plnts uelong M 1, Jingyu Fng 1, iochun Wn 2, Ruyn Hou 2, Wnyou Lio 3 1. College of Life nd Environmentl Sciences, Hungshn University, Hungshn , Chin 2. Key Lortory of Te Biochemistry nd Biotechnology, Anhui Agriculturl University, Hefei , Chin 3. Te Reserch Institute of Anhui Acdemy of griculturl sciences, Hungshn , Chin fjy@hsu.edu.cn, xcwn@hu.edu.cn Astrct: Bckground: Automoile exhust, tire wer nd drifting dust resulted from urniztion re new source of led into te plnt to threten humn helth y food chin. Despite of lrge mount of tmospheric led flls onto the soil, the floting prticle cn lso e sored directly y te leves. Purpose: To evlute the storge of led y te leves when they were contminted nd the element movement in the plnt s well s the possile mechnism referring to the process. Methods: After pplying led solution on leves of Cmelli sinensis (L.) O. Kuntze cultivr Fuyun No. 6 for 10 months, led content in the lef, stem nd root ws determined y GFAAS nd led distriution in different tissues of the three plnt orgns ws investigted y Environmentl scnning electron microscopy (ESEM) coupled with energy dispersive -ry micronlysis (EDA). Results: Led cptured directly y te leves incresed gretly with the ppliction onto the ldes without showing ny morphologicl symptoms or retrdnt growth. Led entered te leves through symplst nd poplst pthwys, nd moved downwrds ctively to the root vi the system. Conclusions Led cptured y te leves constitutes contminnts in the product nd we rgue for the limit of tmospheric led in te grdens. [uelong M, Jingyu Fng, iochun Wn, Ruyn Hou, Wnyou Lio. Led Asorption y Te Leves nd its Distriution in Te Plnts. Life Sci J 2013;10(3): ] (ISSN: ) Keywords: Cmelli sinensis; lef; led uptke; trnsport; distriution 1. Introduction Litertures hve proved tht crops like ryegrss, whet nd rley tht grow in neutrl or lkline soil otin led (P) minly from tmosphere ecuse in such soil P tends to comine with minerl or orgnic compounds to form complex which is insolule to enter the plnts [1-5]. As for Cmelli sinensis (L. ) O. Kuntze (te plnts), scientists elieve soil is the source for P into the plnts ecuse the soil is cidic nd P is relesed to e uptken y plnts roots. Bsed on this thought Chin government hs limited the mount of P in the soil ut never in the ir for te production [6-8]. In resent yers reserchers hve noticed tht P content in te leves rises gretly if te trees re growing close to expresswys [9-11]. According to them, utomoile exhust, tire wer nd drifting dust relese P in the ir, fll onto the soil nd then te plnt roots sor it nd trnsport to the lef [9,10]. Becuse te plnts re evergreen nd hve gret del of orgnic compounds in their leves [12,13], we ssume tht P from the ir might comine directly with the chemicls in leves to form P complex. In ddition, te plnts grow usully for decdes to provide fresh leves s the rw mteril for te everge production, P could ccumulte in leves yer fter yer. However, even though Wng et l. hve recently pplied plstic film to decrese P in young leves y 13%-46% [14], few studies hve reported on the direct effect of tmosphere on lef P rising ecuse the tmospheric P cn lso enter the plnt through the soil. The lck of nucler technology mkes it difficulty to trce P in the system from the ir to the plnt. Since the invention of environmentl scnning electron microscopy (ESEM), plnt structure cn e oserved in vpor t tmospheric pressures neither with dry processing nor gold coting in specimen preprtion due to the unique vcuum system of this equipment. When ESEM is fixed with energy dispersive -ry nlysis (EDA), chemicl nture of structure cn e reveled [15]. Scientists successfully pplied ESEM with EDA to demonstrte silic deposition in rice nd sorghum with fresh, unfixed, hydrted smples [16,17]. This rticle intended to exmine P sorption y te leves on stimultion experiment, in which P ws pplied to te leves s solution insted of gs. It lso pplied ESEM with EDA to investigte the sorption nd storge of P y te leves, s well s the trnsport of P from lef to stem nd root. 2. Mteril nd Methods Plnt culture In erly spring snd ws sifted through 0.3mm sieve, immersed in 10% (v/v) HNO 3 for 24h to remove ckground P, rinsed with tpping wter 1245

2 till ph ck to neutrl. Erthenwre pots were clened nd chrged with 6 kg snd ech. Seeds of Cmelli sinensis (L.) O. Kuntze cultivr Fuyun No. 6 were wshed in detergent, sterilized in 0.1% (w/v) HgCl 2, rinsed nd immersed in constnt dripping wter for 7d. The seeds were covered in the pot with snd nd spryed with wter irregulrly to keep moisture. The experiment ws done in the lortory of te iotechnology in Anhui Agriculturl University, Hefei city. The lortory ws on the sunny side with nturl light, temperture nd humidity. When 2nd lef grew, 4 seedlings with the sme size were remined. Every 7d the pot ws dded 1 L lnced nutrition solution with ph 5.5 [18]. Leves were pplied on the oth ldes y P(CH 3 COO) 2 3H 2 O solution with soft rush 3 times dy. The concentrtion ws 0, 7, 14, 42, 70 or 98 µg L -1 P respectively. New leves were lso treted with P solution. Tretment 98 µg L -1 P hd 7 pots, tretment 0 µg L -1 P hd 3 pots, nd the other tretments hd 6 pots ech. Led determintion y GFAAS After 10 months of P ppliction on te leves, smples of root, stem nd lef were clened with deionized wter, over-dried t 80, ground nd screened through 200 mesh sieve. Lef smple of ech tretment ws lso prepred y the sme procedure s ove ut not clened y deionized wter. Smple of g ws cronted on n electric-hot plte nd dry-shed in mrvl furnce (sx , Fctory of Shnghi Experimentl Electric Oven, Chin) t 500 for 6h. When it ws cool, the smple ws dded 1ml diluted HCl solution (1:1, v/v, 37% HCl to wter) nd heted on the electric-hot plte to oil. This procedure ws repeted untill the smple solution ecme cler. The smple solution ws set to 10ml with deionized wter nd P concentrtion ws determined y GFAAS (SOLAARM6, Thermo Electron Corportion, Americ). Every ssy hd 3 replics of 1 pot ech. The dt ws processed y excel softwre nd correltion nlysis ws done y IBM SPSS Sttistics. Led Detection y ESEM with EDA P reloction in lef, stem nd root ws investigted y n ESEM (T30 ESEM-TMP, Philips, Holnd) fitted with n EDA system (INCA300, Oxford, Englnd). Fresh freehnd sections of lef, stem nd root from 98 µg L -1 P tretment were rinsed with deionised wter nd fixed on copper stge y doule-sided dhesive tpe. ESEM working condition ws 5 to 8 temperture, 20 kv ccelerting voltge, 3.8 τ to 6.1 τ chmer pressure, nd 6.5 nm resolving cpility. P -ry intensity in section ws scnned either in liner or re profile. The contents of P were estimted semiquntittively y -ry counts emitted during the rster scnning. Leves of 0 nd 98 µg L -1 P tretments were lso oserved under Stereoscopy (Motic, SMZ- 143 SERIES, Chin) fitted with cmer (Sony, DSC- F717, Jpn). 3. Results Led injury to lef Although te seedling did not show ny injuries or symptoms in growth or color chnge, stereoscopy oserved drk P compounds gthered in lef veins nd corroded epiderml cells to mke lef surfce extremely rough (Figure 1). Figure 1. Stereoscopic imges of te leves. () lef treted y 98 µg L -1 P solution ws rough nd drk, rrows indicting veins with P in them. () lef treted y H 2 O ws smooth nd right. ESEM reveled tht the upper epidermis of the lef ws dmged much more seriously y P thn the lower (Figure 2). On the upper epidermis frgments of different sizes covered over the whole lde nd no cells could e distinguished [Figure 2()]. While on the lower epidermis only little frgments spotted nd epiderml cells nd gurd cells kept cler cell imge [Figure 2()], indicting tht the lower lde of the te lef hd certin wy to resist P stress. 1246

3 Figure 2. ESEM imges of P contminted te lef. () upper epidermis ws etched nd cells did not hve cler oundries. () lower epidermis hd cler epiderml cells nd gurd cells Led sorption nd storge y leves GFAAS determintion showed tht P content in te leves incresed with the ppliction of P on leves (Figure 3), nd the coefficient correltion ws significntly high (r=0.979, p=0.004). Leves rinsed with deionized wter hd lower P thn those without eing wshed from 5.00% to 17.12%. The results indicte tht te leves cn cpture P y wys of dsorption nd sorption which supported our hypothesis. Figure 3 displys tht P sored y te leves trnsported to the stem nd root especilly t high P ppliction, the correltion coefficients were significnt (stem, r=0.980, p=0.003; root, r=0.973, p=0.005). The dt in Figure 3 suggest tht low concentrtion of outside P tends to store in te leves nd my lso move to the stem nd root when outside P is much. For instnce, P content in the lef ws 3.4 times tht in the root t P 7 µg L -1, ut only out 0.47 t P 98 µg L -1. mesurement P (mg kg -1 ) unwshed lef lef stem root ppliction P (µg L -1 ) Figure 3. P content in lef, stem nd root fter P ppliction on te leves If the concentrtion of P in lef, stem nd root of every tretment ws verged, the rtio of ech orgn to the verge could present the tendency of P prtition mong the three orgns s in Tle 1. Tle 1. Rtio of orgn P to the verge P tretment (µg L -1 ) Lef Stem Root Rtios in tle 1 re reltively stle when the tretment P exceeded certin mount, lthough the exct mount of P is incresing. This might suggest the te leves hve stle storge of P nd will output the excess P to the root. Led distriution in lef Figure 4() nd (c) re the trnsection of P-polluted te lef nd its line scn mtch of P through the min vein. The upper epidermis hd P of 9 counts nd the lower hd 3 counts, showing more P on the upper prt thn on the lower, which explined photos in Figure 2 tht upper epidermis ws corroded while the lower ws not. Prenchym cells ner the upper epidermis lso hd more P thn those ner the lower epidermis. For the tissue, which ws out 280 mm thick, two ptterns of P counts were distinguished. The upper hlf could e nmed the rising pttern nd the lower hlf the smooth pttern. The smooth pttern extended from the lower to the nd l lyer of prenchym cells inside the lower epidermis. Becuse in this typicl vsculr conductive structure occupied the lrgest re nd hd more P thn the, it is possile tht exported P nd imported P. On the lower epidermis lef, P scttered uniformly on epiderml hir, cells, gurd cells nd stomt [Figure 4 () nd (d)], hinting P moved effectively in the lower epidermis. Interesting thing ws tht the lower epidermis ws ner the nd the upper epidermis ner the. These results extended the previous findings tht in te leves P ws stored in the nd trnsported y the conductive system. 1247

4 U P Ph P L c prenchym 部 d Figure 4. P distriution in different tissues of C. Sinensis lef reveled y ESEM with EDA. () Trnsverse section with line scn of P. U, upper epidermis; L, lower epidermis; P, prenchym cells; Ph, ;,. () Epiderml cells, gurd cells nd hir. (c) Line scn mtch of () showing P intensity from upper epidermis to lower epidermis through the min vein. Verticl scle is P counts nd horizontl scle is the distnce from the upper epidermis. (d) Are scn mtch of the rectngle in () showing P intensity in hir, gurd cells nd epiderml cells. c Pe Ph C Pt d e pith f Figure 5. P distriution in different tissues of C. Sinensis stem reveled y ESEM with EDA. () Trnsection of stem with line scn of P from periderm to. Pe, periderm; Ph, ; C, cmium;,. () Trnsection of stem with line scn of P from to pith. Pt, pith. (c). Trnsection of stem showing pith. (d) Line scn mtch of () showing P intensity from periderm to. Verticl scle is P counts nd horizontl scle is the distnce from periderm. (e) Line scn mtch of () showing P counts from to pith. Horizontl scle is the distnce from. (f) Are scn mtch of the rectngle in (c) showing P intensity in storge prticles nd other components in pith cells. 1248

5 Led distriution in stem In the trnssection of the stem there were five tissues including from outside the periderm,, cmium, nd pith (Figure 5, nd c). Becuse the smple ws too lrge in this cse, continuous photos were tken. Two ptterns of P intensity could e seen in the stem [Figure 5 (), (), (d), (e)]. One ws the prol pttern in the of out 80 m thick with low P of 3 counts t oth ends, nd high P of 12 counts in the middle. The other ws the smooth pttern in the nd pith. Arrows in Figure 5() did not show more P in vessel cvities thn the other re indicted tht they could not e the specil reception site of P from the lef, nor did the pith cells in Figure 5(c) nd (f). Becuse of the lrger re, the nd pith might e the storge plce for P in the stem. Led distriution in root Figure 6() showed the trnscetion of the root included tissues from outside epidermis, cortex, endodermis,, cmium nd. The cortex held more thn hlf of the root spce. Figure 6() nd () showed tht P in the cortex ws very dense. Interestingly, endodermis cted s oundry for P distriution outside nd inside. P inside decresed from the to the. The cortex t ws the storge plce of P in the root, insted of the s in the lef nd stem. Ph Ed cortex Ct Ep Figure 6. P distriution in different tissues of C. Sinensis root reveled y ESEM with EDA. () Trnsection of root with line scn of P from t center to periphery endodermis. Ep, epidermis; Ct, cortex; Ed, endodermis; Ph, ;,. () Line scn mtch of () showing P counts from to epidermis. Verticl scle is P counts nd horizontl scle is the distnce from. Dynmic movement of P When the highest P counts in the nd were connected from the lef, stem to the root, dynmic reloction of P sored y lef in te seedlings ws otined (Figure 7). P in the ws reltively stle round 10 counts, while in the it kept incresing drmticlly from 7 counts in lef to 12 in stem nd 25 in root. These results suggest tht in te seedling the could not e the pth of P from the lef to the root, ut the is possile the conducting system. The rising tendency of P counts in the ws entirely consistent with tht of P concentrtions in the lef, stem nd root, indicting the P ccumultion in root from the lef through the. P (mg/kg) exct lef stem root Figure 7. P in lef, stem nd root Reltive P (cps) 4. Discussions The min im of this pper is to estimte the storge of P y te leves. Hn et l. stted tht the 1249

6 tmosphere eside highwy contined P 14.2 µg m -3 nd tht the utomoile exhust contined P 7.3 to 14.0 µg m -3 [11]. In this experiment we set the lest P concentrtion s 7µg L -1 for the considertion tht leves were treted P solution 3 times dy nd this period together ws out 1min. Becuse there re 1440 minutes in dy, P solution in the experiment ws condensed 1000 times to imitte the possile P mount fllen on the lef from the ir. Chinese stte limits P to 5 mg kg -1 in te leves product [19]. In this experiment te leves treted with P 7 µg L -1 ccumulted it mg kg -1. Although this mount ws only 0.365% of the stte limit, it would e 10.95%-14.60% in rel production ecuse te plnts usully grow for out yers, excluding the prt from the root ck to the lef. This figure is in the rnge of Wng et l. s 13% to 46% [14] in rel te grdens. The reson for Wng et l. s greter dt might e tht theirs include the prt from the soil. In ddition, our tretments imply tht more P from the ir would gretly increse the lef cpture of this hevy metl. Therefore it is necessry for the government to propose stndrd of ir P contminnts. Becuse wter wshed wy prt of P cptured y te leves, we concluded tht P entered te leves y poplst nd symplst pthwys. The results of etched upper epiderml nd corrosion resistnt of lower epidermis to P implied tht P might trnsport through the system, which goes with nd the knowledge of elements movement sored y plnt leve [20]. The flow ginst P concentrtion in the system from the lef, stem to the root leds to the ctive mechnism of P movement downwrds. Relince on these mesures must e further confirmed, however, ecuse P tretment in this experiment is not the rel te production ir sitution. It would e eneficil to replicte this study on lrge scles. It would lso e necessry to mesure the flow of P etween the ir nd te plnts over decdes insted of te seedlings in only one yer to estimte more ccurtely the risk of P from the ir. Acknowledgements: This work ws supported y funds from Reserch Fund for the Doctorl Progrm of Higher Eduction of Chin, Soil nd Fertilizer Post Project, Ntionl Te Industry nd Technology System, Ministry of Agriculture, Chin (CARS-23),Sprk Progrm Project, Ministry of Science nd Technology, Chin (2011GA710077, 2012GA710001, 2012GA710037, 2013GA710043). Corresponding Author: Pr. Jingyu Fng College of Life nd Environmentl Sciences, Hungshn University Hungshn, Anhui Province , Chin E-mil: FJY@hsu.edu.cn Pr. iochun Wn Key Lortory of Te Biochemistry nd Biotechnology, Anhui Agriculturl University Hefei, Anhui Province , Chin E-mil: CWAN@hu.edu.cn References 1. Ctldo DA,Wildung RE. Soil nd plnt fctors influencing the ccumultion of hevy metls y plnts. Environmentl Helth Perspectives 1978;27: Tjell JC, Hovmnd MF, Mosek H. Atmospheric led pollution of grss grown in ckground re in Denmrk. Nture 1979;280: Mosek H, Tjell JC, Hovmnd MF. Atmospheric led input to griculturl crops in Denmrk.Chemosphere1989;19(10-11): Dlenerg JW, Vndriel W.Contriution of tmospheric deposition to hevy metl concentrtions in field crops. Netherlnds Journl of Agriculturl Science;1990;38(3): Zho FJ, Adms ML, Dumont C, McGrth SP, Chudri AM, Nicholson FA, Chmers BJ, Sinclir AHP. Fctors ffecting the concentrtions of led in British whet nd rley grin. Environmentl Pollution 2004;131(3): Environmentl requirements for growing re of green food te, NY , Agriculturl industry stndrd of the People's Repulic of Chin, Stndrds press of Chin, Beijing Environmentl requirements for growing re of orgnic te, NY , Agriculturl industry stndrd of the People's Repulic of Chin, Stndrds press of Chin, Beijing, Environmentl requirement for growing re of te, NY/T , Agriculturl industry stndrd of the People's Repulic of Chin, Stndrds press of Chin, Beijing Jin CW, He YF, Zhng K, Zhou GD, Shi JL, Zheng SJ. Led contmintion in te leves nd non-edphic fctors ffecting it. Chemosphere 2005;61(5): Hn WY, Zho FJ, Shi YZ,M LF, Run JY. Scle nd cuses of led contmintion in 1250

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