Original Research The Impact of Short-Term Exposure to Pb and Cd on Flavonoid Composition and Seedling Growth of Common Buckwheat Cultivars

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1 Pol. J. Environ. Stud. Vol. 22, No. 6 (213), Originl Reserch The Impct of Short-Term Exposure to P nd Cd on Flvonoid Composition nd Seedling Growth of Common Buckwhet Cultivrs Mrcin Horowicz 1 *, Henryk Dęski 1, Wiesłw Wiczkowski 3, Dorot Szwr-Nowk 3, Dnut Koczkodj 1, Jonn Mitrus 1, Huert Sytykiewicz 2 1 Deprtment of Plnt Physiology nd Genetics, Siedlce University of Nturl Sciences nd Humnities, Prus 12, 8-11 Siedlce, Polnd 2 Deprtment of Biochemistry nd Moleculr Biology, Siedlce University of Nturl Sciences nd Humnities, Prus 12, 8-11 Siedlce, Polnd 3 Institute of Animl Reproduction nd Food Reserch of Polish Acdemy of Sciences, Deprtment of Chemistry nd Biodynmics of Food, Tuwim 1, Olsztyn, Polnd Received: 1 April 213 Accepted: 19 August 213 Astrct The im of this study ws to compre the tolernce of seedlings of three Polish uckwhet cultivrs (Hruszowsk, Kor, nd Lu) for short-term exposure to P 2+ nd Cd 2+. Seedlings were grown under controlled conditions in Hoglnd nutrient solution, with the ddition of low/high P 2+ or Cd 2+ ions (.1 nd 1. mm, respectively). After 3 dys of treted seedling growth, the levels of totl nthocynins nd content of prticulr flvonoids were mesured. The presence of low concentrtions of oth P 2+ nd Cd 2+ resulted in smll stimultion of the growth of seedlings of ll studied cultivrs, while higher doses inhiit root growth nd, to much lesser extent, tht of shoots. Cdmium (Cd 2+ ) ions were more hrmful for growth of uckwhet seedlings thn P 2+ ions. More resistnt to stress cused y the presence of high concentrtions of P 2+ nd Cd 2+ in the growth medium were seedlings of Hruszowsk nd Lu cultivrs, compred to Kor seedlings. Cotyledons of more resistnt cultivrs (Hruszowsk nd Lu) contined much more flvonoids thn cotyledons of Kor. Keywords: led, cdmium, common uckwhet, seedling growth, flvonoids Introduction While plnts need mny metls such s iron, mgnesium, copper, or zinc, other metls such s led or cdmium re highly toxic. Plnts hve evolved vrious mechnisms to del with toxic metls in the soil [1]. For instnce, specific proteins such s metllothioneins cn effectively ind hevy metls [2, 3]. Led is the most common hevy metl contminnt in the environment. It is ccumulted in dose-dependent *e-mil: mhorowicz@uph.edu.pl mnner in plnts, which results in reduced growth, chnges in chemicl composition, enzyme ctivities, nd lower uptke of mny minerls. Led cn cuse vrious physiologicl nd iochemicl dysfunctions during seed germintion, plnt growth, nd mny physiologicl nd iochemicl processes [4, 5]. Although led trnsport from plnt roots to shoots is usully limited, shoots usully cn ccumulte up to 1/6 of the level in roots [6, 7]. However, common uckwhet elongs to plnts tht ccumulte high led content in leves without significnt dmge [8]. This indictes tht uckwhet is recognized s P hyperccumultor. During 8 weeks of cultivtion, uckwhet leves ccu-

2 1724 Horowicz M., et l. multed c. 2.5-times more P thn the roots, nd 4-times more thn stems [8]. Among the hevy metls, cdmium (Cd) is one of the most toxic elements tken up y plnts [9]. Cdmium is toxic to mny plnt species t low concentrtions [1]. When Cd is present in the nutrient medium, it cuses strong inhiition of growth nd different metolic processes [11]. At the root region Cd competes for sorption of severl essentil elements like clcium nd mgnesium, therefore cusing their deficiency [12]. Cd cn e ccumulted to high concentrtions in roots, wheres only minor mounts cn e trnsported to the ove-ground tissues in the form of metllo-orgnic complexes nd rech tissues of eril prts of the plnts [13, 14]. Root elongtion in plnts ws retrded rpidly within hours of Cd exposure, nd this mens tht interctions with growth regultors my occur [15, 16]. Root growth is more sensitive to hevy metls thn shoot growth [17]. This evidence correltes with the dt tht hevy metls ccumulte predominntly in roots. In mny plnts P 2+ inhiited growth more thn Cd 2+, ut in mize Cd 2+ ws found to exceed P 2+ in its toxicity to roots [18, 19]. In generl, Cd 2+ nd P 2+ inhiits growth, ut t low concentrtions cn promote the growth of root systems nd shoots [2]. Previous studies showed tht led (P 2+ ) nd cdmium (Cd 2+ ) ions, esides growth inhiition, cn distur mny iochemicl processes [21]. For instnce, Cd 2+ induced ctivity of chlcone synthse in soyens [22]. This result suggests involvement of the phenylpropnoid pthwy in the response of legume plnts to metls [23, 24]. The gene encoding phenyllnine mmoni-lyse, key enzyme in the phenylpropnoid pthwy, ws detected mong Cd 2+ - induced genes in Brssic junce [25]. Severl studies hve found tht hevy metls ctivted the phenylpropnoid pthwy nd incresed lignin synthesis in mny plnt species [26-29]. Kováčik et l. [3] oserved n increse of polyphenol oxidse ctivity in roots of Mtricri chmomill y cdmium nd suggested tht the formtion of polymerized phenols could e used to complex Cd ions. Flvonoids re known to form complexes with hevy metls, nd this could led to n effective method of plnt defensive responses to hevy metl stress [31-33]. It hs een confirmed in numerous studies tht flvonoids function s ntioxidnts minly y chelting metl ions. Moreover, metl-flvonoid cheltes re much more powerful free rdicl scvengers thn the prent flvonoids, nd ply prominent role in protecting cells from oxidtive stress [34]. Flvonoids ply n importnt role in limiting metl iovilility nd suppressing metl toxicity y forming complexes. Due to such properties, flvonoids pper to e suitle ntidote for hevy metl poisoning in vivo [35]. Among metl ions, esy-forming complexes with flvonoids re lso P 2+ nd Cd 2+ [35, 36]. Since mny uthors hve found n induction of flvonoids upon hevy metl tretment [33, 37, 38], investigtions on levels of endogenous flvonoids were done. After studies on micropropgted poplr (Populus jcquemontin) plnts, it ws suggested tht flvonoids re le to protect the plnt tissues due to their Cd chelting properties [39]. In these studies Cd tretment cused enhnced ccumultion of flvonoids in poplr seedlings. Evidence tht flvonoids could e n dditionl fctor in hevy metl tolernce in Aridopsis thlin lso hs een found [4]. The im of the present study ws to compre the sensitivity of seedlings of three Polish uckwhet cultivrs (Hruszowsk, Kor, nd Lu) to the short-term exposure of P 2+ nd Cd 2+. Preliminry experiments evluted the effects of different doses of P nd Cd on the growth of uckwhet seedlings. The otined dt were the sis for the selection of the dose of.1 mm of ech metl, which cused slight stimultion of growth. For comprison, concentrtion of 1. mm ws used, which significntly inhiited seedling growth. In the work possile reltion etween flvonoid level nd P or Cd tolernce lso ws investigted in seedlings of common uckwhet cultivrs. Among the studied cultivrs the Hruszowsk is the most widely cultivted in Polnd, Kor is in second plce, while Lu is only grown in experimentl plots. Mteril nd Methods Plnt Mteril nd Growth Conditions Seedlings of uckwhet (Fgopyrum esculentum Moench) cv. Hruszowsk, Lu, nd Kor were used in this study. The germintion process ws crried out in drkness during four dys, s descried previously [41]. Afterwrd, the uckwhet seedlings were exposed for three dys to low (.1 mm) or high (1. mm) concentrtions of P(NO 3 ) 2 or CdCl 2 dissolved in one fifth Hoglnd solution, nd grown in controlled-environment conditions with 16/8 h 24/18ºC dy/night scheme nd light intensity of 1-12 μmol m -2 s -1, which ws provided y 4 W high-pressure sodium lmps [41]. Mesurements of Plnt Elongtion Before nd fter 3-dy period of exposure to P 2+ or Cd 2+, shoot nd min root length were mesured in the seedlings. The differences etween lengths efore nd fter exposure were treted s growth elongtion. Men results of lengths nd their sttisticl evlution were otined from 6-8 seedlings. Determintion of Anthocynins Extrction nd mesurement of nthocynins were crried out using the method descried y Mncinelli [42] (slightly modified [43]). Briefly, hypocotyl or cotyledon tissues were extrcted with cidified (1% HCl, w/v) methnol for 24 h t room temperture, in drkness with occsionl shking. Asornce of the extrcts ws mesured t λ = 53 nm (nthocynins), nd 657 nm (chlorophyll degrdtion products) using Cecil spectrophotometer. The formul A A 657 ws used to compenste for the sorp-

3 The Impct of Short-Term Exposure tion of chlorophyll degrdtion products t 53 nm. Anthocynin content ws clculted s cynidin-3-glucoside, whose moleculr extinction coefficient equls 29,6. Anlyses were crried out for three independent replictes for hypocotyls nd cotyledons seprtely. Determintion of Flvonoids Full detils of flvonoid nlysis were descried erlier [41]. Briefly, freeze-dried smples of uckwhet tissues were extrcted y soniction with mixture of 6% methnol nd.4% trifluorocetic cid. The extrction ws repeted five times. Pooled extrcts were centrifuged nd directly tken to HPLC nlysis. A HPLC system equipped with mm i.d. Cdenz CD-C 18 3 μm column, nd UV-Vis detector set t 35 nm. The flvonoids were eluted in grdient system composed of solvents A (wter/cetonitrile/formic cid, 89:6:5) nd B (wter/cetonitrile/formic cid, 15:8:5). Identifiction of flvonoids y compring the retention times to the ville stndrds ws done. For clculting flvonoid content we used commercil stndrds, except for quercetin-glctosyl-rhmnoside, the contents of which were clculted sed on the stndrd of the rutin. The quercetin-glctosyl-rhmnoside ws identified s descried erlier [41]. Sttistics The results confirm erlier findings tht root growth is more sensitive to hevy metls thn shoots [17]. Only growth elongtion of shoots of Kor cv. ws significntly lower t p<.1. In seedlings of Hruszowsk the elongtion of shoots ws significntly lower t p<.5, nd in the cse of Lu the elongtion decline ws not significnt, even t p<.5. Reduced toxicity of hevy metls to the shoots due to the fct tht plnts hve evolved mechnisms to limit the trnsport of metl ions to the eril prts [6, 7]. It is well known tht the hevy metl ions cn ound to the cell wlls in root cells. Led ions exhiit the high ffinity to the croxyl nd hydroxyl groups of the polyscchrides, which re the min components of cell wlls [5]. For P, the process of inding to cell wll is one of the mjor mechnisms of detoxifiction [45]. It seems tht our results indicted much lower sensitivity uckwhet shoots thn roots to led ions, confirming these findings. Buckwhet tissues re well known s rich source of flvonoids, like glycosides of flvonols nd flvones, s well s nthocynins [41, 43]. Young seedlings of common uckwhet rpidly egin ccumulting nthocynins when exposed to light [46]. Accumultion of nthocynins in young seedlings is stimulted y vrious environmentl stresses [47]. Exposure to stress cused y excess P lso resulted in n incresed ccumultion of nthocynins in seedlings of ll uckwhet cultivrs (Fig. 2). Generlly, the content of nthocynins in the cotyledons nd hypocotyl of In the cse of nthocynins one-wy nlysis of vrince complemented y the lest significnt difference ws used with 5% level of proility (Newmn-Keuls test). In the cse of seedling elongtion nd flvonoid levels, sttisticl nlyses were chieved with the t-test, nd significnt differences in reltion to the control plnts were indicted with *p<.5 nd p<.1. A) Elongtion [ mm ] A * Results nd Discussion Common uckwhet is recognized s P hyperccumultor [8]. It ws found tht uckwhet ccumultes P in the shoots rther thn in the roots. The mechnisms involved in P hyperccumultion in uckwhet shoots remin unknown. Buckwhet is lso le to ccumulte lrge mount of luminum (Al) in the shoots [44]. In Al ccumultion orgnic cids ply n importnt role. There is suggestion tht the orgnic cids my lso ply role in P tolernce nd ccumultion in uckwhet tissues [8]. During our studies low doses of P (.1 mm) in nutrient solution hve stimulted shoot nd root growth of seedlings of ll uckwhet cultivrs, lthough the elongtion ws not sttisticlly significnt (Fig. 1). A similr phenomenon ws lso descried for seedlings of Pinus pine nd Pinus pinster [2]. A high P dose (1. mm) cused the elongtion growth of roots nd shoots to slow down. In the cse of roots the growth inhiition ws quite cler nd sttisticlly significnt in comprison to roots of the control seedlings, t p<.1. Buckwhet seedling root growth ws more sensitive to P tretment compred to tht of shoots. B) Elongtion [ mm ] B Hruszowsk Lu Kor Control.1 mm 1. mm Fig. 1. Effect of 3-dy tretment y low nd high doses of P 2+ in nutrient solution on elongtion of shoots (A) nd roots (B) in seedlings of three uckwhet cultivrs. Mens mrked with one sterisk nd two sterisks were considered sttisticlly significnt in comprison to control t p<.5 nd p<.1, respectively. Lck of sterisk mens no significnt difference in comprison to control. Sttisticl clcultions were crried out for ech cultivr seprtely.

4 1726 Horowicz M., et l. uckwhet ws higher under high P doses (1. mm) rther thn low (.1 mm), nd much higher thn tht in the control seedlings. Among the studied cultivrs of uckwhet, P hd the lest impct on the content of nthocynins in hypocotyls of Kor cv. There is well known reltionship etween exposure to hevy metls nd ccumultion of nthocynins [48, 49]. In red mple (Acer rurum) grown in soil with the ddition of P 2+ the mount of nthocynins in the leves incresed nerly four-fold [5]. Incresed synthesis of nthocynins my e relted to their role in reducing the toxicity of hevy metls on plnts. According to Hle et l. [51], nthocynins cn protect cells from the dmging effects of hevy metls y the formtion of stle complexes with them. After the sorption into plnt tissues, hevy metls cn e chelted in the cytosol, nd finlly llocted within the vcuole [52, 53]. The vcuole is one of the mjor cellulr comprtments involved in the mintennce of tolernce to the incresed mount of hevy metls [52]. As regrds the effects of Cd on plnt development, it induces visile symptoms of phytotoxicity, such s reduced growth [54]. In present studies, unlike in the cse of P, low Cd concentrtions (.1 mm) inhiited elongtion of shoots nd roots of Hruszowsk nd Kor seedlings (Fig. 3). Higher sensitivity of roots nd shoots to cdmium toxicity, compred to led, ws noted erlier for whet nd other plnts [18, 19]. Seedlings of Lu were more resistnt A) Anthocynins [ g x g -1 ] B) Anthocynins [ g x g -1 ] A Hruszowsk Lu Kor B Control.1 mm 1. mm Fig. 2. Effect of 3-dy tretment with low nd high doses of P 2+ in nutrient solution on nthocynin content (on fresh weight sis) in seedling cotyledons (A) nd hypocotyl (B) of three common uckwhet cultivrs. Brs (mens +SD) mrked y the sme letter(s) re not significntly different using Newmn-Keuls test t 5% level of proility. Sttisticl clcultions were crried out for ech cultivr seprtely. A) Elongtion [ mm ] B) Elongtion [ mm ] to this concentrtion of Cd ions, lthough some, (not sttisticlly significnt), inhiition of growth occurred. Significntly greter inhiition of root nd shoot growth ws cused y high dose Cd (1 mm). In the cse of Kor, inhiition of root growth ws lmost complete, nd in the cse of ll cultivrs shoots reched 4-5% s compred to the control seedlings (Fig. 3). Buckwhet elongs to Phyperccumultor plnts, nd therefore hs efficient mechnism of P-detoxifiction [8]. It is possile tht this mechnism lso plys role in flvonoids. The negtive effect of cdmium on plnt growth nd development is well known [1]. Our results confirm strong inhiition of plnt growth y Cd reviewed y Benvides et l. [11] nd recently descried for soyen [55]. Among tested uckwhet cultivrs it hs een found tht Kor seedlings were most susceptile to the toxic effects cused y Cd 2+. Even low dose of those ions (.1 mm) cused growth inhiition of roots nd shoots of the Kor seedlings, which ws significntly lower compred to control t p<.1 (Fig. 3). Also, growth of Hruszowsk seedlings were significntly inhiited y oth low nd high concentrtions of Cd 2+, lthough root elngtion of the seedlings for the 1 mm concentrtion of Cd ws lmost three times higher thn for the roots of the Kor (Fig. 3). Similrly to P, the content of nthocynins in the cotyledons nd hypocotyl of uckwhet ws higher under the high dose Cd (1. mm) thn low (.1 mm), nd much A B Hruszowsk Lu Kor Control.1 mm 1. mm Fig. 3. Effect of 3-dy tretment y low nd high doses of Cd 2+ in nutrient solution on elongtion of shoots (A) nd roots (B) in seedlings of three uckwhet cultivrs. Mens mrked with two sterisks were considered sttisticlly significnt in comprison to control t p<.1. Lck of sterisk mens no significnt difference in comprison to control. Sttisticl clcultions were crried out for ech cultivr seprtely.

5 The Impct of Short-Term Exposure higher thn tht in the control seedlings. Exposure to excess Cd (1. mm) resulted in much higher incresed ccumultion of nthocynins in Hruszowsk seedlings in comprison to P tretment (Figs. 2 nd 4). In studied uckwhet cultivrs, Cd hd smller impct on the content of nthocynins in tissues of Kor nd Lu, thn in Hruszowsk. In the rection of lef tissues to low concentrtions of Cd 2+ nd P 2+, n increse in levels of ll uckwhet flvonoids (glycosides luteolin, pigenin, nd quercetin) ws noted (Tles 1 nd 2). A prticulrly lrge ccumultion ws oserved in the cse of rutin. Stimultion of flvonoid iosynthesis ws noted in previous studies [33, 37, 38]. Recently, it lso ws found tht ctivity of crucil enzyme in flvonoids iosynthesis nd phenylmmoni lyse (PAL), ws incresed in roots of soyen (Glycine mx) nd lupine (Lupinus luteus) seedlings treted with Cd 2+ nd P 2+ [23]. Our results correspond with these findings. When compring the uckwhet cultivrs, it my e noted tht the high content of flvonoids seems to e ssocited with higher resistnce of the seedlings to P nd Cd (Tles 1 nd 2). Totl content of flvonoids in cotyledons of Hruszowsk nd Lu ws c. 1.5-times higher thn in Kor. The low level of flvonoids in Kor cotyledons ws ccompnied y sustntil inhiition of root elongtion. Shoot elongtion of the Kor seedlings ws lso lower thn in the cse of Hruszowsk nd Lu. Thus, A) Anthocynins [ g x g -1 ] B) Anthocynins [ g x g -1 ] c. Hruszowsk Lu Kor Fig. 4. Effect of 3-dy tretment with low nd high doses of Cd 2+ in nutrient solution on nthocynin contents (on fresh weight sis) in seedling cotyledons (A) nd hypocotyl (B) of three common uckwhet cultivrs. Brs (mens +SD) mrked y the sme letter(s) re not significntly different using Newmn-Keuls test t 5% level of proility. Sttisticl clcultions were crried out for ech cultivr seprtely. A B Control.1 mm 1. mm Tle 1. Effect of 3-dy tretment y low nd high doses of P 2+ in nutrient solution on flvonoids levels (mg g -1 DW) in cotyledons of uckwhet seedlings. The dt were nlyzed y onewy nlysis of vrince for ech flvonoid seprtely. Mens mrked with one sterisk nd two sterisks were considered sttisticlly significnt in comprison to control t p<.5 nd p<.1, respectively. Lck of sterisk mens no significnt difference in comprison to control. NA not nlyzed. Flvonoid Control.1 mm 1. mm Hruszowsk Orientin Iso-orientin * Vitexin * 5.54 Iso-vitexin Rutin * 8.95* Totl * 54.4 Lu Orientin 6.6 NA 5.88 Iso-orientin NA 19.7 Vitexin 5.8 NA 4.89 Iso-vitexin NA 1.96* 2.2 NA 2.3 Rutin 9.58 NA 1.13 Totl NA Kor Orientin Iso-orientin * Vitexin Iso-vitexin Quercetin-glctosylrhmnoside Quercetin-glctosylrhmnoside Quercetin-glctosylrhmnoside * Rutin * Totl * 34.97* the detoxifiction mechnism in the uckwhet is proly relted lso to flvonoid levels. The enhnced content of flvonoids proly is responsile for inding of metl ions, nd lso for their deposition within the vcuole [24]. In previous study the level of flvonols ws incresed in primry leves of Phseolus coccineus under hevy metl tretment, nd the level depended on the metl nd its concentrtion [26]. These results otined in our study indicte tht in rection to Cd nd P they cn lso involve other mechnisms, such s the ccumultion of flvonoids,

6 1728 Horowicz M., et l. Tle 2. Effect of 3-dy tretment y low nd high doses of Cd 2+ in nutrient solution on flvonoids levels (mg g -1 DW) in cotyledons of uckwhet seedlings. The dt were nlyzed y one-wy nlysis of vrince for ech flvonoid seprtely. Mens mrked with one sterisk were considered sttisticlly significnt in comprison to control t p<.5. Lck of sterisk mens no significnt difference in comprison to control. Flvonoid Control.1 mm 1. mm Hruszowsk Orientin * Iso-orientin * 12.79* Vitexin * Iso-vitexin * Rutin * 6.99 Totl * 38.63* Lu Orientin Iso-orientin * Vitexin Iso-vitexin * Rutin * 8.26* Totl Kor Orientin * Iso-orientin * 1.27* Vitexin * 4.54* Iso-vitexin * 9.34* Quercetin-glctosylrhmnoside Quercetin-glctosylrhmnoside Quercetin-glctosylrhmnoside Rutin * 6.54 Totl * 35.18* which re metl cheltors, nd cn lso rect with rective oxygen species, converting the peroxyl rdicl into highly stilized rdicl [56]. Significnt correltions were found etween the levels of orientin nd iso-orientin, s well s vitexin nd iso-vitexin in the cotyledons of uckwhet (r=.9944 nd r=.9742, respectively). Also, significnt correltion ws found etween totl content of oth C-glucosides of luteolin nd pigenin (r=.9133). It proly mens tht iosynthesis of these flvonoids involves the sme enzyme, or set of enzymes. No significnt correltion ws found etween the content of oth quercetin glycosides: rutin nd quercetinglctosyl-rhmnoside (r=.2733). Conclusions Low concentrtions of oth P 2+ nd Cd 2+ dded to nutrient solution resulted in smll stimultion of the growth of seedlings of ll studied cultivrs of common uckwhet, while higher doses inhiit root growth, nd to much lesser extent, of shoots. Cd 2+ ions were more hrmful for growth of uckwhet seedlings thn P 2+ ions. More resistnt to stress cused y the presence of high concentrtions of P 2+ nd Cd 2+ in the growth medium ws seedlings of Hruszowsk nd Lu cultivrs, in comprison to seedlings of Kor. Cotyledons of more resistnt cultivrs (Hruszowsk nd Lu) contined much more flvonoids thn cotyledons of less resistnt seedlings of Kor. It suggests tht the detoxifiction mechnism of led nd cdmium in uckwhet seedlings proly involve flvonoids. To the est of our knowledge, this is the first report uncovering the impct of genetic vriility of flvonoid contents within seedlings of vrious uckwet genotypes on the ility to detoxify led nd cdmium ions. This hypothesis, however, requires further studies, which my explin the detiled mechnism of mentioned ion detoxifiction. Acknowledgements This project ws prtly founded y the Ministry of Agriculture nd Rurl Development of Polnd No. HORhn-81-6/12. References 1. KOCHIAN L. V., PENCE N. S., LETHAM D. L. D., PINEROS M. A., MAGALHAES J. V., HOEKENGA O. A., GARVIN D. F. Mechnisms of metl resistnce in plnts: luminum nd hevy metls. Plnt Soil 247, 19, COBBETT C. S. Phytocheltins nd their roles in hevy metl detoxifiction. Plnt Physiol. 123, 825, WOJAS S., CLEMENS S., HENNIG J., SKLODOWSKA A., KOPERA E., SCHAT H., BAL W., ANTOSIEWICZ D. M. Overexpression of phytocheltin synthse in tocco: distinctive effects of AtPCS1 nd CePCS genes on plnt response to cdmium. J. Exp. Bot. 59, 225, SHARMA P., DUBEY R. S. Led toxicity in plnts. Brz. J. Plnt Physiol. 17, 35, SEREGIN I. V., KOSEVNIKOVA A. D. Roles of root nd shoot tissues in trnsport nd ccumultion of cdmium, led, nickel, nd strontium. Russ. J. Plnt Physiol. 55, 1, HUANG J. W., CUNNINGHAM S. D. Led phytoextrction: species vrition in led uptke nd trnsloction. New Phytol. 134, 75, SHU X., YIN L., ZHANG Q., WANG W. Effect of P toxicity on lef growth, ntioxidnt enzyme ctivities, nd photosynthesis in cuttings nd seedlings of Jtroph curcs L. Environ. Sci. Pollut. Res. 19, 893, 212.

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