Sunflower Plants as Bioindicators of Environmental Pollution with Lead (II) Ions

Senso s 2009, 9, 5040-5058; doi:10.3390/s90705040 senso s ISSN 1424-8220 www.mdpi.com/jou nal/senso s A icle Sun lowe Plan s as Bioindica o s o En i onmen al Pollu ion wi h Lead (II) Ions Olga K ys o o a 1, Viole a Shes i ska 1,2, Michaela Galio a 3, Ka el No o ny 3, Joze Kaise 4, Jose Zehnalek 1, Pe Babula 5, Radka Opa ilo a 5, Voj ech Adam 1,6 and Rene Kizek 1,* 1 Depa men o Chemis y and Biochemis y, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 2 Depa men o Plan Biology, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic 3 Depa men o Chemis y, Facul y o Science, Masa yk Uni e si y, Ko la ska 2, CZ-611 37 B no, Czech Republic 4 Ins i u e o Physical Enginee ing, Facul y o Mechanical Enginee ing, B no Uni e si y o Technology, Technicka 2896/2, CZ-616 69 B no, Czech Republic 5 Depa men o Na u al D ugs, Facul y o Pha macy, Uni e si y o Ve e ina y and Pha maceu ical Sciences, Palackeho 1-3, CZ-612 42 B no, Czech Republic 6 Depa men o Animal Nu i ion and Fo age P oduc ion, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e and Fo es y, Zemedelska 1, CZ-613 00 B no, Czech Republic * Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]uni.cz Recei ed: 31 May 2009; in e ised o m: 22 June 2009 / Accep ed: 24 June 2009 / Published: 25 June 2009 Abs ac : In his s udy, he in luence o lead (II) ions on sun lowe g ow h and biochemis y was in es iga ed om a ious poin s o iew. Sun lowe plan s we e ea ed wi h 0, 10, 50, 100 and/o 500 µM Pb-EDTA o eigh days. We obse ed al e a ions in g ow h in all expe imen al g oups compa ed wi h non- ea ed con ol plan s. Fu he we de e mined o al con en o p o eins by a B ad o d p o ein assay. By he eigh h day o he expe imen , o al p o ein con en s in all ea ed plan s we e much lowe compa ed o con ol. Pa icula ly no iceable was he loss o app ox. 8 µg/mL o 15 µg/mL in shoo s o oo s o plan s ea ed wi h 100 mM Pb-EDTA. We also ocused ou a en ion on he ac i i y o alanine ansaminase (ALT), aspa a e ansaminase (AST) and u ease. Ac i i y o he enzymes inc eased wi h inc easing leng h o he ea men and applied concen a ion OPEN ACCESS Senso s 2009, 9 5041 o lead (II) ions. This inc ease co esponds well wi h a highe me abolic ac i i y o ea ed plan s. Con en s o cys eine, educed glu a hione (GSH), oxidized glu a hione (GSSG) and phy ochela in 2 (PC2) we e de e mined by high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion. Cys eine con en declined in oo s o plan s wi h he inc easing ime o ea men o plan s wi h Pb-EDTA and he concen a ion o oxic subs ance. Mo eo e , we obse ed en imes highe con en o cys eine in oo s in compa ison wi h shoo s. The obse ed educ ion o cys eine con en p obably ela es wi h i s u iliza ion o biosyn hesis o GSH and phy ochela ins, because he con en o GSH and PC2 was simila in oo s and shoo s and inc eased wi h inc eased ea men ime and concen a ion o Pb- EDTA. Mo eo e , we obse ed oxida i e s ess caused by Pb-EDTA in oo s whe e he GSSG/GSH a io was abou 0.66. In shoo s, he oxida i e s ess was less dis inc i e, wi h a GSSG/GSH a io 0.14. We also es ima ed he a e o phy ochela in biosyn hesis om he slope o linea equa ions plo ed wi h da a measu ed in he pa icula expe imen al g oup. The highes a e was de ec ed in oo s ea ed wi h 100 µM o Pb-EDTA. To de e mine hea y me al ions many analy ical ins umen s can be used, howe e , mos o hem a e only able o quan i y o al con en o he me als. This p oblem can be o e come using lase induced b eakdown spec oscopy, because i is able o p o ide a high spa ial-dis ibu ion o me al ions in di e en ypes o ma e ials, including plan issues. Da a ob ained we e used o assemble 3D maps o Pb and Mg dis ibu ion. Dis ibu ion o hese elemen s is concen a ed a ound main ascula bundle o lea , which means a ound mid ib. Keywo ds: phy o emedia ion; hea y me als; sun lowe ; lead ions; high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion; spec ome y; lase induced b eakdown spec oscopy 1. In oduc ion En i onmen al emedia ion deals wi h he emo al o con aminan s om soil, g oundwa e , sedimen , su ace wa e e c. o he gene al p o ec ion o human heal h and he en i onmen [1]. Remedia ion p ocesses can be expensi e, as he a e mos ly ex-si u me hods in ol ing exca a ion o impac ed soils and subsequen ea men a he su ace. The e o e new, e icien , inexpensi e and non- en i onmen ally dis up i e echnologies a e s ill de eloping. One o he g oups o such new echnologies is called bio emedia ion. I in ol es he ea men o en i onmen al p oblems h ough o ganisms. Mic oo ganisms (e.g., Desul omonile, Clos idium, Pseudomonas, Acine obac e ) and plan s (e.g., Be ula, Populus) a e mos commonly used o hese pu poses [2-7]. I plan s a e used, we call his p ocess phy o emedia ion [1,8-10]. A ange o p ocesses media ed by plan s a e use ul in ea ing en i onmen al p oblems. Plan s can chemically modi y oxic subs ances as a di ec esul o plan me abolism (phy o ans o ma ion), can educe he mobili y o subs ances in he en i onmen (phy os abiliza ion) o up ake and concen a e subs ances om he en i onmen in o he plan biomass (phy oex ac ion). The scheme o a ious ways how o a plan me abolizes o deposi he pollu an is shown in Figu e 1. Senso s 2009, 9 5042 Figu e 1. Phy o emedia ion can occu h ough a se ies o complex in e ac ions be ween plan s, mic obes, and he soil, including accumula ion, hype accumula ion, exclusion, ola iliza ion, and deg ada ion o he a ge pollu an . Plan s also s abilize mobile con amina ed sedimen s by o ming dense oo ma s inside soil. Vola iliza ion T anspi a ion Ino ganics O ganics Accumula ion Hea y me als, Radionuclides, Me aboli es Rhizosphae ic me abolism Me als, O ganics, Radionuclides Hea y me als, Radionuclides Chlo ina ed hyd oca bons Me cu y, Selenium Vola iliza ion T anspi a ion Ino ganics O ganics Accumula ion Hea y me als, Radionuclides, Me aboli es Hea y me als, Radionuclides, Me aboli es Rhizosphae ic me abolism Me als, O ganics, Radionuclides Hea y me als, Radionuclides Chlo ina ed hyd oca bons Me cu y, Selenium Mos oxic subs ances (o ganic pollu an s, hea y me als) come om an h opogenic ac i i ies such as mining, a ic, hea y indus y, e c. [11-13]. Con a y o o ganic pollu an s, hea y me als canno be deg aded o des oyed. To a small ex en hey en e ou bodies ia ood, d inking wa e and ai . As ace elemen s, some hea y me als (e.g., coppe , selenium, zinc) a e essen ial o main ain he me abolism o he human body. Howe e , o he s such as cadmium, lead, and me cu y a e oxic a all. A highe concen a ions bo h g oups o hea y me als ( oxic and essen ial) can lead o poisoning [13]. Hea y me als a e also dange ous because hey end o bioaccumula e. Lead is one o he mos dange ous and oxic hea y me als. Le els o lead in he en i onmen a e no s able and a y acco ding o indus ial p oduc ion, u baniza ion, clima e changes and many o he ac o s [14]. The le els o lead in he en i onmen a y be ween 4 and 20 mg/g o dus . Uncon amina ed wa e s con ain lead in concen a ions anging om 0.001 o 0.06 mg/L. In soils, le els o lead each 5 o 30 mg pe kg o soil. When lead is added in o pe ol as an addi i e, he highes lead le els a e de e mined on he su aces o lea es, om whe e lead en e s he ood chain, as well as soil o wa e . Lead is p esen in soils as sal s in soluble as well as insoluble o ms. Lead con amina ion in he soil is known o inhibi seed ge mina ion [15,16]. The inhibi ion o ge mina ion by exogenously supplied Pb2+ is a possible e ec o in e e ence wi h some impo an enzymes in ol ed in he p ocess. Pho osyn hesis is conside ed as one o he me abolic p ocesses mos sensi i e o Pb2+ oxici y [17]. Closing o he s oma a, dis up ion o he chlo oplas ic o ganiza ion, change in he me aboli es o pho osyn hesis and eplacemen o essen ial ions like magnesium a e he main e ec s on pho osyn hesis o lead oxici y [14,18-21]. The Senso s 2009, 9 5043 me al has also been epo ed o inhibi pho osyn hesis in isola ed chlo oplas s. The e ha e been also published da a epo ing on inhibi ion o enzymes c ucial o ni ogen assimila ion [14]. Figu e 2. Expe imen al a angemen o LIBS: 1 – Nd:YAG lase , 2 – modula o o second ha monic equency, 3 – pe iscope, 4 – CCD came a, 5 – abla ion chambe , 6 – ib e op ic sys em, 7 – monoch oma o , 8 – ICCD came a. As we men ioned abo e o he pa icula example o lead ions, he e a e many mechanisms and pa hways which can be a ec ed by hea y me als [22-25]. P o ec i e mechanisms o a plan cell agains he oxic e ec s o hea y me als mainly in ol e syn hesis o compounds ich in cys eine called phy ochela ins. Thei syn hesis comes om he mos abundan hiol – educed glu a hione. To de ec hese compounds many a ious me hods and echniques ha e been employed [23,24,26-30], including senso s and biosenso s [31-33]. Howe e , up ake and anspo as well as s o age o hea y me als h ough plan issues emain s ill unclea . To conside whe he a speci ic plan species is able o no able o emedia e he pollu ed en i onmen , no only hea y me als con en in he plan issues, bu also he dis ibu ion o such me al ions in he issues mus be analysed. Analy ical me hods and ins umen s o de ec ion o lead (II) ions ha e been e iewed se e al imes [34-38]. The diagnos ic echniques enabling moni o ing high spa ial- and la e al-dis ibu ion o elemen s wi hin di e en plan s uc u es include mainly X- ay imaging me hods [39-41]. X- ay mic oscopy and mic o- adiog aphy in es iga ions usually make use o so X- ays gene a ed by plasma lase , mic o ocus X- ay sou ces and synch o on adia ion [42]. Al hough he X- ay adia ion based me hods a e ela i ely high-cos and a ailabili y o he expe imen al appa a us is limi ed due o possibili y o “in-si u” analysis only, i o e s new aspec s o s udying he dis ibu ion o hea y me als. Howe e , X- ay imaging me hods a e in ensi ely in es iga ed in ou labo a o ies; ecen ly we ha e been ocusing also on he ealiza ion o spa ially- esol ed spec o-chemical analysis by u ilizing lase -abla ion based echniques. Lase induced b eakdown spec oscopy (LIBS, Figu e 2) is a ype o a omic emission spec oscopy which u ilises a highly ene ge ic lase pulse as he exci a ion sou ce and is able o p o ide high spa ial- dis ibu ion o me al ions in di e en ypes o ma e ials [43-45]. The cha ac e o he abla i e p ocess Senso s 2009, 9 5044 depends on he ea u es o he lase used (wa eleng h, pulse du a ion, powe and ene ge ic p o ile o he ays), su ounding a mosphe e and he ea u es o he sample i sel (ma ix, abso p ion cha ac e is ics, i s s uc u e) [46]. LIBS me hod is one o analy ical ins umen s which makes quali a i e and quan i a i e analysis and also moni o ing o elemen dis ibu ion in di e en ypes o samples possible. The main ad an age o his me hod is ha i equi es no, o minimal sample p e- ea men and enables mul i-elemen al analysis wi h high h ee-dimensional esolu ion. A limi ing ac o is especially he diame e o lase ay. In his s udy, he in luence o lead (II) ions on sun lowe plan s (Figu e 3) was in es iga ed om a ious poin s o iew. We aimed ou s udy a common g ow h pa ame e s, mo phological changes, o al p o ein con en , ac i i y o ce ain enzymes, le el o s ess induced hiols and spa ial dis ibu ion o lead. Figu e 3. Pic u es o sun lowe plan s in he second, six h and eigh h expe imen al day a e Pb-EDTA applica ion (0, 10, 50, 100 and 500 µM). 2nd day 0 µM 10 µM 50 µM 100 µM 500 µM 6 h day 8 h day 10 cm Senso s 2009, 9 5045 2. Resul s and Discussion 2.1. Mo phological changes Lead is a poisonous me al ha has many ad e se e ec s on plan s and animals. Sun lowe plan s we e ea ed wi h 0, 10, 50, 100 and/o 500 µM Pb-EDTA o eigh days. We obse ed al e a ions in g ow h in all expe imen al g oups compa ed wi h non- ea ed con ol plan s. Plan s exposed o lead (II) ions g ew as e in compa ison wi h con ol plan s, excep o he highes applied concen a ion. This phenomenon p obably ela es o he s imula o y e ec s o he p esence o Pb-EDTA, because con ol plan s we e cul i a ed in dis illed wa e only, whe e no o he nu ien s a e p esen . In addi ion we obse ed chlo osis on plan s ea ed wi h he highes concen a ion (Figu e 3). When we compa ed he esh weigh o plan s ea ed wi h lead (II) ions wi h he non- ea ed expe imen al g oup, i was possible o clea ly no ice he e ec o applied Pb-EDTA concen a ion on he ae ial pa s o plan s, excep o he highes applied concen a ion (Figu e 4 A). Figu e 4. Changes o (A) esh and (B) d y weigh s o sun lowe plan s exposed o Pb- EDTA. D y mass was ob ained by d ying o he cons an weigh a 105 °C in he o en. All da a we e ob ained by sub ac ion om con ol plan s. The expe imen was ca ied in iplica es. AB Leng h o ea men (d) Leng h o ea men (d) -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 2468 -0.025 -0.020 -0.015 -0.010 -0.005 0.000 0.005 0.010 2468 0.0 0.1 0.2 0.3 0.4 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Shoo Roo -0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 D y weigh (g) F esh weigh (g) Shoo Roo 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Senso s 2009, 9 5046 Ne e heless, he ad e se e ec o lead (II) ions is shown on dependence o d y weigh on leng h o he ea men and applied concen a ion (Figu e 3B). De e mined change is p obably connec ed wi h inc eased wa e up ake o plan s exposed o s ess caused by hea y me als. This hypo hesis is suppo ed by esul s epo ing on nuclea magne ic esonance analysis o ea ly soma ic emb yos clus e s [29]. In he case o change in esh weigh o oo s, inc eases by he six h and eigh h day we e de ec ed, excep o he highes applied concen a ion. D y weigh o oo s dec eased, excep on he second day o he ea men o all expe imen al g oups (Figu e 4 A,B). 2.2. To al p o ein con en Hea y me als aken up by plan s o induce s ess eac ions, which mani es as enhancemen s o he le els o ce ain molecules. Fi s ly, he le el o mRNA is enhanced wi h subsequen changes in p o ein p o ile. The e o e, we de e mined o al con en o p o eins by a B ad o d p o ein assay. To al con en o p o eins sligh ly inc eased in bo h ae ial pa s and oo s in he second day. F om he ou h day o he ea men , a dec ease o o al con en o p o eins occu ed. In eigh h day o he expe imen his loss was app ox. 8 µg/mL o 15 µg/mL in shoo s o oo s o plan s ea ed wi h 100 mM Pb-EDTA (Figu e 5). To al con en o p o eins is d ama ically educed hanks o he hea y me al ions. This end ma ches well wi h he d y weigh dependence (Figu es 4 and 5). Figu e 5. Changes in o al p o ein con en in sun lowe plan s exposed o Pb-EDTA. All da a we e ob ained by sub ac ion om con ol plan s. The expe imen s we e ca ied ou in iplica e. Leng h o ea men (d) -20 -15 -10 -5 0 5 10 2468 -10 -8 -6 -4 -2 0 2 To al p o ein con en (mg/ml) Shoo Roo 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ Senso s 2009, 9 5047 2.3. De e mina ion o plan enzymes’ ac i i y The e is s ill no much a ailable in o ma ion abou he signi icance o some commonly analyzed enzymes as ma ke s o s ess eac ions in plan s. In se e al pape s, we ha e demons a ed ha some enzymes (such as amino ans e ases o u ease) can pa icipa e in plan s ess eac ions [22,26,47-51]. Thus, we ocused ou a en ion on he ac i i y o alanine ansaminase (ALT), aspa a e ansaminase (AST) and u ease. T ansaminases ca alyze he ans e o he amino g oups o amino acids o 2-oxo- acids. In plan s, ansaminases pa icipa e e y e ec i ely in ans o ma ions o ni ogen compounds. They a e impo an o he syn hesis o amino acids om oxo-acids in he ci a e cycle and o o he c ucial biochemical pa hways. They also play key oles in he syn hesis o seconda y me aboli es as well as chlo ophyll. In oo s AST and ALT ac i i ies we e inc eased du ing he expe imen s in compa ison o con ol plan s (Figu e 6). This inc ease co esponds well wi h he highe me abolic ac i i y. U ease ac i i y was enhanced in ae ial plan pa s as well as in oo s wi h inc easing leng h o exposi ion and applied concen a ion sligh ly (da a no shown). Figu e 6. Changes o AST and ALT ac i i ies in sun lowe plan s exposed o Pb-EDTA. All da a we e ob ained by sub ac ion om con ol plan s. The expe imen was ca ied in iplica es. Leng h o ea men (d) Leng h o ea men (d) -0.8 -0.6 -0.4 -0.2 0 0 1 2 3 4 5 2 4 6 8 -10 -8 -6 -4 -2 0 AST Shoo -4 0 4 8 12 16 2468 Roo Ca aly ic ac i i y (µka /l) Shoo Roo ALT Ca aly ic ac i i y (µka /l) 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 2.4. Con en o low molecula mass hiols Low molecula mass compounds ich in cys eine moie ies play a e y impo an ole in he abili y o wi hs and o e en hype accumula e hea y me als ions. Due o his, we paid a en ion o such compounds. Pa icula ly, he con en s o cys eine, educed glu a hione (GSH), oxidized glu a hione Senso s 2009, 9 5048 (GSSG) and PC2 we e de e mined by high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion (Figu e 7). Con en s o cys eine di e ed ma kedly in shoo s and oo s. Cys eine con en declined in he oo s o plan s as he ime o he ea men o plan s wi h Pb-EDTA and concen a ion o oxic subs ance inc eased. Mo eo e , we obse ed en imes highe con en o cys eine in oo s in compa ison wi h shoo s. The obse ed educ ion o cys eine con en p obably ela es o i s u iliza ion o he biosyn hesis o GSH and phy ochela ins. Con en o GSH was simila in oo s and shoo s and inc eased wi h inc easing ime o he ea men and concen a ion o Pb-EDTA (Figu e 7). We plo ed he dependence wi h linea eg ession o es ima e he a e o syn hesis o GSH. The a e exp essed as he slope o he linea equa ion was 0.531x and 0.635x o oo s and shoo s, espec i ely, whe e “x” is concen a ion o applied Pb-EDTA. Figu e 7. Changes o cys eine, GSH, GSSG and PC2 con en s in sun lowe plan s exposed o Pb-EDTA. All da a we e ob ained by sub ac ion om con ol plan s. The expe imen was ca ied ou in iplica e. Cys Cys eine con en (µg/g) shoo oo GSH GSSG PC2 Reduced glu a hione con en (µg/g) Phy ochela in2 con en (µg/g) 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8 Leng h o ea men (d) Leng h o ea men (d) Leng h o ea men (d) Leng h o ea men (d) Oxidized glu a hione con en (µg/g) 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ 10 µM Pb 2+ 50 µM Pb 2+ 100µM Pb 2+ 500µM Pb 2+ I is a common knowledge ha hea y me als induce gene a ion o ee oxygen species, which subsequen ly damage cell compa men s (memb anes, nucleic acids). Oxygen adicals can be sca enged by a ious mechanisms inside a cell [52]. Low molecula mass hiols a e able o eac wi h oxygen adicals ia o ma ion o disulphides. One o he mos s udied and well known eac ions o such ype is he edox cycling o GSH in o GSSG [53]. In ou expe imen , GSSG le els g adually inc eased (Figu e 7). We obse ed oxida i e s ess caused by Pb-EDTA in oo s when he GSSG/GSH a io was abou 0.66. In shoo s, he oxida i e s ess was less dis inc i e, wi h a GSSG/GSH a io o Senso s 2009, 9 5055 15. Sawidis, T. E ec o cadmium on pollen ge mina ion and ube g ow h in Lilium longi lo um and Nico iana abacum. P o oplasma 2008, 233, 95–106. 16. Pandey, S.; Gup a, K.; Mukhe jee, A.K. Impac o cadmium and lead on Ca ha an hus oseus – A phy o emedia ion s udy. J. En i on. Biol. 2007, 28, 655–662. 17. Doume , S.; Lampe i, L.; Checchini, L.; Azza ello, E.; Mugnai, S.; Mancuso, S.; Pe uzzelli, G.; Bubba, M. 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