PHYSIOLOGICAL AND PROTEOMIC RESPONSES OF TOBACCO SEEDLINGS EXPOSED TO SILVER NANOPARTICLES

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1 PHYSIOLOGICAL AND PROTEOMIC RESPONSES OF TOBACCO SEEDLINGS EXPOSED TO SILVER NANOPARTICLES Rent Bi Deprtment of Biology, Fculty of Science, University of Zgre

2 INTRODUCTION Nnoprticles (NPs) Silver nnoprticles (AgNPs) three dimensions etween 1 nd 1 nm dvnced electricl, chemicl nd physicl properties gret ntiteril nd ntifungl properties medicl ppliction nd devices, textiles, food pckging, helthcre nd household products toxic effects shown on teri, niml cells, lge nd plnts Model orgnism toco (Nicotin tum L.) seedlings Is AgNP toxicity relted to nnoprticle-specific effects or is it result of ionic silver relesed from AgNPs? Figure 1. TEM imge of AgNP suspension.

3 MATERIALS AND METHODS AgNPs/AgNO 3 Determintion of silver content ICP - MS Oxidtive stress prmeters 25 μm 5 μm 75 μm 1 μm ROS levels - DHE test lipid peroxidtion protein cronyl quntifiction DNA dmge - Comet ssy 1 μm Antioxidnt enzymes ctivites SOD, PPX, APX, CAT Microscopy nlyses AgNP locliztion morphologicl chnges - light microscopy ultrstructurl chnges - electron microscopy Proteome chnges 2-D electrophoresis MALDI TOF/TOF protein identifiction using NCBIprot

4 Ag (μg g -1 DW) SILVER CONTENT AgNP AgNO de cd de cd e cd Figure 1. Silver content in toco seedlings treted with AgNPs nd AgNO 3. Vlues re mens ± SE of three different experiments, ech with three replics. Vlues mrked with different letters represent significnt difference (p.5) ccording to Duncn test.

5 % til DNA ROS content s % of control MDA content (nmol g -1 fw) OXIDATIVE STRESS PARAMETERS A AgNP AgNO3 c B c d AgNP AgNO3 d C cronyl content (μmol mg -1 protein),9,8,7,6,5,4,3,2,1 AgNP AgNO3 d d cd d D AgNP AgNO3 e cd de cd Figure 2. Content of ROS (A), MDA (B), protein cronyl (C) nd % til DNA (D) in toco seedlings treted with AgNPs nd AgNO 3. Vlues re mens ± SE of three different experiments, ech with three replics. Vlues mrked with different letters represent significnt difference (p.5) ccording to Duncn test.

6 APX ctivity CAT ctivity SOD ctivity PPX ctivity ANTIOXIDANT ENZYME ACTIVITY A (U mg -1 protein) AgNP AgNO3 d d c B (μmol product min -1 mg -1 protein) AgNP AgNO3 C AgNP AgNO3 D AgNP AgNO3 (μmol product min -1 mg -1 protein),14,12,1,8,6,4,2 c (μmol H 2 O 2 min -1 mg -1 protein),3,25,2,15,1,5 c Figure 3. Specific ctivities of SOD, PPX, APX nd CAT in toco seedlings treted with AgNPs nd AgNO 3. Vlues re mens ± SE of three different experiments, ech with three replics. Vlues mrked with different letters represent significnt difference (p.5) ccording to Duncn test.

7 MICROSCOPY ANALYSES Morphologicl chnges A AgNP locliztion B Figure 4. AgNP locliztion in the root cells of the 1 um AgNP-treted toco seedlings (A) nd right field imge (B). Ultrstructurl chnges Figure 5. Root length of toco seedlings (A). Semithin sections of root from control (B), 1 μm AgNP-treted (C) nd 1 μm AgNO 3 -treted (D) toco seedlings (r = 33.1 μm) nd lef from control (E), 1 μm AgNP-treted (F) nd 1 μm AgNO 3 -treted (F) toco seedlings (r = 3.6 μm). RC root cp, AP picl meristem, RE region of elongtion, EP epidermis, UE upper epidermis, LE lower epidermis, PP plisde prenchym, SP spongy prenchym. Pehrec Štefnić et l. / Chemosphere 29 (218) Figure 6. Ultrstructure of root cells nd lef chloroplsts. Root cells of control (A), 1 μm AgNP-treted (B) nd 1 μm AgNO 3 - treted (C) toco seedlings (r = 2 μm). Chloroplsts in lef cells of control (D), 1 μm AgNP-treted nd 1 μm AgNO 3 - treted toco seedlings (r = 1 μm). N nucleus, V vcuole, Mt mitochondrion, Pt plstid, PG plstogloules.

8 PROTEOMIC CHANGES A B C Figure 7. Functionl ctegoriztion of the differentilly expressed proteins of toco seedlings treted with 1 μm AgNPs (A) nd 1 μm AgNO 3 (B). Anlysis of responsiveness of differentilly expressed proteins of toco seedlings exposed to AgNPs nd AgNO 3 (C).

9 CONCLUSIONS higher Ag content ws mesured in seedlings exposed to AgNPs compred to AgNO 3 of the sme concentrtion otined results on oxidtive stress prmeters reveled tht in generl higher toxicity ws recorded in AgNO 3 -treted seedlings compred to those exposed to nnosilver presence of silver in the form of nnoprticles ws confirmed in the root cells, which my explin the lower toxicity of AgNPs proteomic study showed tht oth AgNPs nd AgNO 3 cn ffect photosynthesis, nd tht is in correltion with the oserved ultrstructurl chnges of chloroplsts mjority of the proteins involved in the primry metolism were upregulted fter oth types of tretments, indicting tht enhnced energy production, which cn e used to reinforce defensive mechnisms, enles plnts to cope with silver-induced toxicity

10 ACKNOWLEDGEMENTS Plnt proteomics l: Biljn Blen Petr Pehrec Štefnić Petr Cvjetko Mirt Tklec Assocites: An-Mrij Domijn Ilse Letofsky-Ppst Sndr Šikić Mrio Cindrić