Life Cycle Approaches To Understand the Interactions between Crops And Engineered Nanoparticles at Molecular Level

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1 Life Cycle Approaches To Understand the Interactions between Crops And Engineered Nanoparticles at Molecular Level Achintya Bezbaruah Civil and Environmental Engineering North Dakota State University

2 Plant-Nanoparticle Interactions: Transdisciplinary Research at Molecular Level

3 Acknowledgements AFRI-NIFA USDA Grant# National Science Foundation Grant #CMMI Environmental and Conservation Sciences (ECS) Civil and Environmental Engineering Research Group Members and Collaborators

4 Engineered Nanomaterials (ENMs) Carbon Nanotubes (CNT) Fullerenes TiO 2 CeO 2 ZnO Ag Au SiO 2 Quantum Dots NZVI Ceria NM < 100 nm

5 Dangers(?) of ENMs Bioaccumulation Enter food chain ENMs behaves differently from bulk material Nanoparticles help in plant growth

6 Objectives Understanding the molecular level interactions of engineered nanomaterials (ENMs) with plants - Information about uptake and translocation of ZnO and CNT in crops - Genomic responses in plants exposed to ENMs - Modeling studies Relate experimental results to 1) Food security and Environment 2) Development of smarter ENMs Genomic Studies In vivo Studies Characterization Modeling In vitro Studies

7 In-vivo Studies

8 In-vivo Studies Analysis of CNT and ZnO Nanoparticle uptake by plant at macroscale level Effects of Nanoparticles on plant growth and performance

9 Ti µmol g-1 dry weight Evidence of Uptake Metal concentrations Proxy for metal nanoparticle uptake Not clear if indicates nanoparticle uptake or biotransformation surface + internal internal a b c Photo Carl Farmer 1 Aug 2002 Portree, SkyeCurly dock Jacob, Bezbaruah et al. Int. J. Phytoremediation, 15: , 2013 (0) (0.125) (0.375) Ti nanoparticles mg L-1 (mm Ti L -1 equivalent) 9

10 Ti µmol g -1 dry weight Evidence of Uptake Translocation to shoots Rumex crispus (curly dock) c b a Photo Carl Farmer 1 Aug 2002 Portree, Skye Curly dock Jacob, Bezbaruah et al. Int. J. Phytoremediation, 15: , (0) (0.125) (0.375) Ti nanoparticles mg L -1 (mmol Ti L -1 equivalent) 10

11 Iron Nanoparticle Uptake Experimental setup 11

12 Length, cm Bioavailability Results: Shoot and Root Lengths Roots Shoots 15 Control 1 Blank Spent NZVI 10 5 Control 1: All nutrients Blank: All nutrients but (PO 4 3- and Fe) Spent NZVI: All nutrients but (PO 4 3- and Fe) + Used NZVI after PO 4 3- adsorption Statistically significant 0 Control Blank Spent NZVI Almeelbi and Bezbaruah, under prep, 2013 Almeelbi and Bezbaruah, J. Nanopart. Res., 14:1-14,

13 Biomass, mg Bioavailability Results: Shoot and Root Biomass Roots Shoots 60 Control 1 Blank Spent NZVI Control Blank Spent NZVI Control 1: All nutrients Blank: All nutrients but (PO 4 3- and Fe) Spent NZVI: All nutrients but (PO 4 3- and Fe) + Used NZVI after PO 4 3- adsorption Statistically significant Almeelbi and Bezbaruah, under prep,

14 mg/kg-dry weight mg/kg-dry weight mg/kg-dry weight Bioavailability Elemental Analysis Fe Control Spent NZVI P Control Spent NZVI Stem Leaf 0 Stem Leaf Fe P All statistically significant Almeelbi and Bezbaruah, under prep, Control Spent NZVI

15 In-vitro Studies

16 In-vitro Studies Analysis of cell wall pore diameter in presence of NPs Understanding the mechanism of internalization of NPs Study of change in mechanical properties of plant cells Study of genetic changes in cells exposed to NPs

17 Allium Studies Onions (Allium cepa) are grown in a nanoparticle suspension Chromosomal abnormalities in root cell mitosis studied after 2 days Original Allium Test Aerated Allium Test

18 Normal Cells

19 ZnO NP Effects Multipolar anaphase Vagrant Chromosomes Sticky Metaphase

20 ZnO NP Effects Multipolar anaphase Vagrant Chromosomes Sticky Metaphase

21 SWCNT Effects Sticky metaphase Multipolar Anaphase Telophase without cytokinesis Chromosome fragments

22 SWCNT Effects Sticky metaphase Multipolar Anaphase Telophase without cytokinesis Chromosome fragments

23 SWCNT Effects Sticky Anaphase Micronucleus Disturbed metaphase

24 Summary Chromosomal abnormalities in root cells grown with CNT suspensions: Multipolar anaphase Micronucleaus Sticky anaphase Binucleated cells Sticky Metaphase Chromosomal fragments Nano-ZnO suspensions: Multipolar anaphase Sticky metaphase Vagrant chromosomes

25 Modeling Studies

26 Modeling Studies Application of molecular dynamics and simulations to understand DNA-NP interactions, cell wall protein-np interactions Relate experimental findings with simulation data

27 Objectives To understand how carbon nanotube affects plant DNA. To elucidate NP transport(s) in plants (root to leaves/seeds, leaves to roots)

28 Model Building DNA structure Rice Species: Oryza sativa subsp. japonica 48 bp used for model building Length = 17 nm Carbon nanotube structure VMD 1 nm diameter and 17 nm length

29 Initial Structures SWCNT = 2100 atoms DNA = 3070 atoms 48 bp used for model building Length = 17 nm

30 DNA-SWCNT Solvated Model after 0.2 ns (Room temperature and pressure) Solvated Molecules atoms

31 DNA-SWCNT Initial model SWCNT DNA After 0.2 ns (room temperature and pressure). Conformation of DNA has changed due to the presence of CNT. Water molecules have been removed for visualization purposes

32 DNA-SWCNT SWCNT DNA-SWCNT interaction after 0.2 ns (room temperature and pressure). DNA Water molecules have been removed for visualization purposes

33 Non-bonded Interaction Energy Non-bonded interactions between Non-bonded Interaction Energy (kcal/mol) With SWCNT(distance between DNA and SWCNT = 6Å) Without SWCNT (ELE) (VDW) (ELE+VDW) (ELE) (VDW) (ELE+VDW) Water and DNA Water and ADE Water and CYT Water and THY Water and GUA Interactions between DNA and water have changed due to change in number of water atoms near DNA

34 Conclusions Molecular model of rice DNA-SWCNT hybrid is build. Change in DNA conformation indicates interaction between DNA and SWCNT in the presence of SWCNT. Interactions between DNA and water have changed due to change in number of water atoms near DNA

35 On going work We are now increasing the simulation time to understand the structural and conformational changes in DNA due to the longterm presence of SWCNT. NP transport modeling for the whole plant.

36 Characterization

37 Characterization Characterization of NPs in plant system by: - AFM Imaging - TEM and EELS - FTIR Spectroscopy - Others?

38 Genomic Studies

39 Genomic Studies Use of genomic techniques to integrate observations at the macro, cellular and molecular level Quantification of differential gene expression using sequencing

40 Biological Network overlaid with Gene Expression Data

41 Our Mega Nano Group! 41

42 Thank you

43 Simulation Method MD software NAMD was used for simulation. The time step used for the simulation was 0.5fs (10-15 s). Solvated model was equilibrated at zero pressure and 0 K temperature to minimize the initial constraint energy involved in the model. The temperature was raised to room temperature, 300 K in 3 steps. The pressure was raised in four steps 0.25, 0.50, 0.75, and 1.0 atm. For DNA-CNT solvated models, energy minimization followed by molecular dynamics simulation with temperature and pressure was run for sufficiently long time of 400 ps, to ensure the convergence of energy. VMD software is used for visualization and interactive studies

44 Computational Resources Center for Computationally Assisted Science & Technology (CCAST) cluster at North Dakota State University Nodes=6, Processors=8 (total processors used = 48) Walltime for each simulation: hours (8-10 days)