Supporting Information for

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1 Supporting Information for CuO Nanoparticle Interaction with Arabidopsis: Toxicity, Parent-Progeny Transfer and Gene Expression Zhenyu Wang,, Lina Xu, Jian Zhao,,, Xiangke Wang, Jason C. White, and Baoshan Xing, Institute of Costal Environmental Pollution Control, and Ministry of Education Key Laboratory of Marine Environment and Ecology, Ocean University of China, Qingdao 2661, China Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 26671, China School of Environment and Chemical Engineering, North China Electric Power University, Beijing 1226, China Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT 654, United States Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 13, United States Pages: 13 Figures: 1 Tables: 7 Corresponding author Tel.: address: bx@umass.edu (Dr. Baoshan Xing); jzhao@ouc.edu.cn (Dr. Jian Zhao) S1

2 A B Figure S1. TEM micrographs of CuO NPs (A) and BPs (B)..15 Cu2+ concentration (mg L-1).1.5 CuO NPs CuO BPs Figure S2. Time dependent dissolution of CuO NPs (5 mg L -1 ) and CuO BPs (5 mg L -1 ). S2

3 Germination rate (%) Col- Bay- Ws-2 2 Control.15 Cu 2+ 2 NPs 5 NPs Treatment 5 BPs Figure S3. The germination rates of seeds after exposure to distilled water (control), distilled water with.15 mg L 1 Cu 2+ ions, 2 mg L 1 CuO NPs (2 NPs), 5 mg L 1 CuO NPs (5 NPs), and 5 mg L 1 CuO BPs (5 BPs) for 2 days. The values were given as mean ± SD (standard deviation). For a given ecotype, there was no significant difference among different treatments. (p <.5, LSD, n=3). S3

4 Root fresh weight (mg/plant) Control.15 Cu 2+ 2 NPs 5 NPs 5 BPs A Shoot fresh weight (mg/plant) Control 2 NPs 5 BPs.15 Cu 2+ B 5 NPs Figure S4. The inhibition of CuO NPs on the growth of root (A) and shoot (B) of Col- seedlings (fresh weight). Col- seedlings were cultured for 96 h in distilled water, 2 mg L -1 CuO NPs (2 NPs), 5 mg L -1 CuO NPs (5 NPs),.15 mg L -1 Cu 2+ ions (.15 Cu 2+ ) or 5 mg L -1 CuO BPs (5 BPs). The values were given as mean ± SD (standard deviation). At 96-h exposure, significant difference between control and other treatments was marked with (p <.5, T test, n=3). S4

5 Root fresh weight (mg/plant) Control 2 NPs 5 BPs.15 Cu 2+ A 5 NPs Shoot fresh weight (mg/plant) Control.15 Cu 2+ 2 NPs 5 NPs B 5 BPs Figure S5. The inhibition of CuO NPs on the growth of root (A) and shoot (B) of Bay- seedlings (fresh weight). Bay- seedlings were cultured for 96 h in the control (distilled water), distilled water with 2 mg L -1 CuO NPs (2 NPs), 5 mg L -1 CuO NPs (5 NPs),.15 mg L -1 Cu 2+ ions (.15 Cu 2+ ) or 5 mg L -1 CuO BPs (5 BPs). The values were given as mean ± SD (standard deviation). At 96-h exposure, significant difference between control and other treatments was marked with (p <.5, T test, n=3). S5

6 Root fresh weight (mg/plant) Control.15 Cu 2+ A 2 NPs 5 NPs 5 BPs Shoot fresh weight (mg/plant) Control.15 Cu 2+ B 2 NPs 5 BPs 5 NPs Figure S6. The inhibition of CuO NPs on the growth of root (A) and shoot (B) of Ws-2 seedlings (fresh weight). Ws-2 seedlings were cultured for 96 h in the control (distilled water), distilled water with 2 mg L -1 CuO NPs (2 NPs), 5 mg L -1 CuO NPs (5 NPs),.15 mg L -1 Cu 2+ ions (.15 Cu 2+ ) or 5 mg L -1 CuO BPs (5 BPs). The values were given as mean ± SD (standard deviation). At 96-h exposure, significant difference between control and other treatments was marked with (p <.5, T test, n=3). S6

7 A B C D E F Figure S7. SEM observations of Arabidopsis (Col-) after CuO NPs exposure (5 mg L -1 ). (A) unexposed roots; (B), (C) different regions of CuO NPs-treated roots. Images D, E, F are the magnified views of image A, B and C, respectively. In Panels C and F, the white boxes indicated that the roots were thickened after CuO NPs exposure. A B C Figure S8. Photos of pollen obtained from Arabidopsis (Col-) grown in distilled water (A), 5 mg L 1 CuO NPs (B), and.15 mg L 1 Cu 2+ ions (C). S7

8 Col- Bay- Ws Figure S9. An example for the different expression of gene bands during DDRT-PCR analysis of Arabidopsis seedlings (Col-, Bay-, and Ws-2) after exposure to CuO NPs. On the top of the figure, the number 1-3 represents the seedlings treated with CuO NPs (5 mg L 1 ) for 24, 48 and 96 h while 4-6 for that treated with Cu 2+ (.15 mg L 1 ) for 24, 48 and 96 h, respectively. The number represented the untreated seedlings. In addition, the genes marked with blue box and red box are ones up-regulated and down-regulated, respectively. The up- and down-regulation of the genes was indicated by the increased or decreased expression quantity of genes in comparison with the un-exposed control. The darker color of bands indicated the higher expression quantity in the gel photo. Based on DDRT-PCR analysis, 18 differently expressed genes were identified among all the genes of Arabidopsis. S8

9 Thylakoid membrane Figure S1. Schematic diagram of ROS generation sites in chloroplast electron transport chain after seedlings exposed to CuO NPs. In photosystem II (PS II), after accepting the light energy, P68 changes from its normal state to excited state (P68), P68 as the reductant, release the electron to pheophytin (pheo) (primary electron acceptor). The electron passes along quinone A (QA), quinone B (QB), plastoquinone (PQ), cytochrome bf (cty bf) to plasto cynin (PC). In photosystem I (PS I), P7 is excited by light, an electron frees from P7, and transport from iron-sulfur centers (Fe-S) to Ferredoxin (Fd). Under the actin of feerrdoxin-nadp+ redutase (Fp), an electron passes to NADP +, and finally forms NADPH. CuO NPs could block the electron transport from QA to QB and from PQ to cytochrome b6f in chloroplast, and thus caused excessive free electrons to produce ROS. S9

10 Table S1. The composition of dicotyledonous nutrient solution. Name Concentration (mmol L -1 ) Name Concentration (mmol L -1 ) K 2 SO 4.75 MnSO 4 H 2 O.1 Ca(NO 3 ) 2 4H 2 O 2. CuSO 4 5H 2 O MgSO 4 7H 2 O.65 ZnSO 4 7H 2 O.1 KH 2 PO 4.25 (NH 4 ) 6 Mo 7 O KCl.1 Fe-EDTA.1 H 3 BO 3.1 Table S2.Sequences of the gene-specific primers used in the semi-quantitative PCR experiment. Gene name 18S rrna Forward Primer Sequence 5'-GTTGCAGTTAAAAAGCTCGT-3' Reverse Primer Sequence 5'-TTGATTTCTCATAAGGTGCC-3' C-1 5'-TAACTGGAAGAAGCGGACC-3' 5'-CCATCCTCAGAAACCTATTCAC-3' C-2 5'-AAGCGAGTCTTCATGGGA-3' 5'-CTCACCAATGTTTGCGTTAC-3' C-3 5'-ATAAGGCGTGAACACCGAG-3' 5'-AAAGACCTCTACTACCCTGTCG-3' Table S3. Sequences of the AXR2/IAA7, AXR3/IAA17 and SLR1/IAA14 primers used in the quantitative RT-PCR analysis. Gene name Forward Primer Sequence Reverse Primer Sequence 18S rrna 5'-GTTGCAGTTAAAAAGCTCGT-3' 5'-TTGATTTCTCATAAGGTGCC-3' AXR2/IAA7 5'-AACATGATCGGCCAACTTA-3' 5'-GAAGCCTCTCTTGCTTCC-3' AXR3/IAA17 5'-TAATAATGATGGGCAGTGTCG-3' 5'-CCTCTCTTGTTTCCGGTTAC-3' SLR1/IAA14 5'-TACTCTTGTTTGGTCAACGAAT-3' 5'-GATAGTTGCCTTTGGAGTCT-3' S1

11 Table S4. Characterization of CuO NPs and CuO BPs Particle Purity Surface Area Size Zeta potential Cu 2+ (%) (m 2 g -1 ) (nm) a (mv) Concentration (mg L 1 ) Nano-CuO 99.9% Bulk-CuO 99.9% a Size observed from TEM micrographs. Table S5. Arabidopsis ecotypes used in this study. Name Stock Number a Place of origin a Phenotype a Water resistance b Heavy metal resistance c Columbia Cs6673 Columbia in Small rosette; Medium Medium (Col-) USA height = cm Bayreuth Cs668 Bayreuth in Large rosette; Low Low (Bay-) Germany height = cm Wassilewski Cs22659 Wassilewskija Medium rosette; High High ja in Russia height = 3-35 cm (Ws-2) a taken from b taken from Vashisht et al. (21); c taken from Murphy and Taiz (1995). References: Vashisht, D.; Hesselink, A.; Pierik, R.; Ammerlaan, J.; Bailey-Serres, M.; Visser, E.; Ppedersen, O.; Zanten, M.; Vreugdenhil, D.; Jamar, D.C.L.; Voesenek, L. A. C. J.; Sasidharan R. Natural variation of submergence tolerance among Arabidopsis thaliana accessions. New Phytol. 21, 19, Murphy, A.; Taiz, L. Comparison of metallothionein gene expression and nonprotein thiols in ten Arabidopsis ecotypes (correlation with copper tolerance). Plant Physiol. 1995, 19, S11

12 Table S6. Effects of CuO NPs on root morphology of Arabidopsis after 96 h exposure. Index Control.15 mg L -1 2 mg L -1 NPs 5 mg L -1 NPs 5 mg L -1 BPs Cu 2+ Root Col ± 1.7 a ±.56 a 9.69 ±1.35 b 7.85 ±.33 c ±.35 a length Bay ± 1.6 a 1.52±.98 b 9.67±.83 bc 8.2±.38 c ± 1.55 a (cm) WS ±.1 ab ±.58 a ±.13 b 11.1 ±.8 3 b ±.21 ab Root Col-.99 ±.6 b 1. ±.11 b 1.11 ±.1 ab 1.18±.6 a 1.4 ±.6 ab diameter Bay- 1.6 ±.6 a.95 ±.12 a 1.1 ±.12 a 1.13 ±.5 a 1.4 ±.17 a (mm) WS-2 1.6±.4 b 1.11 ±.7 ab 1.11 ±.1 ab 1.21 ±.8 a 1.4 ±.6 b Root Col ±.17 a 1.16±.14 a 1.7 ±.9 ab.92±.8 b 1.27±.7 a Surfer Area Bay ±.17 a 1.1 ±.21 ab.97 ±.7 bc.93±.3 c 1.35 ±.38 ab (cm 2 ) WS ±.13 a 1.56 ±.26 a 1.3±.18 a 1.34±.7 a 1.31 ±.9 a Root Col-.86 ±.4 a.85±.6 a.65 ±.3 b.36±.6 c.94 ±.6 a volume Bay-.9 ±.3 a.84 ±.1 ab.71 ±.13 b.45±.14 c.94 ±.7 a (cm 3 ) WS-2 1.2±.11 a 1.3 ±.8 a.93±.5 ab.78±.12 b 1.1±.3 a Root tips Col ± a ± a ± 22. b ± c 25.7 ± 9.75 a (NO.) Bay- 27.8± 9.18 a 28.73± a ± ab ± 2.84 b ± 13.2 a WS ± 14.3 a 26.8 ± 9.12 a ± b ± b ± a Note: The values were given as mean ± SD. Significant difference was marked with different letters (p <.5, LSD, n=3). S12

13 Table S7.The known expressed sequence tags of significant homology with the 6 positive expression genes in CuO NPs-exposed Arabidopsis thaliana seedlings. Clone name a Sequence (bp) Accession number Gene description Identities C AT1G128/NP_ Auxin signaling F-box protein C AT3G2475.1/NP_ DNA mismatch repair protein MSH5 93% 73% C-3 33 AT5G511.1/NP_ Fe superoxide dismutase 94% B No significant similarity found B-2 33 No significant similarity found B No significant similarity found a C-1, C-2, C-3: 3 positive expression genes of Col-; B-1, B-2, B-3: 3 positive expression genes of Bay-. S13