Supporting Information for

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Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis thaliana: Effect of Cytochrome P450 enzymes and Role of Fe. Zunwei Chen†,‡, Jia Wang†, Hui ...

Supporting Information for Enantioselective Phytotoxicity of Dichlorprop to Arabidopsis thaliana: Effect of Cytochrome P450 enzymes and Role of Fe

Zunwei Chen†,‡, Jia Wang†, Hui Chen†, Yuezhong Wen†,* Weiping Liu†



MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College

of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China ‡

Department of Veterinary Integrative Bioscience, Texas A&M University, College

Station, Texas 77843, United States

Summary of the Supporting Information: Pages: 15; Figures: 5; Tables: 4; Texts: 4.

*

Corresponding author:

Tel.: +86-571-8898-2421. Fax: +86-571-8898-2421. E-mail address: [email protected] (Y.W.) S1

Content Text S1. Details for Plants Cultivation and Herbicide Treatments Text S2. Fluorescence Intensity Measurement by Using ImageJ Text S3. Details for Detection of Fe Distribution in A. thaliana Leaves Text S4. Details for Determination of DCPP concentrations by UPLC-QTOF-MS Table S1. Primers Sequence of Genes Tested Table S2. Chlorophyll contents of Arabidopsis thaliana seedlings treated with dichlorprop (DCPP) and 1-aminobentriazole (ABT) Table S3. The Pearson Correlation Coefficients of Gene Expression between Fe Aggregation (ISU1& ISU3) and ACCase (CAC1, CAC2, CAC3 and aseI) in A. thaliana Table S4. Effects of ABT on the Enantioselective Difference (ED)* Induced by 0.2 µmol·L-1 DCPP in A. thaliana Figure S1. Effects of ABT on the fresh weight of A. thaliana Figure S2. Effects of ABT on the production of ROS in A. thaliana roots induced by 0.2 µmol·L-1 DCPP. (A) Laser confocal images: (a-c) without ABT; (d-f) 20 µmol·L-1 ABT; (g-i) 40 µmol·L-1 ABT. The white stick equals to 50 µm. (B) Statistical results Figure S3. UPLC-Q-TOF MS evidences for the determination of DCPP. (A) C9H8Cl2O3 XIC from standard sample of 5 ppm DCPP (Q-TOF MS (100-1500): 234.992 +/-0.005 Da, positive ion mode); (B) C9H8Cl2O3 XIC from standard sample of 5 ppm DCPP (Q-TOF MS (100-1500): 232.977 +/-0.005 Da, negative ion mode); (C-F) C9H8Cl2O3 XIC from samples treated with 0.2 µM (R)-DCPP (C), 0.2 µM (S)-DCPP (D), 0.2 µM (R)-DCPP+40 µM ABT (E) and 0.2 µM (S)-DCPP+40 µM ABT (F) (Q-TOF MS (100-1500): 232.977 +/-0.005 Da); (G) C9H8Cl2O3 XIC from control plant (Q-TOF MS (100-1500): 232.977 +/-0.005 Da) Figure S4. Gene expression of Fe transportation. (A) IRT1; (B) IRT2; (C) FRO2; (D) FRO3; (E) NRAMP1; (F) NRAMP3; (G) NRAMP4 Figure S5. The gene expression correlation between Fe aggregation (A: ISU1; B: ISU3) and ACCase (CAC1, CAC2, CAC3 and aseI) in A. thaliana S2

Text S1. Details for Plants Cultivation and Herbicide Treatments Arabidopsis thaliana (ecotype Columbia) was selected as the model plant for the short life cycle and obvious phenotypic change when exposed to contaminants. Seeds of A. thaliana were sterilized with 4% (v/v) sodium hypochlorite for 1 min, followed by three-time rinse with 75% ethanol and repeated with sterile water. Then the seeds were planted in 24-well culture cluster, containing 1 mL Murashige and Skoog (MS) medium mixed with dichloroprop (DCPP) and 1-aminobenzotriazole (ABT) in each cell. In preliminary experiments, to choose proper exposure concentration, the concentration of DCPP enantiomers and race mate ranged from 0.1 to 0.3 µmol·L-1, and for ABT, the concentration were set at 0.5, 1, 10, 20 and 40 µmol·L-1. After three-day vernalization, culture clusters were put in an incubator at 23 ± 2°C with 16 h light (4000 lx) and 8 h dark illuminating cycles for 20 days until analysis.

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Text S2: Fluorescence Intensity Measurement by Using ImageJ. The fluorescence capture was performed by using laser-scanning confocal microscope and the images were automatically obtained by ZEN 2010 software. Then the images were further analyzed by ImageJ software. First of all, images were transformed into 8-bit/16-bit type to be monochrome ones; then subtract background at 50; intensity was set as measurements area and pixels were set as scale; finally the images were inverted and then find at least 15 black light dots (guard cells) to measure the intensity.

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Text S3. Details for Detection of Fe Distribution in A. thaliana Leaves Distribution of Fe in A. thaliana leaves was determined with a high-resolution chemical imaging method based on synchrotron X-ray microfluorescence (µ-XRF) with a beamline BL15U1 at Shanghai Institute of Applied Physics, Chinese Academy of Science. Firstly, samples were thoroughly washed with distilled water. Then samples were fixed on the tape for analysis. Then beam size of 2×2 µm2 was used. The majority of maps were focused on the entire leaf blade. Color-coded composite chemical maps of the target element in the leaves of A. thaliana were performed by Igor Pro 6.0 software (IGOR).

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Text S4. Details for Determination of DCPP concentrations by UPLC-QTOF-MS ACQUITY UPLC BEH-C18 column (1.7 µm, 2.1×50 mm, Waters Corp.) was used in all the chromatographic experiments. The mobile phase were 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B). The linear gradient programs were as follows, 0/5, 5/5, 25/95 and 26/5 (min/B%). Sample injection volume was 3 µL, column oven temperature was 30 °C, flow rate was 0.4 mL/min and UV detector was set at 254 nm. As for optimal mass spectrometry (MS) conditions, scan range m/z was 100-2,000; The source voltage was -4.5 kV and temperature was 550 °C in the negative ion mode. The pressure of Gas1 (Air) and Gas2 (Air) were set to 50 psi and the pressure of Curtain Gas (N2) was set to 30 psi. Injection volume was set at 10 µL; flow rate was 0.2 mL/min; maximum allowed error was set to ± 5 ppm; Declustering potential (DP) was 100 V and collision energy (CE) was 10 V. As for the MS/MS acquisition mode, the IDA-based auto-MS2 was performed on the 8 most intense metabolite ions, the parameters were almost the same except that CE was set at -40 ± 20 V, ion release-delay (IRD) was set at 67, ion release width (IRW) was at 25. In a cycle of full scan (1s), the scan range of m/z of precursor ion and product ion were set as 100-2,000 Da and 50-1,500 Da, respectively. The exact mass calibration was performed automatically before each analysis employing the Automated Calibration Delivery System.

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Table S1. Primers Sequence of Genes Tested Function

Name

Primer Sequence

IRT1

Forward: 5’-CTATACACCAGCAAGAACG-3’ Reverse: 5’-AGATAATCCAATGACCACC-3’

IRT2

Forward: 5’-GCCACAATCATAGTCACG-3’ Reverse: 5’-AGCAAGCATTTAACAGTCC-3’

NRAMP1

Forward: 5’-CATTACTCAAAGCCAGACC-3’ Reverse: 5’-GCAAGCTTCCTTGATACC-3’

Fe transportation

NRAMP3

Forward: 5’-GAAACGAAGAGGAGGACG-3’ Reverse: 5’-AACCAAAAGACCCATTGC-3’

NRAMP4

Forward: 5’-TCGACGAAGAAGAAGACG-3’ Reverse: 5’-ATAATCCAAAGCACCATCC-3’

FRO2

Forward: 5’-CTCCAACATCTTCTCCTACC-3’ Reverse: 5’-GAACATATTTCCGCAACC-3’

FRO3

Forward: 5’-ATCACTCCTCAATCACTTCC-3’ Reverse: 5’-AACCCCAAGCATATATTCC-3’

ISU1

Forward: 5’-TCCGTCTAGAAAAAGTTACTTCCGAAACCC-3’ Reverse: 5’-TCCGGGTACCGGTTGTATGTATCATCCTCTTC-3’

Fe Aggregation

ISU2

Forward: 5’-TCCGTCTAGACAAGCTCCATAGAGAGAAGCG-3’ Reverse: 5’-TCCGGGTACCTCTGTTTGGGTCATAAAACATC-3’

ISU3

Forward: 5’-TCCGTCTAGACTGCCCTTGATTCCGGCAAAGAG-3’ Reverse: 5’-TCCGGGTACCCTTGGGATCTGGGTCACATGC-3’

CAC1

Forward: 5’-GCTTAACGTGTGCCATTGATT-3’ Reverse: 5’-TTCTCATGGTGCCGATTCTAC-3’

CAC2

Forward: 5’-GCAATGGGAGAGAAACTTCG-3’ Reverse: 5’-TATTCTGCCAGGTCCAGGTC-3’

ACCase

CAC3

Forward: 5’-AAGCTGGTTGTTTCCCAATCT-3’ Reverse: 5’-AGGAAGGAAGAGAAAGCATCG-3’

accD

Forward: 5’-CTTGCCATTGGATGTGCTAAT-3’ Reverse: 5’-GGTCTTCTGTGCTCAACTTGG-3’

`aseI

Forward: 5’-ACAGGCTACAATGAACGATGG-3’ Reverse: 5’-CAGCATTATGGGCATTCAGTT-3’

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Table S2. Chlorophyll contents of Arabidopsis thaliana seedlings treated with dichlorprop (DCPP) and 1-aminobentriazole (ABT) a

Chlorophyll a (mg/g plant)

Chlorophyll b (mg/g plant)

Treatments

Control

(R)-DCPP

(S)-DCPP

Without ABT

0.55 ± 0.07a

0.16 ± 0.03c

0.50 ± 0.07a

20 µM ABT

0.47 ± 0.05a

0.26 ± 0.06b

0.42 ± 0.09ab

40 µM ABT

0.41 ± 0.10ab

0.31 ± 0.03b

0.35 ± 0.09ab

Without ABT

0.17 ± 0.02a

0.06 ± 0.01d

0.16 ± 0.023a

20 µM ABT

0.15 ± 0.03ab

0.09 ± 0.01c

0.14 ± 0.02ab

40 µM ABT

0.15 ± 0.03ab

0.13 ± 0.01b

0.13 ± 0.03ab

a

The data presented consist of average values ± standard deviation of three independent batches. Different letters in the columns represent statistically significant differences (p