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Supplementary materials for

Variations of dissolved organic matter and Cu fractions in rhizosphere soil induced by the root activities of castor bean

Guoyong Huang1,2, Xiupei Zhou1, Guagguang Guo1, Chao Ren1, Md Shoffikul Islam1,3, Muhammad Shahid Rizwan1,4, Hongqing Hu,1†

1Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtse River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University;Wuhan 430070, China;

2 SCNU Environmental Research Institute, Guangdong Provincial Key Laboratory of Chemical Pollution and Environmental Safety & MOE Key Laboratory of Theoretical Chemistry of Environment, South China Normal University, Guangzhou 510006, China

3Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur, Bahawalpur, 63100, Pakistan

4 Department of Soil Science, University of Chittagong, Chittagong 4331, Bangladesh;

Number of Pages: 9

Number of tables：2

Number of Figures：3

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Fig.S1 The photos illustrating the experiment design, plant cultivation and the rhizosphere soil in pots.

Analysis of LMWOAs and amino acids

Analysis of LMWOAs was preformed according to the methods described by Huang et al. (2016). High Performance Liquid Chromatography (HPLC) system (Agilent Technologies 1200, USA) equipped with a UV–visible detector was used to analyze the LMWOAs and the measurement conditions were as follow: the mobile phase 0.01M K2HPO4 (pH 2.90), the flow rate 1 ml/min, the temperature of the chromatographic column 30 °C, the injection volume was 20 μL and the detection wave length 210 nm. Identification of LMWOA was performed by comparison of retention time with the standard organic acids.

Quantification of amino acids was performed by HPLC (Agilent Technologies 1200, America) coupled with a triple quadrupole mass spectrometer (AB Sciex, API 5500 Qtrap System, America) according to the methods described by Dong et al. (2018). The AB Sciex AAA C18 column (4.6 mm × 150 mm, 5 µm) was used for the separation of analyses and the temperature was thermostatted at 30 °C. The flow rate was 0.8 mL/min and the injected volume was 10 µL. Tandem mass (MS/MS) detection was performed in a positive mode using multiple reactions monitoring (MRM). The source parameters were: entrance potential (EP): 10 V; spray voltage: 5500 V; curtain gas: 20 psi; desolvation temperature (TEM): 600 °C; ion source gas1 and gas2 were set at 60 psi. High purity nitrogen was used as the gas source and curtain gas. Identification of amino acids was performed by comparison of retention time with the standard amino acids.

Extraction and fraction of Cu

The sequential extraction method was used to determine the metal partitioning as described by Huang et al. (2019) in details, and the main extract reagents and the operations were listed in Table S1. Accurate 0.5000 g dried soil sieved by 0.15-mm from each pot was used for the Cu sequential extraction. After each extraction step, the remained soil in the centrifuge tube was washed twice with the distilled water and the extracts of the washing steps was discarded. The sediment from step 3 was washed with distilled water and then air-dried soil was digested with a combined, concentrated HCl-HNO3-HClO4 (v:v:v=1:3:1) on a hot plate. The Cu concentrations in all the extract were analyzed with AAS (Spector-AA 220FS, Varian, USA).

Table S1 BCR sequential extraction procedures for Cu fractions in soil with 0.5000g dry soil

|Steps |Fraction |Extract reagents |Working conditions |

|I |Acid exchangeable |20 mL 0.11 M HOAc |Shake for16 h under25±1 °C |

| |fraction | | |

|II |Reducible fraction |20 mL 0.5 M NH2OH·HCl（HNO3） |Shake for16 h under25±1 °C |

|III |Oxidizable fraction |①5 mL 30% H2O2 |①Intermittent shake for 1 h at room temperature |

| | |②3 mL 30% H2O2 |② Intermittent shake for 1 h at 85±2 °C |

| | |③25 mL 1.0 M NH4OAc (pH 2.0±0.1) |③Shake for16 h under25±1 °C |

|IV |Residual fraction |HCl-HNO3-HClO4 (v:v:v=3:1:1) |Digestion |

The response of plant growth to different Cu levels

Plant growth of castor bean was inhibited under the high Cu stress (Fig. S2). There were no visible toxicity symptoms observed in the castor bean under the low concentration of Cu (≤ 200 mg kg-1) in the soil, but the cotyledon of castor bean in Cu400 was turning yellow with the exposure time, and the growth of the first two euphylla was slower than that in the other treatments. The dry weight of whole plants was dramatically decreased with the increasing Cu dose in the soil (p ≤ 0.05) except in the Cu100 treatment. The dry weight of root and shoot were 2.69 g and 7.72 g in control treatment (described as CK in the text), respectively. However, the dry weight of root and shoot were 0.45 g and 0.85 g in Cu400 treatment, respectively. The proportion of dry weight of shoot and that of plant was 0.74 in CK, and it decreased to 0.64, 0.69, 0.62 and 0.65 in Cu50, Cu100, Cu200 and Cu400 respectively. Precious studies showed the castor bean could grow well in the natural contaminated soil (de Souza Costa et al., 2012; Olivares et al., 2013; Kang et al., 2015), but the growth of the castor bean in this study was greatly inhibited (Fig. S2). The reason for this phenomenon might be due to the difference on the soil properties and the bioavailability of soil Cu. The soil pH values ranged from 6.6 to 7.5 and the concentration of Cu extracted with diethylenetriamine pentaacetic acid (DPTA) ranged from 2.74 to 20.83 mg kg-1 in most sites of the mine tailing in the previous report (Olivares et al., 2013). However, the acidic soil (pH < 5.0) with high concentration of available Cu was observed in the Cu treated soil (Tables 1 and 2), thus inhibiting the growth of castor bean.

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Fig.S2 Dry weight of castor bean grown in the soil treated with various dosages of Cu. CK was short for the control. Data were presented as mean value ±standard deviation (n=9). The different lower-case letters above the column indicated significant difference (p ≤ 0.05) among the different Cu treatments.

The structure of DOM in soil

Fig. S3 demonstrated that the same peak wavelength was found in either emission or excitation spectra (occurring at 410 nm and 337 nm, respectively) of the rhizosphere DOM and non-rhizosphere DOM, indicating that there no much differences between the components of DOM. However, the fluorescence intensity of DOM from rhizosphere soil was higher than that of non-rhizosphere soil, which was probably ascribed to the accumulation of simple structural components with a small molecular weight, e.g. LMWOAs and amino acids. The fluorescence intensity was related to the structure of DOM closely (D’ Orazio et al., 2014; Xu et al., 2018).

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Fig. S3 Fluorescence emission (A) and excitation (B) spectra of DOM in rhizosphere and non-rhizosphere soil in the Cu200 treatment.

|Table S2 Results of Paired t-test for the data of pH, TC, TN and Cu fractions between the rhizosphere and|

|non-rhizosphere soil |

|Items |t-value |df |95% confidence interval |Sig. |

| | | |lower limit |upper limit |(both sides, p=0.05)|

|pH |5.672 |14 |0.02985 |0.06615 |0.000 |

|TN |0.000 |14 |-0.07251 |0.07251 |1.000 |

|TC |4.846 |14 |0.16352 |0.42315 |0.000 |

|Exchangeable Cu |1.112 |14 |-1.88618 |5.94485 |0.285 |

|Reducible Cu |4.856 |14 |5.08332 |13.12468 |0.000 |

|Oxidizable Cu |6.053 |14 |7.4443 |15.61427 |0.000 |

|Residual Cu |3.229 |14 |4.82974 |23.94226 |0.006 |

Reference

de Souza Costa, E. T., Guilherme, L. R. G., de Melo, É. E. C., Ribeiro, B. T., Euzelina dos Santos, B. I., da Costa Severiano, E., Hale, B. A. 2012. Assessing the tolerance of castor bean to Cd and Pb for phytoremediation purposes. Biol. Trace Elem. Res. 145(1), 93-100.

Dong, M., Qin, L., Xue, J., Du, M., Lin, S. Y., Xu, X. B., Zhu, B. W. 2018. Simultaneous quantification of free amino acids and 5’-nucleotides in shiitake mushrooms by stable isotope labeling-LC-MS/MS analysis. Food Chem. 268, 57-65.

D’Orazio, V., Traversa, A., Senesi, N. 2014. Forest soil organic carbon dynamics as affected by plant species and their corresponding litters: a fluorescence spectroscopy approach. Plant soil 374(1-2), 473-484.

Huang, G., Guo, G., Yao, S., Zhang, N., Hu, H. 2016. Organic acids, amino acids compositions in the root exudates and Cu-accumulation in castor (Ricinus communis L.) under Cu stress. Internat. J. Phytoremediat. 18(1), 33-40.

Huang, G., Gao, R., You, J., Zhu, J., Fu, Q., Hu, H. 2019. Oxalic acid activated phosphate rock and bone meal to immobilize Cu and Pb in mine soils. Ecotox. Environ. Safe. 174, 401-407.

Kang, W., Bao, J., Zheng, J., Hu, H., Du, J. 2015. Distribution and chemical forms of copper in the root cells of castor seedlings and their tolerance to copper phytotoxicity in hydroponic culture. Environ. Sci. Pollut. R. 22(10), 7726-7734.

Olivares, A. R., Carrillo-González, R., González-Chávez, M. D. C. A., Hernández, R. M. S. 2013.Potential of castor bean (Ricinus communis L.) for phytoremediation of mine tailings and oil production. J. Environ. Manage. 114, 316-323.

Xu, P., Zhu, J., Fu, Q., Chen, J., Hu, H., Huang, Q. 2018. Structure and biodegradability of dissolved organic matter from Ultisol treated with long-term fertilizations. J. Soil Sediment. 18(5), 1865-1872.

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