Collection of fungal specimens and experimental design
E. pisciphila was isolated from the roots of Arundinella bengalensis (authenticated by Prof. Shugang Lu from Yunnan University, and the voucher specimen was preserved in Yunnan University), naturally growing in an old mine smelting site in Huize County, Yunnan Province, Southwest China (103°630 E, 26°550 N), and preserved in the Agricultural Culture Collection Center of China (accession number ACCC32496). The fungus was first incubated in Melin-Norkrans (MMN) liquid medium at 28 °C and 180 rpm for 7 d. Then, E. pisciphila was pretreated by incubating in the medium supplemented with 0, 50.0, 100.0, or 200.0 μM melatonin for 1 d. Exogenous melatonin dissolved in the medium can be absorbed by organisms as a small amphipathic molecule . E. pisciphila was then incubated in the media supplemented with or without Cd2+ (111.2 mg L− 1), Cu2+ (100.0 mg L− 1), Zn2+ (1010.0 mg L− 1), and Pb2+ (800.0 mg L− 1) for 2 d.
Determination of malondialdehyde (MDA), oxygen free radical (OFR), and superoxide dismutase (SOD)
A total of 0.5 g fresh hyphae were ground into fine powder with liquid nitrogen in a mortar. The detection of different parameters was performed by Malondialdehyde (MDA) Detection Kit (A003–1-2), Oxygen Free Radical (OFR) Detection Kit (A052–1-1), and Superoxide Dismutase (SOD) Detection Kit (A001–1-2) (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.
Determination of Cd, Cu, Zn, and Pb concentrations in E. pisciphila
Hyphae were collected and washed three times with deionized water. Subsequently, samples (0.5 g) were oven-dried at 80 °C, then digested with HClO4 and HNO3 mixture (1:4 v/v) at 260 °C. The concentrations of Cd, Cu, Zn, and Pb were analyzed using an atomic absorption spectrophotometer (AA240; Shimadzu Co., Kyoto, Japan).
Quantification of melatonin by high-performance liquid chromatography (HPLC) in E. pisciphila
Hyphae (0.1 g) were ground in liquid nitrogen and extracted with 1.0 mL chloroform for 1 h at room temperature before melatonin quantification. Chloroform extracts (200.0 μL) were completely evaporated and dissolved in 0.1 mL 40% (v/v) methanol, and 10.0 μL aliquots were subjected to HPLC using a fluorescence detector system (Waters, Milford, MA, USA). The samples were separated on a 4.6 × 150 mm Sunfire C18 column (Waters, Milford, MA, USA) using the following gradient elution profile: from 42% (v/v) to 50% (v/v) methanol in 0.1% (v/v) formic acid for 27 min, followed by isocratic elution with 50% (v/v) methanol in 0.1% (v/v) formic acid for 18 min at a flow rate of 0.15 mL min− 1. Melatonin was detected at 280 nm (excitation) and 348 nm (emission). All measurements were taken in triplicate.
Identification and phylogenic tree construction of EpTDC1, EpSNAT1, and EpASMT1
Based on the transcriptome database of E. pisciphila , the nucleic acid sequences of putative EpTDC1, EpSNAT1, and EpASMT1 were obtained. The open reading frame (ORF) nucleotide and amino acid sequences of these unigenes were predicted using Open Reading Frame Finder in National Center for Biotechnology Information (NCBI). Then BLAST search was performed using the EpTDC1, EpSNAT1, and EpASMT1 amino acid sequences. Phylogenetic trees were generated from various amino acid sequences after alignment with Clustal X (version 1.83), and phylograms were constructed using the Neighbor-Joining algorithm (MEGA 5.0) with branch length.
Expression analyses of EpTDC1, EpSNAT1, and EpASMT1
Total RNA was extracted from hyphae using the RNAiso Plus 9108 (TaKaRa, Japan) according to the manufacturer’s instructions. The isolated RNA (1.0 μg) was used to synthesize cDNA via the PrimeScriptII 1st Strand cDNA Synthesis Kit 6210A (TaKaRa, Japan). The quantitative real-time PCR (qRT-PCR) analysis was performed with a LightCycler® 480 II Real-Time PCR Detection System (Roche, Basel, Swiss) using SYBR Premix Ex Taq RR820A (TaKaRa, Japan) and β-tubulin gene as the internal control. Primers used in this study are listed in Table S2. All the reactions were performed with three biological and technical replicates independently.
Overexpression of EpTDC1 and EpASMT1 in E. coli and A. thaliana
The ORF sequences of EpTDC1 and EpASMT1 were amplified by PCR using the specific primer set containing the restriction endonuclease enzyme sites. For PCR amplification of EpTDC1, F: 5′-CGGGATCCATGCTCTGCTTGAGAGGC-3′ (BamHI restriction site is underlined) and R: 5′-GGGTTCGAACTTTGGCATGGCCATTC-3′ (HindIII restriction site is underlined) as the primer. For EpASMT, F: 5′-CGGAATTCATGATGCTAGACAACAAAG-3′ (EcoRI restriction site is underlined) and R: 5′- GGGTTCGAACATTAGACCCATCCGTC-3′ (HindIII restriction site is underlined) as the primer. The purified PCR products were ligated to the expression vector pET28a (Invitrogen, Carlsbad, CA, USA). Then the final recombinant vectors (pET28a-EpTDC1 and pET28a-EpASMT1) were transformed into E. coli BL21(DE3) cells (TaKaRa, Japan).
To obtain transgenic A. thaliana plants, EpTDC1 and EpASMT1 were amplified by PCR with primers F: 5′-CGGGATCCATGCTCTGCTTGAGAGGC-3′ (BamHI restriction site is underlined)/R: 5′-GCTCTAGATTTGGCATGGCCATTC − 3′ (XbaI restriction site is underlined) and F: 5′-GGGGTACCATGATGCTAGACAACAAAG-3′ (KpnI restriction site is underlined)/R: 5′- GCTCTAGAATTAGACCCATCCGTC-3′ (XbaI restriction site is underlined) respectively. The PCR products were inserted into the plant expression vector pCAMBIA1304 (Invitrogen, Carlsbad, CA, USA). The recombinant vectors pCAMBIA1304-35S::EpTDC1 and pCAMBIA1304-35S::EpASMT1 were introduced into Agrobacterium tumefaciens GV3101 (Invitrogen, Carlsbad, CA, USA) and then transformed into WT A. thaliana Columbia-0 (Col-0) (stock CS70000, purchased from the Arabidopsis Biological Resource Center http:// www.arabidopsis.org/abrc) through the floral dip method. The seeds were screened on ½ strength MS supplemented with 25.0 mg L− 1 kanamycin in Petri plates. The transgenic Arabidopsis plants were confirmed by PCR. The homologous T3 transgenic lines named EpTDC1–1/EpTDC1–2 and EpASMT1–1/EpASMT1–2 were selected for further analysis.
Growth assessment in transgenic E. coli and A. thaliana treated with heavy metals
Control (pET28a) and transgenic (pET28a-EpTDC1/pET28a-EpASMT1) E. coli BL21(DE3) strains were inoculated into 100.0 mL Luria Broth (LB) liquid media with 1.0 mM isopropyl β-D-thiogalactopyranoside (IPTG). The final concentrations of 50.0 mg L− 1 Cd2+, 100.0 mg L− 1 Zn2+, 70.0 mg L− 1 Cu2+, and 100.0 mg L− 1 Pb2+ were adjusted in the media separately. The optical density at 600 nm (OD600) of the E. coli strains was measured at 4 h intervals for different times as described in the corresponding figure legends. Each treatment was conducted independently four times.
Seeds of transgenic A. thaliana plants were sown on ½ strength MS medium supplemented with 0, 5.0, and 10.0 μM Cd2+ and 0, 10.0, and 30.0 μM Cu2+ respectively with the WT A. thaliana as the control. After 1 d of stratification at 4 °C, the seedlings were grown for 10 d in a growth chamber at 25 °C, a photosynthetic photon flux density (PPFD) of 100 μmol m − 2 s − 1, and a photoperiod of 16/8 h (day/night). The plants were photographed, and the root length and fresh weight were measured immediately. Each experiment was conducted independently six times.
Determination of cd accumulation in A. thaliana
Seeds of transgenic A. thaliana and WT plant were germinated on ½ MS medium in Petri plates. When the second true leaves were fully expanded, seedlings were transferred into the soil fortified with 0, 20.0, 40.0 mg kg− 1 Cd2+. Plants grown in the soil for 30 d were collected and the Cd concentrations in the whole plant, shoots, and roots were detected by the same method used in E. pisciphila. Each experiment was conducted independently four times.
Independent-Samples t-test or one-way ANOVA with Duncan’s multiple range test (SPSS 16.0) was used to evaluate the significant differences between means. The data were presented as the mean ± standard deviation (SD). For statistically significant difference, P-values < 0.05 were considered statistically significant.