Plant materials and growth conditions
One-year-old seedlings of Populus euphratica were obtained from the Chinese Academy of Forestry (Beijing, China). P. euphratica plantlets were micro-propagated in a growth room maintained at 25°C with 18 h of light per day. To examine PePCR2 expression patterns in after Cd treatment, two-week-old P. euphratica plantlets were exposed to water supplemented with 50 µM CdCl2 for 0, 3, 6, 12, and 24 h. The seedlings were collected after Cd treatment and stored at -80°C for later RNA extraction.
P. alba×P. glandulosa (84K poplar) seedlings were used for transformation, and 84K poplar seedlings were cultured on woody plant medium (WPM) supplemented with 0.02 mg/mL 1-naphthaleneacetic acid (NAA) and 0.05 mg/mL indole-3-butyric acid (IBA). The seedlings were grown in a greenhouse at 25°C with a photoperiod of 16/8 h light/dark.
Nicotiana tabacum L. was used for transient transformation. Seeds were germinated on 1/2 Murashige and Skoog medium (MS) medium for 7 days and transferred into soil at 25°C with a photoperiod of 16/8 h light/dark. Four-week-old seedlings were used for localization analysis of the PePCR2 protein.
Poplar Rna Isolation And Real-time Quantitative Polymerase Chain Reaction (Rt-qpcr)
Total RNA was extracted using a rapid TRIzol-based two-step method (Meng et al., 2010), and first-strand cDNA was synthesized from 0.5-1 µg of purified RNA using the Hifair II 1st Strand cDNA Synthesis Kit with gDNA Digester (YEASEN, Shanghai, China). Levels of mRNA expression were quantified by RT-qPCR with TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). For analysis of PePCR2 (XP_011023881) expression patterns in P. euphratica, PeActin2 (He et al. 2018) was used as an internal control. For the analysis of the expression of other transporter genes in 84K poplar, Actin (Wang et al. 2021) was used as an internal control. The 2−ΔΔCt method was used to calculate the relative expression levels of PePCR2 and other transporter genes. Other heavy metal transporter sequences in poplar were searched from the 84K poplar transcriptome database (PRJNA716488) was searched for other poplar heavy-metal transporter sequences (Wang et al. 2021). All primers used are listed in Supplemental Table S1. The RT-qPCR experiment procedure was referred as the Bustin et al. 2009.
Protein Domain Identification And Subcellular Localization
Homologous amino acid sequences of PePCR2 were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/), and BioEditv7.1 was used to analyze amino acid sequences and conserved structures. The secondary structure of PePCR2 was predicted by a membrane-protein prediction system SOSUI (https://harrier.nagahama-i-bio.ac.jp/sosui/). The cDNA of PePCR2 was cloned from P. euphratica using primers PePCR2-F and PePCR2-R. PePCR2 fused to GFP was cloned into the pCAMBIA1300:eGFP vector, resulting in a PePCR2-GFP construct. This construct was used to transform Agrobacterium tumefaciens GV3101, and transient transformation assays were conducted in tobacco leaves, as described previously (Gui et al. 2015). GFP was excited at 488 nm, captured at 505–555 nm, and imaged using confocal laser scanning microscopy (Leica TCS SP5, Mannheim, Germany).
Heterologous expression of PePCR2 in yeast and analysis of heavy-metal tolerance in yeast
The entire coding region of PePCR2 was cloned into the pYES2.0 vector and transformed into yeast strain cells (BY4741 and ΔycfI) using the LiOAc/PEG method (Gietz and Schiestl 2007). Transgenic yeasts were identified by screening on SD-Ura medium (FunGenome, Beijing, China). Yeast lines expressing empty pYES2 and PePCR2 of the same initial concentration (OD600 = 0.5) were grown on YPG (yeast extract 10 g/L, peptone 20 g/L, and 20% galactose) solid medium in the absence or presence of CdCl2, CoCl2, NiSO4, ZnSO4, MnSO4, FeSO4, Fe2(SO4)3, Pb(NO3)2, and CuSO4 at 30°C for 3–7 d.
Yeast Growth Curve And Cd Concentrations
To understand yeast growth under heavy-metal stress, Cd-sensitive yeast cells (ΔycfI) were transformed with PePCR2, and pre-cultured in 20 mL SD-Ura liquid medium with shaking at 180 rpm at 30°C overnight. The OD600 values were then diluted to 0.1, and the cells were grown in SD-Ura liquid medium with 60 µM CdCl2 with shaking at 180 rpm at 30°C. The OD600 values were determined after 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h. In addition, to measure the accumulation of Cd in the transgenic ΔycfI yeast overexpressing PePCR2, the transgenic yeasts were cultured in SD-Ura liquid medium for 48 h followed by treatment with 60 µM CdCl2 for 48 h. The empty-vector and transgenic yeasts were collected by centrifugation at 10,000 rpm for 10 min, and the yeast cells were washed with 10 mM Na2EDTA and deionized water. The yeast samples were dried in an oven at 60°C for 2 d. After drying, all yeast samples were weighed and then digested with HNO3 using a microwave digestion instrument (Milestone, Sorisole, Italy), and the Cd concentration of samples were measured by ICP-MS (PerkinElmer, Waltham, MA, USA).
Generation Of Transgenic Poplar
The pCAMBIA1300-PePCR2-GFP vector was introduced into Agrobacterium tumefaciens EHA105 using the freeze-thaw method (Holsters et al. 1978). Transformation of 84K poplar was carried out using the previously described leaf disk transformation method (He et al. 2018) with modifications. The leaves were incubated on WPM substrate (pH = 6.0) containing 0.060 mg/L 6-benzylaminopurine (6-BA), 0.001 mg/L thidiazuron (TDZ), 0.010 mg/L IBA, 200 mg/L cefotaxime, 200 mg/L timentin, 3 mg/L hygromycin, and 0.80% (w/v) agar for shoot induction and selection. The regenerated shoots were grown on selective rooting medium (1/2 MS medium containing 200 mg/L cefotaxime, 200 mg/L timentin, 3 mg/L hygromycin, and 0.80% (w/v) agar). We identified transgenic PePCR2 84K poplars by PCR using primers PePCR2-F and GFP-R, and RT-qPCR was used to analyze PePCR2 expression levels in the transgenic poplars.
Measurement Of Electrolyte Leakage And Nitrotetrazolium Blue Chloride Staining
To induce Cd stress, transgenic 84K poplars overexpressing PePCR2 and wild-type (WT) seedlings were grown for 6 d in 1/2 MS medium and then transferred to mediums containing 0 µM, 300 µM, and 400 µM CdCl2 for 12 d. The seedlings were then collected and used for electrolyte leakage (EL) analysis, and the roots were used for nitrotetrazolium blue chloride (NBT) staining. EL analysis was performed according to the method described by Zhang et al. (2019). For histochemical detection of O2 accumulation, the roots were stained with nitroblue tetrazolium (NBT; Biotopped, Beijing, China) as described by Sun et al. (2018). The stained roots were bleached in acetic acid–ethanol (1:3, v/v) for 5 h and then stored in glycerol-ethanol (1:4, v/v). NBT staining was imaged using an Olympus DP80 CCD camera (Japan).
Cd Flux Recordings With A Non-invasive Microtest Technique
Seven-day-old poplar seedlings (WT and PePCR2) were transferred to water containing 0 µM and 400 µM CdCl2 for 12 h. Net Cd2+flux was measured in the roots by the non-invasive microtest technique (NMT100 Series; Amherst, MA, USA). The Cd2+ selective electrodes were prepared and calibrated as described previously (Wu et al. 2019).
Expression Of Other Transporters In Transgenic Poplar Lines
To understand the correlation between PePCR2 and other transporters, the expression levels of eight genes encoding transporters, including ATP-binding cassette (ABC) G-type transporter 29 (ABCG29), P1B-type ATPase transporter 5 (HMA5), natural resistance-associated macrophage protein 6 (NRAMP6), pleiotropic drug resistance 2 (PDR2), yellow stripe-like 3 (YSL3), YSL7, Zn/Fe-regulated transporter-related protein 1 (ZIP1), and ZIP11 were analyzed in transgenic PePCR2 poplar. These sequences were retrieved from the 84K transcriptome database (Wang et al. 2021), and primer sequences were designed. The 35SPro::PePCR2 transgenic and WT 84K poplar seedlings were grown on 1/2 MS solid medium with 50 µM CdCl2 for 6 h. The relative gene expression levels in transgenic and WT 84K poplar were detected and analyzed by RT-qPCR.
Statistical analysis
Experimental data were analyzed using SPSS software. The statistical significance between two means was determined using one-way analysis of variance (ANOVA) followed by post-hoc t tests. Two significance levels were applied: *0.01 < P < 0.05 and **P < 0.01. Results are represented as means ± SD (standard errors) of three replicates.