The ZM variety of V. sativa was used in this study, the culture process of plants was described as previously (Rui et al., 2016). For VsPCS1 expression analysis, 7-day-old plants were treated with 0, 5, and 50 μM Cd for different days.

Sterile seeds of Arabidopsis wild-type (Columbia, WT), mutant atpcs1, transgenic plants were surface sterilized and germinated on 1/2 Murashige & Skoog (MS) agar medium (pH 5.8) containing the different concentrations of Cd. After 3 days at 4°C in the dark, seeds were germinated in a growth chamber (22/18°C day/night temperatures, 16/8 h day/night photoperiod) for 8 days, the length of plants roots, fresh weight, and dry weight were measured.

To analysis GHS and PCs content and Cd transport, the seeds of WT, atpcs1, 35S::VsPCS1, and 35S::VsPCS1/atpcs1 were first grown on half-strength MS agar plates for 2 weeks, then the seedlings were grown hydroponically in 1/4 strength Hoagland’s nutrient solution (Liu et al., 2017) for 2 weeks, after, the plants was placed in the solution with or without 10 μM CdCl₂ (Remans et al., 2008; Cuypers et al., 2011) for 24 h to detect the expression level of AtABCC1, 3 [C-type ATP-binding cassette (ABC) transporter], AtCAX2 (Calcium exchanger 2), AtNRAMP3 (Natural resistance-associated macrophage proteins 3), and for 5 days to analyze NPT content and intracellular Cd localization.

The nucleic acid sequences of other plant phytochelatin synthases were searched from NCBI database¹. The sequences were analyzed using DNAMAN software (Version 6.0.3.99 LynnonBioSoft, Foster City, CA, United States). Primers were designed according to the highly conserved region of PCS1 for VsPCS1 isolation. Total RNA was isolated by total RNA extraction Kit (TakaRa) from V. sativa. The first strand cDNA was synthesized by RT-PCR using PrimeScript (TaKaRa) and oligo (dT) primers, PCR amplifications were performed with VsPCS1 primers. The PCR products were sequenced, then the sequences of VsPCS1 were tested by BLAST².

Wild-type Arabidopsis (Col-0) and its mutant, atpcs1 (SAIL_650_C12), with T-DNA insertion in an exon of AtPCS1 gene was available from public repositories³ for this study. Homozygous atpcs1 lines were identified by the method of Yi and An (2013).

pCAMBIA1304 was used as the plant expression vectors. CDS of VsPCS1 was cloned into pCAMBIA1304 using primers of PCS1-F2 and PCS1-R2. The confirmed plasmid were transformed into Arabidopsis WT Col-0 and the mutant atpcs1 plants via standard floral dip transformation using Agrobacterium tumefaciens (Clough and Bent, 1998), which were named 35S::VsPCS1 and 35S::VsPCS1/atpcs1, respectively. Homozygotic transgenic lines of T3 progeny were used for subsequent study.

RNA isolation and cDNA synthesis followed the procedure above mentioned. The quantitative RT-PCR was performed with SYBR pre-mix EX Taq (TaKaRa) by a Real-time PCR system (Eppendorf, Mastercycler ep realplex, Germany) with VsPCS1 primers presented in Supplementary Table 2. V. sativa gene Actin11 was used as an internal control and relative expression levels of genes which were calculated by 2^-∆∆^C_T method (Gao et al., 2016). The primers for AtABCC1, 3, AtCAX2, AtNRAMP3 genes presented in Supplementary Table 1, and AtActin2 gene was used as an internal control.

The mesophyll protoplasts of WT, atpcs1, 35S::VsPCS1, and 35S::VsPCS1/atpcs1 were isolated by enzyme solution containing 0.25% w/v macerozyme R-10 (Yakult), 1% w/v cellulase R-10 (Yakult), 0.4 M D-mannitol, 20 mM MES (pH 5.7), 10 mM CaCl₂, 20 mM KCl, and 0.1% w/v bovine serum albumin (Yoo et al., 2007). The isolated cells were purified using 21% sucrose and counted on a hemacytometer.

V. sativa protoplasts were prepared from 14-days-old leaves using the same modifications as described (Wu et al., 2009). The isolated cells were purified and concentrated using 30% sucrose.

35S (CaMV35S) promoter-driven expression clones were generated in the pSGFP vectors, resulting in N-terminal green fluorescent protein (GFP)-protein fusions. The CDS of V. sativa PCS1 gene amplified using the primers of PCS1F1 and PCS1R1. PCR products were cloned into pSGFP vector and confirmed by sequencing. 35S::VsPCS1-GFP plasmid were transiently expressed in V. sativa mesophyll protoplasts via polyethylene glycol (4000)-calcium transfection (Yoo et al., 2007). Incubating at room temperature for 16 h, transformed cells were observed with a uitraviewvox confocal microscope (PerkinElmer, United States).

The plant materials were washed with 5 mM CaCl₂ for half an hour, and then dried at 105°C for 15 min and 48 h at 80°C in an oven. The digestion of dried samples according to the method of Rui et al. (2016). Cd concentrations were determined using atomic absorption spectrophotometer (novAA^® 400; Analytik Jena, Jena, Germany).

For Cys, GSH and PCs measurement, they were extracted by 1 ml TFA (0.1%) and 6.3 mM diethylene triaminepentaacetic acid, put the homogenate on ice for 15 min. Following centrifugation (13200 g, 30 min, 4°C), the supernatant were derivatized based on the methods of Kühnlenz et al. (2015). UPLC system (Agilent 1290 Infinity, Germany) was equipped with a ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm) used for the separation of the mBBr-labeled thiols. Thiols were detected using the fluorescence detector set at excitation and emission wavelengths of 380 and 470 nm. Quantification was performed via authentic Cys, GSH, PC₂, PC₃, and PC₄ standards.

The leaf protoplasts with or without Cd treatment from WT, atpcs1, 35S::VsPCS1, and 35S::VsPCS1/atpcs1 were loaded with 0.04% v/v Leadmium Green AM dye (Molecular Probes, Invitrogen, Carlsbad, CA, United States). Intracellular Cd localization was observed according to the method of Park et al. (2012) using a uitraviewvox confocal microscope (PerkinElmer, United States). The intensity of green fluorescence signal was quantified by ImageJ software.

The data were analyzed by one-way analysis of variance, followed by multiple comparisons with the least significant difference (LSD) test (P < 0.05), using SPSS software (ver. 17.0; SPSS, Inc., Chicago, IL, United States), different letters indicate significant differences among treatments.

The complete full-length phytochelatin synthase cDNA (complementary DNA) was amplified from V. sativa cDNA using a pair of degenerate primers. VsPCS1 coding DNA sequence (CDS) contains 1500 base pairs that encodes 500 amino acids (Supplementary Data 1, 2). Sequences analysis of amino acids showed that PCS1 is highly conserved between V. sativa (VsPCS1) and Medicago truncatula (MtPCS1) with 89.4% identity and 94% similarity. Twenty Cys residues are present in VsPCS1. Seven Cys residues are conserved in plant kingdom, and one Cys specific residue is in VsPCS1 (Supplementary Figure 1). In the aspect of catalytic activity, VsPCS1 holds the same catalytic active sites as other plant PCS1. Phylogenetic analysis for PCS1 demonstrated that V. sativa was grouped with leguminous plants. These data suggested that the VsPCS1 obtained is a V. sativa PCS1 homolog (Figure 1).

PCS homologs have been characterized as crucial factors for the detoxification of metals in organisms (Pomponi et al., 2006; Liu et al., 2011; Shukla et al., 2012; Kühnlenz et al., 2014, 2015). To test whether VsPCS1 hold the function to detoxify the heavy metals in V. sativa, we firstly determined the expression of VsPCS1 in response to excess Cd among different tissues, including leaves, stems, and roots. Through employing reverse transcript-polymerase chain reaction (RT-PCR) and quantitative RT-PCR, no significant difference on the VsPCS1 transcripts was detected in all the detected tissues under Cd-free and 5 μM Cd treatments. However, 50 μM Cd treatment dramatically induced the increase of VsPCS1 transcripts in roots, being about 4.2 times of Cd-free treatment (Figures 2A,B). Such Cd induced increase was not observed in leaves and stems under 50 μM Cd treatment for 24 h. Time-course analysis demonstrated that VsPCS1 in the roots rapidly and consistently responded to 50 μM Cd, showing a significant increase of transcript from 12 to 96 h (Figures 2C,D).

To investigate the subcellular localization of VsPCS1, a fused protein of VsPCS1 with GFP was transiently expressed in mesophyll protoplasts of V. sativa. GFP signal mainly accumulated in the cytoplasm of VsPCS1-GFP transformed cells, whereas GFP signal is diffused everywhere in GFP transformed cells (Figure 3).

To test whether VsPCS1 is a functional homolog of Arabidopsis PCS1, we conducted a complementation experiment by ectopic expression of VsPCS1 in Arabidopsis PCS1 deficient mutant atpcs1 (designated as 35S::VsPCS1/atpcs1). Consistent with previous report (Nahar et al., 2014), atpcs1 was hypersensitive to Cd. Such Cd hypersensitivity of atpcs1 was restored when constitutively expressed VsPCS1 (Figure 4).

To further determine the function of VsPCS1 in detoxifying Cd, VsPCS1 was also ectopically expressed in wild-type Arabidopsis (Col-0). Fifteen independent 35-VsPCS1 lines were obtained by using hygromycin screen; six out of seven independent transgenic lines showed VsPCS1 overexpression; and two transgenic lines (#2 and #5) were selected for further analysis (Supplementary Figure 2). Ectopic expression of VsPCS1 has no effect on the levels of AtPCS1 in Arabidopsis (Supplementary Figure 3). To investigate Cd tolerance of these VsPCS1-expressing lines, Arabidopsis seeds of WT and homozygotic transgenic lines were germinated and grown on solid 1/2 MS medium contained 0, 25, 50, and 75 μM Cd for 8 days. No significant difference could be observed in the growth between WT and all of transgenic lines under Cd-free treatment (Figure 4A). When treated with 25 and 50 μM Cd, 35S::VsPCS1 (#2 and #5) had a longer root length and more fresh weight than WT (Figures 4B,C). These results demonstrate that overexpression of VsPCS1 increase Cd tolerance in 35S::VsPCS1.

Cd tolerance is tightly related to the Cd uptake from growth medium (Lin and Aarts, 2012). Since Cd tolerance of both 35S::VsPCS1 and 35S::VsPCS1/atpcs1 significantly increased, we investigated the effects of VsPCS1 overexpression on Cd concentration. Under control condition, Cd concentration was undetectable in all Arabidopsis lines (data not shown). After 8-day treatment with 25 and 50 μM Cd, a comparable Cd concentration was detected in WT and 35S::VsPCS1. Similarly, no difference in Cd concentration was observed between atpcs1 and 35S::VsPCS1/atpcs1 (Figure 5).

Apart from Cd uptake, the tolerance is also affected by cellular distribution of Cd. To examine whether the enhanced Cd tolerance conferred by overexpression of VsPCS1 resulted from the increased vacuolar sequestration, the cellular distribution of Cd was analyzed in the leaves of WT, atpcs1, 35S::VsPCS1 (#2 and #5), and 35S::VsPCS1/atpcs1 (#1 and #10) using a Cd-indicator dye, by which Cd concentration could be indicated by green fluorescence. In the absence of Cd, leaf cells from WT showed a negligible Leadmium^TM Green fluorescence signal (Figures 6A–C). After Cd treatment, observed green fluorescence was emitted in all of the detected lines. As expected, the green fluorescence signal mainly emitted from the vacuole of cells in wild-type (Figures 6D–F) and transgenic lines (35S::VsPCS1 #2, 35S::VsPCS1#5, 35S::VsPCS1/atpcs1#1, and 35S::VsPCS1/atpcs1#10) (Figures 6J–U). However, the green fluorescence signal in atpcs1 was detected mainly in cytoplasm and chloroplasts, being an orange-green signal when green Leadmium^TM Green and red chlorophyll auto-fluorescence were merged (Figures 6G–I). Quantitative analysis showed that average intensity of Cd-fluorescence was significantly lower in the vacuole and higher in the cytosol of atpcs1 than 35S::VsPCS1/atpcs1, demonstrating that VsPCS1 could rescue the defects of PC-based sequestration of Cd into the vacuole in atpcs1. Surprisingly, no difference in average intensity of Cd-fluorescence was observed between WT and 35S::VsPCS1 (Figure 6V).

The PC-Cd complexes sequestrating to vacuole were mainly mediated by two ABCC proteins, AtABCC1 and AtABCC3 in Arabidopsis (Park et al., 2012; Brunetti et al., 2015). To exclude the possibility that the rescued Cd vacuole transport by overproducing VsPCS1 is owing to the increased ABCC proteins in Arabidopsis, we measured their expression in different Arabidopsis lines. As shown in Figure 7, the expression of AtABCC1 and AtABCC3 had the same transcription level in all lines under control condition. Although the transcript level of AtABCC3 increased in all lines after 10 μM Cd treatment, no significant increase were observed between VsPCS1 overproducing lines and WT. Unexpectedly, a higher AtABCC3 transcript was detected in the atpcs1 than other lines. Cd treatment did not affect the expression of AtABCC1.

Phytochelatin synthases are well-known to synthesize PCs from reduced glutathione (Noctor et al., 2011). To test whether PCS1 hold the catalytic activity for PCs synthesis, we measured the GSH and its high-order products PCs in the lines we used. As expected, atpcs1 mutant had the highest contents of GSH among all the detected lines. Consistently, a relative reduction of PCs was observed in atpcs1 compared with WT. Such change could be bounced back by introducing VsPCS1 into atpcs1 (Figure 8). Total PCs content in 35S::VsPCS1/atpcs1 was about twofold of that in the mutant atpcs1. Surprisingly, overexpressing VsPCS1 in Arabidopsis dramatically increase high-order products, especially PC₄ in 35S::VsPCS1.