## Generation and Selection of the Arabidopsis *PSP* Knock-Down Mutant
The RNAi construct for the generation of PSP knock-down plants was designed according to Guo et al. (2003) using the intron-containing vector pSK-int as intermediate vector for cloning. The 660 bp long DNA fragments encoding for the sense and antisense RNA of the *Arabidopsis thaliana PSP* gene (At1g18640, nt 3-663 on cDNA) were generated using primer pairs P1/P2 and P3/P4, respectively, and were integrated into the pSK-Int vector via internal restriction sites (Supplementary Table S1). The complete construct was cloned into the final plant vector pUT-Kan encoding an Ubiquitin 10 promoter for expression of the construct and a kanamycin resistance gene for plant selection (Supplementary Figure S1A). Transformation in *Arabidopsis thaliana* Col-0 wildtype plants was carried out using the floral dip method (Clough and Bent, 1998). Transformed plants were selected on half-strength Murashige-and-Skoog medium plates containing 50 μg/ml kanamycin. Correctness of insert was verified via PCR on genomic DNA using primer P5 and P6. Selection was done out of 8 independent lines and was based on reduction of *PSP* transcript abundance. Knockdown of *PSP* mRNA amount was measured by RT-PCR on cDNA of T1 plants using primer P7 and P8. RT-PCR of *psp* mutant lines revealed a strong reduction of the *PSP* transcript abundance in mutant line #17, while mutant line #18 showed a weaker reduction in *PSP* transcript abundance. Detailed results of *PSP* knockdown amount for mutant line #17 and #18 in comparison to the WT at day and night generated via transcriptome analysis are shown in Supplementary Figure S1B. Homozygous T3 plants of *psp* mutant lines #17 and #18 were used for further characterization of the mutants.
For the characterization of the mutant, chlorine gas surface-sterilized seeds of *Arabidopsis thaliana* Col-0 wildtype and *psp* mutant lines #17 and #18 were stratified at 4°C for 2 days. Germination was carried out on half-strength Murashige-and-Skoog (MS) medium (Duchefa Biochemie) containing 0.8% agar (w/v) in growth chambers (12 h light/12 h dark, 22°C day temperature/18°C night temperature, 100 μmol photons m-2 s-1 light intensity, ambient air). After pre-incubation of *psp* mutant plants and Col-0 wildtype control plants on ½ MS plates for 14 days, plants were transferred to soil. Further plant growth occurred in growth chambers under long day conditions (16 h light/8 h dark, 22°C day temperature/18°C night temperature, 100 μmol photons m-2 s-1 light intensity) in high CO2 (3000 ppm CO2). The growing condition allowed us to compare results of *psp* mutants with the *bou-2* mutant. 24 h before harvest, plants were shifted to ambient air conditions. Other growth parameters remained unchanged.
To examine the significance of the phosphorylated pathway of Ser biosynthesis for Cys synthesis under normal conditions and upon Cd treatment, *Arabidopsis thaliana* Col–0 and *psp* mutant line #17 were used. Prior to cultivation, seeds were surface sterilized with 70% (v/v) ethanol containing 1% (v/v) Triton X-100, twice with 100% ethanol, sown on soil, and incubated at 4°C for at least 2 days to break dormancy. After incubation, seeds were transferred to growth chambers and kept under controlled conditions (12 h light/12 h dark, 22°C day temperature/19°C night temperature, 100–150 μmol photons m-2 s-1, light intensity, 60% relative humidity), at ambient CO2. After emergence of two fully developed leaves, each plant was transferred into a separate pot with new soil substrate and cultivated 4 weeks under the conditions indicated above. One week before harvest, one set of plants was watered with 50 mL 2mM CdCl2 solution in one dose. Experiments were performed with 5-week old plants.
