Sandi Paulisic et al The EMBO Journal Expanded View Figures A B C Figure EV1. Characterization of RNAi-HFR1 and chfr1 mutants in Cardamine hirsuta. A, B Relative expression levels of ChHFR1 gene, normalized to EF1a in ChWT, (A) two RNAi-HFR1 lines (#01 and #21) and (B) the two chfr1 mutants of C. hirsuta. Seedlings were grown for 7 days in W. Expression values are the mean  SE of three independent biological replicates relative to ChWT. Asterisks mark significant differences (Student’s t-test: **P-value < 0.01) relative to ChWT. C The two identified chfr1-1 and chfr1-2 mutants have one nucleotide insertion at position 420 of the ChHFR1 ORF, which leads to a frame shift and a premature stop codon. ◀ Figure EV2. Alignments of HFR1, PIF4, PIF5, and PIF7 partial DNA sequences in Arabidopsis thaliana and Cardamine hirsuta. A Location of shared primers and amplicons used for comparison of expression levels by RT-qPCR between species. B Transcript abundance of PIF4 and PIF5, normalized to YLS8, SPC25, and EF1a in ChWT and AtWT grown as in Fig 2. Expression values are the means  SE of three ▸ independent biological replicates relative to the data of AtWT grown in continuous W at day 3. Asterisks mark significant differences (2-way ANOVA: **P-value < 0.01, ***P-value < 0.001) between ChWT and AtWT when grown under W (black asterisks) or W + FR (red asterisks). ª 2020 The Authors The EMBO Journal 40: e104273 | 2021 EV1 The EMBO Journal Sandi Paulisic et al A B Figure EV2. EV2 The EMBO Journal 40: e104273 | 2021 ª 2020 The Authors Sandi Paulisic et al The EMBO Journal A Figure EV3. ChHFR1 and AtHFR1 complement the Arabidopsis thaliana hfr1-5 mutant long hypocotyl phenotype. A Cartoon of HFR1 promoters from A. thaliana (pAtHFR1) and C. hirsuta (pChHFR1). These promoters cover 2,000 bp from the beginning of the translation start of the two HFR1 genes. The positions of G-boxes (CACGTG) are indicated with arrows. B GUS staining of representative A. thaliana seedlings expressing GUS under the pAtHFR1 (line #03). Seven-day-old W-grown seedlings were treated B with W + FR for the indicated amount of time. C Correlation between HypW+FR-HypW (means  SE of at least four biological replicates, data shown in Fig 3C) and relative levels of ChHFR1 or AtHFR1 expression (means  SE of three biological replicates, data shown in Fig 3B). The estimated regression equations and the R2 values are shown for each plot. C ª 2020 The Authors The EMBO Journal 40: e104273 | 2021 EV3 The EMBO Journal Sandi Paulisic et al A B C Figure EV4. ChHFR1 protein accumulates in high W. A Alignment of AtHFR1 and ChHFR1 protein sequences. Putative COP1 interacting motifs, defined in AtHFR1, are highlighted with a light gray box. VP motifs are highlighted with blue letters. Amino acid sequences inside the blue line rectangles correspond to the synthetic AtHFR1, ChHFR1, and At/ChHFR1 VP peptides used in the microscale thermophoresis assays (Appendix Table S3). B Cartoon representing the light treatments given to seedlings to estimate relative HFR1-3xHA levels. Seedlings grown for 7 days in low W (~ 20 µmol/m2s1, R: FR  6.4) were first moved to high W (~ 100 µmol/m2s1, R:FR  3.9) for 3 h and then either transferred to high W (control) or high W + FR (R:FR  0.06) for 3 h. Seedling samples were collected at the time points indicated with asterisks. C Relative HFR1-3xHA protein levels of hfr1>35S:ChHFR1 seedlings (line #16) grown as indicated in B, with a representative immunoblot in a lower panel. Relative protein levels are the mean  SE of three independent biological replicates relative to the data point of 0 h in high W (0 h W). Asterisks mark significant differences in protein levels (Student’s t-test: **P-value < 0.01; *P-value < 0.05) relative to the 0 h W value. Source data are available online for this figure. EV4 The EMBO Journal 40: e104273 | 2021 ª 2020 The Authors Sandi Paulisic et al The EMBO Journal A B C D E F Figure EV5. Microscale thermophoresis (MST) experimental traces and analysis. A–F Raw MST traces and analysis of AtCOP1 WD40 with different peptides in triplicates (duplicates for At/ChHFR1 VP). The concentration of AtCOP1 WD40 is fixed at 0.15 lM mixed with 16 serially diluted peptide concentrations at 1:1 ratio. Panels A, C, and E show the normalized MST traces. The blue box area illustrates the fluorescence before activation of the infrared (IR) laser and red box area illustrates average fluorescence after activation of the IR laser. Average values  SD (error bars) were subsequently used for fluorescence normalization. kD fit displaying fraction bound as a function of ligand concentration is shown in adjacent right panels B, D, and F. (A) Raw MST traces for AtHFR1 (in blue) and ChHFR1 (in light-brown) VP peptides. Individual concentrations that showed slight aggregation or precipitation are shown in gray and were excluded from the kD fit calculation. (B) Fitted data over a concentration range from 0.032 to 500 lM for AtHFR1 VP (blue dots) and 0.032 to 1,000 lM for ChHFR1 VP (light-brown dots) were used to derive the corresponding dissociation constant kD. (C) Raw MST traces for At/ChHFR1 VP peptide (in orange). One concentration that showed slight precipitation or aggregation is shown in gray. A concentration range of 0.0154 to 506 lM was used for the At/ChHFR1 VP. (D) No kD was determined, as no binding between COP1 WD40 and the At/ChHFR1 VP peptide (orange dots) was detected. (E) A concentration range from 0.0076 to 250 lM for HsTRIB1 (in red) and AtCRY1 (in green) peptides was used. Raw MST traces show no aggregation or precipitation effects during this binding. One AtCRY1 VP outlier is shown in gray. (F) The kD for HsTRIB1 (brown dots) and AtCRY1 (green dots) VP peptides was calculated using the normalized traces. ª 2020 The Authors The EMBO Journal 40: e104273 | 2021 EV5