110 kDa kDa His-XLG1 His-XLG2 His-XLG3 kDa His-GPA1 His-T475N His-XLG2C His-RTV1 TRX His-XLG1C His-XLG2C His-XLG3C XLG1C XLG2C XLG3C kDa 207 No tag A Full-length Supplemental Fig. 1. Purification of XLG, GPA1, and RTV1 proteins. A. His TRX tagged-GPA1, XLG2C, XLG2C(T475N), RTV1 and His-TRX were all expressed in E. coli as N-terminal 6  His- tagged proteins and purified by Ni 2+ -NTA agarose column chromatography. Purified proteins were separated by 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue. B. 6X His TRX-tagged XLG C-termini (amino acids 486 to 888 for XLG1, 464 to 862 for XLG2, 433 to 849 for XLG3) and (C) 6xHis-tagged XLG full-length proteins were purified. All His-tagged proteins were fused to thioredoxin (TRX), which is about kDa, between the 6X His tag and the main protein. D. Purification of XLG C-termini proteins lacking a tag. N-terminus GST fusion XLG C-terminus proteins were purified and then GST was cleaved by thrombin (Sigma). Arrows indicate designated proteins. B C D

XLG2C (no tag) Time (min) XLG2 full GDP ▶ GTP ▶ Time (h) GTP  S GDP ATP GDP ▶ GTP ▶ A B GTP remaining (%) Time (min) C Supplemental Fig. 2. Like XLG2C 6X His TRX-tagged protein, XLG2 full-length 6X His TRX- tagged and XLG2C (no tag) proteins have GTPase activities in the presence of Ca 2+. A. Substrate competition test of XLG2C in the presence of Ca 2+. Reactions were performed as described in Fig. 2E in the presence of competitors (3  M unlabeled GTP  S, GDP or ATP). B. GTPase activities of full-length XLG2 and XLG2C (no tag) in the presence of Ca 2+, demonstrating that neither truncation nor tag is required for GTPase activity. C. GTPase activity of GPA1 in the presence of 10 mM Mg 2+ or Ca 2+.

None Competitor GTP  S GDP ATP GTP hydrolysis (%) C XLG1C - Mg 2+ Mn 2+ Ca Time (h) A ◀ GTP ◀ GDP Supplemental Fig. 3. XLG1 has GTP binding and GTPase activities in the presence of Ca 2+. A. [ 35 S]GTP  S binding to XLG1C, its mutant and GPA1 in the presence of CaCl 2 or MgCl 2. Amount of [ 35 S]GTP  S binding to XLG2, its mutant form XLG1C(S497N) and GPA1 was measured after a 2 hour incubation in the presence of CaCl 2 or MgCl 2. B. GTPase activity of XLG1C in the presence of different cofactors, showing preference for Ca 2+. C. GTPase activity of full-length XLG1, XLG1C and XLG1C(S497N) in the presence of Ca 2+. D. Substrate competition test of XLG1C in the presence of Ca 2+, showing specificity for GTP. 2 h XLG1 full S497N XLG1C B Ca 2+ Mg 2+ GTPγS binding (%) 2 h ◀ GTP ◀ GDP D

XLG3C - Mg 2+ Mn 2+ Ca Time (h) A None GTP  S GDP ATP GTP hydrolysis (%) C Supplemental Fig. 4. XLG3 has GTP binding and GTPase activities in the presence of Ca 2+. A. [ 35 S]GTP  S binding to XLG3C, its mutant form XLG3C(S444N) and GPA1 in the presence of CaCl 2 or MgCl 2. Amount of [ 35 S]GTP  S binding to XLG3C, its mutant and GPA1 was measured after 2 hour-incubation in the presence of CaCl 2 or MgCl 2. B. GTPase activity of XLG3C in the presence of different cofactors, showing preference for Ca 2+. C. GTPase activity of full-length XLG3, XLG3C and XLG3C(S444N) in the presence of Ca 2+. D. Substrate competition test of XLG3C in the presence of Ca 2+, showing specificity for GTP. ◀ GTP ◀ GDP 2 h XLG3 full S444N XLG3C B ◀ GTP ◀ GDP GTPγS binding (%) Ca 2+ Mg 2+ D

Supplemental Fig. 5. RTV1 mRNA expression is up-regulated by vernalization. Expression of RTV1 and XLG2 under cold treatment. RTV1 and XLG2 transcript levels were normalized to ACTIN2 levels using the formula C T = C T (each gene) – C T (ACTIN2). One-week old Col seedlings on plates were treated with the indicated duration of cold treatment (4  C). Real-time PCR was used to determine the level of RTV1 mRNA expression. Relative fold changes were shown compared between cold-treated and no cold-treated Col plants with no cold-treated plants set equal to 1. RD29A was used as a positive control for a cold responsive gene (Zhu et al., 2004) and the repression of FLC was used as a positive control for a vernalization responsive gene (Sung and Amasino, 2004) Fold change Cold treatment (Time)

Supplemental Fig. 6. RTV1 binds to a DNA fragment from the DFR promoter, which is not flowering-related. Electrophoretic mobility shift assay (EMSA) of RTV1 using 400 bp DFR promoter-derived DNA fragment. RTV1 protein (0-28 nM; 0-1  g) and 400-bp radiolabelled double-stranded DFR probe were incubated and then separated on a 5% tris-borate EDTA- polyacrylamide gel. RTV1 (µg) Free probe Protein-DNA complex