Supplementary Figure 1. jmj30-2 and jmj32-1 produce null mutants. (a) Schematic drawing of JMJ30 and JMJ32 genome structure showing regions amplified

Transcription

1 Supplementary Figure 1. jmj30-2 and jmj32-1 produce null mutants. (a) Schematic drawing of JMJ30 and JMJ32 genome structure showing regions amplified by primers used for mrna expression analysis. Gray boxes indicate the 5 and 3 UTR. Black boxes indicate exons, while blue boxes indicate exons coding for the JmjC domain. White boxes with a line indicate introns. T-DNA insertion mutants are marked by downward pointing arrowheads above the schematic diagrams, whereas smaller arrowheads below denote regions amplified by primers used for RT-qPCR. Scale bar, 500 bp. (b) JMJ30 mrna expression in WT and jmj30-2. Relative fold changes to WT are presented. Bars indicate s.d. of three biological replicates; two-tailed Student s t-test, **P<0.01. (c). JMJ32 mrna expression in WT and jmj32-1. Relative fold changes to WT are presented. Bars indicate s.d. of three biological replicates; two-tailed Student s t-test, **P<0.01.

2 Supplementary Figure 2. jmj30 jmj32 double mutant accelerates flowering under short day conditions. (a) jmj30 jmj32 double mutant showed early-flowering phenotype when grown under SD 29 C conditions. Representative 40-day-old plants were shown as comparison. Scale bar, 1 cm. (b) Total primary rosette leaves before bolting in WT and jmj30-2 jmj32-1 plants grown under SD 29 C conditions were counted (2 independent experiments with plants each). Bars indicate s.d.; twotailed Student s t-test, **P<0.01.

3 Supplementary Figure 3. efp browser view of JMJ30 and JMJ32 gene expression during Arabidopsis development. (a,b) The expression pattern analysis of (a) JMJ30 and (b) JMJ32 was performed using the Arabidopsis efp browser ( 44. Data are normalized by the GCOS method.

4 Supplementary Figure 4. JMJ30 and JMJ32 are ubiquitously expressed and can localize to the nucleus. (a) Semi-quantitative RT-PCR of JMJ30 and JMJ32 in different tissues types. Tip-41 like is shown as an internal control. S, seedlings; RL, rosette leaves; CL, cauline leaves; St, stems; In, inflorescences. (b) Transient over-expression of JMJ30-VENUS and JMJ32-VENUS in tobacco leaves. DAPI was used to visualize the chromatin. Scale bar, 25 µm. (c) Immunolocalization of JMJ30-HA in nuclei extracted from the leaves of jmj30-2 pjmj30::jmj30-ha transgenic lines grown under LD 22 C and 29 C conditions. Anti-HA antibody and DAPI were used to visualize JMJ30-HA proteins and chromatin, respectively. Scale bar, 5 µm.

5 Supplementary Figure 5. pjmj30::jmj30-ha complements the jmj30 jmj32 mutations. (a) Compared to WT, jmj30 jmj32 showed accelerated flowering when grown under 29 C LD conditions. However, introduction of an endogenous pjmj30 promoter driven JMJ30-HA construct rescued the early-flowering phenotype of jmj30 jmj32 at 29 C LD. Representative 23-day-old plants were shown as comparison. Scale bar, 1 cm. (b) Total primary rosette leaves before bolting in WT, jmj30 jmj32 and jmj30 jmj32 pjmj30::jmj30-ha plants grown under 29 C LD conditions were counted (2 independent experiments with plants each). Bars indicate s.d.; two-tailed Student s t-test, **P<0.01.

6 Supplementary Figure 6. Over-expression of JMJ30 and JMJ32 caused decreased H3K27me3 levels. Immunostaining of H3K27me3 and (a,b) JMJ30-HA or (c,d) JMJ32-HA in nuclei extracted from Arabidopsis leaf protoplasts transiently over-expressing (a) JMJ30-HA or (c) JMJ32-HA or their respective mutated version of JMJ30H326A-HA and JMJ32H174A-HA. Over-expression of JMJ30-HA and JMJ32-HA reduced H3K27me3 levels. However, mutations of the conserved Fe(II)- binding histidine residue in the JmjC domain of JMJ30H326A-HA and JMJ32H174A-HA showed no effect on H3K27me3 demethylation. Anti-HA antibodies, anti-h3k27me3 antibodies and DAPI were used to visualize JMJ30/32-HA proteins, H3K27me3 marks and chromatin, respectively. Arrows indicate transformed nuclei over-expressing the JMJ-HA proteins. Arrowheads indicate control non-transformed nuclei. Scale bar, 10 µm. (b,d) The intensities of at least 50 pairs of closelylocated JMJ-HA-transformed and non-transformed nuclei were analyzed using ImageJ. Data were presented as a relative intensity of transformed/non-transformed nuclei. (b) Statistical analysis of a. (d) Statistical analysis of c. Bar indicates s.d.; two-tailed Student s t-test, **P<0.01.

7 Supplementary Figure 7. Over-expression of JMJ30 produces a late-flowering phenotype. (a) 35S::JMJ30-HA transgenic plants showed a late-flowering phenotype when grown at LD 22 C. Representative 27-day-old plants were shown as comparison. Scale bar, 1 cm. Plants in the genotypes of WT, jmj30-2, jmj30-2 jmj32-1, 35S::JMJ30-HA line #15-5 and 35S::JMJ30-HA line #2-6 were removed from the soils and aligned to take the photo. (b) Total primary rosette leaves before bolting in WT, jmj30-2, jmj30-2 jmj32-1 and 35S::JMJ30-HA plants grown under LD 22 C conditions were counted (2 independent experiments with plants each). Bars indicate s.d.; twotailed Student s t-test, *P<0.05. (c) Relative JMJ30-HA protein levels over 16 h examined by western blot using anti-ha-hrp antibody. Total proteins from 35S::JMJ30-HA seedlings grown under 22 C LD conditions were used. Ponceau Red-stained membrane is shown as a loading control. Numbers indicate relative band intensity compared to 22 C 0h sample. Two biological replicates were performed and one set of the data is shown here. (d) Relative expression of JMJ30 transcripts in WT, jmj30-2, and two independent lines of 35S::JMJ30-HA. Relative fold changes to WT are presented. Bars indicate s.d. of three biological replicates; two-tailed Student s t-test, **P<0.01.

8 Supplementary Figure 8. JMJ30 binds the FLC locus directly. ChIP analysis of JMJ30-HA enrichment at the FLC locus in 8 DAG 35S::JMJ30-HA seedlings grown under LD 22 C conditions. A mouse IgG was used as control. Bar indicates s.e.m. of three biological replicates; two-tailed Student s t-test, *P<0.05, **P<0.01.

9 Supplementary Figure 9. Temperature-dependent stabilization of JMJ30-HA protein. The jmj30-2 pjmj30::jmj30-ha plants grown at LD 22 C were pre-treated for 30 min with the protein synthesis inhibitor cycloheximide (CHX). After adding the proteasome inhibitor MG132 (+MG132) or DMSO (-MG132) at 0 hours (h) (right before the beginning of dark photoperiod), the plants were transferred to 22 C or 29 C. Samples were harvested at 1h, 4h, and 8h. JMJ30-HA protein levels were examined by western blot using anti-ha-hrp antibody. Ponceau Red-stained membrane is shown as a loading control. Numbers indicate relative band intensity compared to 29 C 0h or 22 C 1h sample. Two biological replicates were performed and showed similar expression patterns. One set of the data is shown here.

10 Supplementary Figure 10. FRI jmj30 jmj32 showed a modest reduced expression of FLC when grown at elevated temperature. Relative expression of FLC in 1-week (W1), 2-week (W2) and 3- week (W3) old seedlings of FRI and FRI jmj30 jmj32 grown at LD 29 C. The FLC expression ratio of FRI jmj30 jmj32/fri is shown below the graph. Bars indicate s.d. of three biological replicates; two-tailed Student s t-test, *P<0.05.

11 Supplementary Figure 11. jmj30 and jmj32 do not enhance ref6 mutation. ref6-1 shows a lateflowering phenotype when grown under LD 22 C conditions, but the flowering phenotype of ref6-1 was not significantly different from ref6-1 jmj30-2 and ref6-1 jmj32-1 double mutants or the ref6-1 jmj30-2 jmj32-1 triple mutant. Scale bar, 1 cm.

12 Supplementary Figure 12. Dot blot showing the substrate specificity of the different antimodified histone antibodies. Different amount of biotin-labeled histone peptides (unmodified histone H3 Residue 1-21 and H3 Residue 21-44, modified histone H3K4me3, H3K9me3, H3K27me3 and H3K36me3) were blotted to a nitrocellulose membrane. Western blot were then performed using the anti-histone antibodies indicated on the top. The Strepavidin-HRP probed membrane is shown as a control.

13 Supplementary Figure 13. Uncropped immunoblot used in main figures and supplementary figures. The cropped regions were highlighted in red boxes.

14 Supplementary Table 1. Genotyping primers used in this study. Primer name Primer sequence Description jmj30-1_gt_f TGTTGGTCTCCTCTGAAGCTC Genotyping primers for jmj30-1 (SAIL_811_H12) jmj30-1_gt_r GTTCATTTATCTGCCCATTCG jmj30-2_gt_f CAAACTCTGCTGCAATCGATTTC Genotyping primers for jmj30-2 (GK-454C10) jmj30-2_gt_r GAAAATGTCACAAGCTCTTGCTTC jmj32-1_gt_f GACTGAGAAAACCTGAACTCAGC Genotyping primers for jmj32-1 (SALK_003313) jmj32-1_gt_r GTCGTGTAAAGGACTGAAGGTTG flc-3_gt_f TAGAAAGAAATAAAGCGAGAAA Genotyping primers for flc-3 flc-3_gt_r TATCGCCGGAGGAGAAGC Supplementary Table 2. Primers used in plasmid construction. Primer name Primer sequence Description XhoI-JMJ30_F GGCTCGAGACTTTCCCCAACTCATCATCAC Primers used for Bsp120I-JMJ30-R CCGGGCCCCGAGCTAGAAGATTCTGCTTCA 35S::JMJ30-HA construct XhoI-JMJ32_F GGCTCGAGGTCAATGGCTAAAGAGATAGAGAATTTATGG Primers used for Bsp120I-JMJ32-R CCGGGCCCGGGAGCAATCTCTGCATCACTG 35S::JMJ32-HA construct JMJ30-H326A-F TGGGACAGTTACTCCGTTAGCCCATGATCCACATCATAAT Primers used to JMJ30-H326A-R ATTATGATGTGGATCATGGGCTAACGGAGTAACTGTCCCA mutagenize 35S::JMJ30-HA to 35S::JMJ30H326A-HA JMJ32-H174A-F GACTCCGTGACTTCATTTGCTAAAGATCACTATGAGA Primers used to JMJ32-H174A-R TCTCATAGTGATCTTTAGCAAATGAAGTCACGGAGTC mutagenize 35S::JMJ32-HA to 35S::JMJ32H174A-HA JMJ30-FL_F GCCAGATCCTGAAACAATTCTC Primers used to clone JMJ30-FL_R CTGATTTTGGGGTCTTTGGATTC JMJ30 genomic fragment JMJ30-mut_F GCAGAATCTTCTAGCTCGGGCGCCTAGCGAATGGATTTATTT Primers used to mutagenize JMJ30 JMJ30-mut_R AAATAAATCCATTCGCTAGGCGCCCGAGCTAGAAGATTCTGC fragment with insertion of SfoI restriction site JMJ32-FL_F GAGATCAGTTAAGCCAAGCCAAG Primers used to clone JMJ32-FL_R CATGATACACACATCAAAAAGACGG JMJ32 genomic fragment JMJ32-mut_F JMJ32-mut_R GTCTCAGATTGCTCCCGGCGCCTGATTTAATGGTGGAAC GTTCCACCATTAAATCAGGCGCCGGGAGCAATCTGAGAC Primers used to mutagenize JMJ32 fragment with insertion of SfoI restriction site

15 Supplementary Table 3. Primers used in expression analysis and ChIP assays. Primer name Primer sequence Description JMJ30-sqRT_F ACTTGGACTACCTCAATGCTGTTG semi qpcr primer for JMJ30 JMJ30-sqRT_R TCATGGTGTAACGGAGTAACTGTC JMJ32-sqRT_F GTTAGGCATGTACCTTGGTCTAGTG semi qpcr primer for JMJ32 JMJ32-sqRT_R TCCAAGAAAGAACTGGATTGAGTG Tip41-sqRT_F CATTATAGGTTTGGCGAAGATGAG semi qpcr primer for TIP41-like (AT4G34270) Tip41-sqRT_R TGAAACCACCACAATAAGTCAGTG JMJ30-RT-P1_F GATTCTGTTTTGTTGGTCTCCTC RT-qPCR primer for JMJ30 P1 JMJ30-RT-P1_R GATTAGCCAAAACATGTCTCACC JMJ30-RT-P2_F GAATCACTTGGACTACCTCAATGC RT-qPCR primer for JMJ30 P2 JMJ30-RT-P2_R CATTGGAGACGATTTATTGGTCC JMJ30-RT-P3_F CAAGACGAACTTTACCCTTACTCTG RT-qPCR primer for JMJ30 P3 JMJ30-RT-P3_R GGATGTACAACATTTCACCTTCTTC JMJ32-RT-P1_F CTCCAAAGCTATTACTCATTGGC RT-qPCR primer for JMJ32 P1 JMJ32-RT-P1_R GAAACAAAGATCACTATCTCCGG JMJ32-RT-P2_F GTTTCATTGTACTGTCAAGGCTGG RT-qPCR primer for JMJ32 P2 JMJ32-RT-P2_R CATACTTGATGTCAAACTGCATGTC JMJ32-RT-P3_F CTTCACTCAATCCAGTTCTTTCTTG RT-qPCR primer for JMJ32 P3 JMJ32-RT-P3_R GAACATATTGAACCAAACACAGCC FLC-RT_F CCGAACTCATGTTGAAGCTTGTTGAG RT-qPCR primer for FLC FLC-RT_R CGGAGATTTGTCCAGCAGGTG FT-RT_F CTTGGCAGGCAAACAGTGTATGCAC RT-qPCR primer for FT FT-RT_R GCCACTCTCCCTCTGACAATTGTAGA SOC1-RT_F AGCTGCAGAAAACGAGAAGCTCTCTG RT-qPCR primer for SOC1 SOC1-RT_R GGGCTACTCTCTTCATCACCTCTTCC SVP-RT_F CAAGGACTTGACATTGAAGAGCTTCA RT-qPCR primer for SVP SVP-RT_R CTGATCTCACTCATAATCTTGTCAC CO-RT_F TCAGGGACTCACTACAACGACAATGG RT-qPCR primer for CO CO-RT_R TTGGGTGTGAAGCTGTTGTGACACAT AGL24-RT_F GAGGCTTTGGAGACAGAGTCGGTGA RT-qPCR primer for AGL24 AGL24-RT_R AGATGGAAGCCCAAGCTTCAGGGAA Tip41-RT_F GTGAAAACTGTTGGAGAGAAGCAA RT-qPCR primer for TIP41-like (AT4G34270) Tip41-RT_R TCAACTGGATACCCTTTCGCA FLC-ChIP-P1_F GCATTAGGTTGTTCCCTCCAAAC ChIP RT-qPCR primer for FLC P1 FLC-ChIP-P1_R GCCCTACCCATGACTAACGTGAG FLC-ChIP-P2_F TGTTCTCAATTCGCTTGATTTCTAGT ChIP RT-qPCR primer for FLC P2 FLC-ChIP-P2_R GCCCGACGAAGAAAAAGTAGATAG FLC-ChIP-P3_F CATCTCTCCAGCCTGGTCAAG ChIP RT-qPCR primer for FLC P3 FLC-ChIP-P3_R GGGCTATGAAAATTGCGGTATG FLC-ChIP-P4_F CCTTGGATAGAAGACAAAAAGAGAAAGTG ChIP RT-qPCR primer for FLC P4 FLC-ChIP-P4_R AGGTGACATCTCCATCTCAGCTTC MU-ChIP_F GATTTACAAGGAATCTGTTGGTGGT ChIP RT-qPCR primer for MU MU-ChIP_R CATAACATAGGTTTAGAGCATCTGC