JMJ14-HA. Col. Col. jmj14-1. jmj14-1 JMJ14ΔFYR-HA. Methylene Blue. Methylene Blue
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1 Fig. S1 JMJ14 JMJ14 JMJ14ΔFYR Methylene Blue Col jmj14-1 JMJ14-HA Methylene Blue Col jmj14-1 JMJ14ΔFYR-HA Fig. S1. The expression level of JMJ14 and truncated JMJ14 with FYR (FYRN + FYRC) domain deletion in different JMJ14 complementary lines. JMJ14-HA and JMJ14ΔFYR-HA indicates the P JMJ14 ::JMJ14-HA jmj14-1 and P JMJ14 ::JMJ14ΔFYR-HA jmj14-1 transgenic lines respectively.
2 Fig. S2 A DAPI H3K4me1 H3K4me2 H3K4me3 JMJ14 -YFP-HA Methylated histone Relative intensity B WT JMJ14-YFP-HA JMJ14 -YFP-HA H3K4me3 H3K4me2 H3K4me1 Fig. S2. Deletion of FYR (FYRN + FYRC) domain does not influence the demethylase activity of JMJ14. (A) Overexpression of JMJ14 -YFP-HA reduced H3K4me3, H3K4me2 and H3K4me1. (B) The Statistical analysis of enzymatic activity of JMJ14 -YFP-HA compared with JMJ14-YFP-HA.
3 Fig. S3 A At1g72460 JMJ14-HA anti-ha 160 anti-ha JMJ14ΔFYR-HA anti-ha jmj14-1 anti-ha Relative fold enrichment B At2g32510 jmj14-1 JMJ14-HA JMJ14ΔFYR-HA JMJ14-HA anti-ha 35 anti-ha JMJ14ΔFYR-HA anti-ha jmj14-1 anti-ha Relative fold enrichment C At5g45810 jmj14-1 JMJ14-HA JMJ14ΔFYR-HA JMJ14-HA anti-ha 40 anti-ha JMJ14ΔFYR-HA anti-ha jmj14-1 anti-ha Relative fold enrichment jmj14-1 JMJ14-HA JMJ14ΔFYR-HA Fig. S3. The anti-ha ChIP-seq results of JMJ14-HA, JMJ14ΔFYR-HA and jmj14-1 on genome browser (left) and the ChIP-qPCR validation results (right) of At1g72460 (A), At2g32510 (B) and At5g45810 (C). JMJ14-HA and JMJ14ΔFYR-HA indicates the P JMJ14 ::JMJ14-HA jmj14-1 and P JMJ14 ::JMJ14ΔFYR-HA jmj14-1 transgenic lines respectively. Two independent lines of each transgenic plants were used in the qpcr. and indicate the 5 and 3 regions of the genes which were showed in the left. FYR represents FYRN and FYRC domains.
4 Fig. S4 NAC050 1 : MGRESLAVVSSPPSATAPSTAVSATSLAPGFRFHPTDEELVSYYLKRKVLGKPVRFDAIG : 60 NAC052 1 : MGRESVAVVTAPPSATAPGTASVATSLAPGFRFHPTDEELVSYYLKRKVLGQPVRFDAIG : 60 NAC : EVDIYKHEPWDLAVFSKLKTRDQEWYFFSALDKKYGNGARMNRATNKGYWKATGKDREIR : 120 NAC : EVDIYKHEPWDLAVFSRLKTRDQEWYFYSALDKKYGNGARMNRATNRGYWKATGKDREIR : 120 NAC : RDIQLLGMKKTLVFHSGRAPDGLRTNWVMHEYRLVEYETETNGSLLQDAYVLCRVFHKNN : 180 NAC : RDILLLGMKKTLVFHSGRAPDGLRTNWVMHEYRLVEYETEKNGNLVQDAYVLCRVFHKNN : 180 NAC : IGPPSGNRYAPFMEEEWADGGGALIPGIDVRVRVEALPQANGNNQMDQEMHSASKDLINI : 240 NAC : IGPPSGNRYAPFMEEEWADDEGALIPGIDVKLRLEPPPVANGNDQMDQEIQSASKSLINI : 240 NAC : NELPRDATPMDIEPNQQNHHESAFKPQESNNHSGYEEDEDTLKREHAEEDERPP-SLCIL : 299 NAC : NEPPRETAPLDIESDQQNHHENDLKPEEHNNNNNYDENEETLKREQMEEEERPPRPVCVL : 300 NAC : NKEAPLPLLQYKRRRQNESNNNSSRNTQDHCSSTITTVDNTTTLISSSAAAATNTAISAL : 359 NAC : NKEAPLPLLQYKRRRQSESNNNSSRNTQDHCSSTTTTVDNTTTLISSS-AAATNTAISAL : 359 NAC : LEFSLMGISDKKENQQ------KEETSPPSPIASPEEKVNDLQKEVHQMSVERETFKLEM : 413 NAC : LEFSLMGISDKKEKPQQPLRPHKEPLPPQTPLASPEEKVNDLQKEIHQMSVERETFKLEM : 419 NAC : MSAEAMISILQSRIDALRQENEELKKKNASGQAS : 447 NAC : MSAEAMISILQSRIDALRQENEELKKNNANGQ-- : 451 Fig S4. Sequence alignment shows that NAC050 and NAC052 share 88% identity of their protein sequences.
5 Fig. S5 A B SD/-T-L SD/-T-L JMJ14 AD JMJ14 -BD JMJ14 AD JMJ14 -BD JMJ14 AD JMJ14 -BD AD -BD 53-BD T-AD Fig. S5.-
6 Fig.S6 A NAC050-CFP JMJ14-YFP DAPI Merged P 35S ::JMJ14-YFP-HA P 35S ::NAC050-CFP-HA F1 NAC052-CFP JMJ14-YFP DAPI Merged P ::JMJ14-YFP-HA P ::NAC052-CFP-HA F1 35S 35S B C D P ::JMJ14g-GFPGUS-UTR P ::NAC050g-GFPGUS-UTR P ::NAC052g-GFPGUS-UTR JMJ14 JMJ14 NAC050 NAC050 NAC052 NAC052 Fig. S6. JMJ14 and NAC050/052 colocalize in subcellular and tissue level. (A) JMJ14, NAC050 and NAC052 localize in nuclei. (B-D) JMJ14, NAC050 and NAC052 express in same tissues including meristem, vascular tissue, and stigma.
7 Fig. S TSS 500 Mid -500 TTS 500 NAC050/052 binding level NAC050-HA NAC052-HA Col Fig. S7. The binding of NAC050 and NAC052 to their direct target genes in NAC050-HA, NAC052-HA and Col plants. The tag counts were normalized in each bin according to the total number of reads. NAC050-HA and NAC052-HA indicates the P NAC050 ::NAC050-HA nac050-1 and P NAC052 ::NAC052-HA nac052-2 transgenic lines respectively.
8 Fig.S8 A negative regulation of molecular function negative regulation of catalytic activity B Biological processes protein amino acid phosphorylation phosphate metabolic process phosphorus metabolic process phosphorylation negative regulation of molecular function negative regulation of catalytic activity C Biological processes protein amino acid phosphorylation phosphate metabolic process phosphorus metabolic process phosphorylation negative regulation of molecular function negative regulation of catalytic activity Biological processes protein amino acid phosphorylation Molecular functions identical protein binding serine-type endopeptidase activity adenyl nucleotide binding purine nucleoside binding nucleoside binding Molecular functions electron carrier activity UDP-glucosyltransferase activity protein kinase activity glucosyltransferase activity tetrapyrrole binding adenyl nucleotide binding purine nucleoside binding protein serine/threonine kinase activity identical protein binding nucleoside binding adenyl nucleotide binding purine nucleoside binding nucleoside binding Molecular functions Fig. S8. Significantly enriched biological processes and molecular functions identified by Gene Ontology (GO) analysis of the common target genes of JMJ14, NAC050 and NAC052 (A), the target genes of JMJ14 (B) and the common target genes of NAC050 and NAC052 (C) (FDR < 0.01).
9 Fig. S9 A P: GTCAGAACCTTGTGGAGCAAGCCAACGATG M1: GTCAGAACGGGGTGGAGCAAGCCAACGATG M2: GTCAGAACCTTGTGGAGCCCCCCAACGATG M3: GTCAGAACCCCCTGGAGCCCCCCAACGATG M4: GTCAGAACGGGGTGGAGGGGGCCAACGATG M5: GTCAGAACGGGGTGGAGCCCCCCAACGATG M6: GTCAGAACCAAGTGGAGCTTGCCAACGATG M7: GAGTCAGAACCTTGGCAAGCCAACGATGGA M8: AGTCAGAACCTTGGGACAAGCCAACGATGG M9: TCAGAACCTTGATGGAGACAAGCCAACGAT M10: CAGAACCTTGAATGGAGAACAAGCCAACGA Density C JMJ14 JMJ14&NAC NAC B Protein: GST GST-NAC050 GST GST-NAC052 Probe: P P M1 M2 M3 M4 M5 M6 P P M1 M2 M3 M4 M5 M6 Protein: GST GST-NAC050 GST GST-NAC052 Probe: P P M7 M8 M9 M10 P P M7 M8 M9 M Number of motif within binding region / kb Fig. S9. (A) The DNA probe used for EMSA. P was the original sequence from At1g72460, a common target gene of NAC050, NAC052 and JMJ14. M1 to M10 contain different mutations of the core binding sequence. (B) The binding assay to different probes using 50ng of GST or GST-fused recombinant NAC050 and NAC052 showing that mutations of CTTGNNNNNCAAG core sequence reduce the binding affinity of NAC050 and NAC052. (C) The CTTGNNNNNCAAG motif density in the binding regions of JMJ14 alone (red), JMJ14 and NAC overlapping (green) and NAC alone (blue), respectively.
10 Fig. S10 Input IP with no antibody IP with anti-nac P 35S::NAC050-CFP-HA P 35S::NAC052-CFP-HA WB with anti-ha Fig. S10. The identification of the NAC antibody by immunoprecipitation using transgenic plants.
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