Supplemental Data. Tang et al. Plant Cell. (2012) /tpc

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1 Supplemental Figure 1. Relative Pchlide Fluorescence of Various Mutants and Wild Type. Seedlings were grown in darkness for 5 d. Experiments were repeated 3 times with same results. Supplemental Figure 2. Non-covalently Bound Heme Contents in fhy3, far1, fhy3 far1 Mutants and Wild Type. Seedlings were grown in darkness for 5 d. FW: fresh weight. Mean ± SD, n=4. 1

2 Supplemental Figure 3. Expression of Genes Involved in the Tetrapyrrole Biosynthetic Pathway. (A) Chlorophyll and heme biosynthesis pathways. Genes encoding the enzymes in each step are in italics. Glu, glutamate. Glu trna, glutamyl-trna. Glu SA, glutamate-1-semialdehyde. ALA, 5-aminolevulinic acid. PBG, porphobilinogen. Proto IX, protoporphyrin IX. Mg-proto, Mg-protoporphyrin IX. Mg-proto ME, Mg-protoporphyrin IX monomethyl ester. Pchlide, divinyl protochlorophyllide. Chlide, divinyl chlorophyllide. Chlide a, monovinyl chlorophyllide a. Chl a, chlorophyll a. Chl b, chlorophyll b. HEMA, encodes glutamyl-trna reductase. GSA, encodes glutamate-1-semialdehyde aminotransferase. HEMB, encodes 5-aminolevulinic acid dehydratase. HEMC, encodes porphobilinogen diaminase. HEMD, encodes uroporphyrinogen III synthase. HEME, encodes uroporphyrinogen III decarboxylase. HEMF, CPO, encodes coproporphyrinogen III oxidase. HEMG, enodes protoporphyrinogen IX oxidase. FC, encodes ferrochelatase. HO, encodes heme oxygenase. HY2, encodes phytochromobilin synthase. CHLD, CHLH and CHLI, encode Mg-chelatase. CHLM, encodes SAM Mg-protoporphyrin IX methyltransferase. CRD1, encodes Mg-protophyrin IX monomethylester cyclase. POR, encodes protochlorophyllide oxidoreductase. CHLG, encodes chlorophyll synthetase. CAO, encodes chlorophyll a oxygenase. 2

3 (B) Relative expression of genes involved in tetrapyrrole biosynthesis in the fhy3 far1 double mutant compared with No-0 WT seedlings grown in darkness for 5 d. The expression levels are normalized with an endogenous UBQ gene control. Mean ± SD from three biological replicates. Supplemental Figure 4. Confirmation of ALAD Antibody and Protein Level. (A) His-ALAD recombinant fusion protein was expressed in E. coli (top panel). Arrow indicates a specific band of approximately 55 kda. Both anti-his antibody and anti-alad antibody specifically recognized recombinant proteins induced by IPTG (lower panels). No band was found in the cell samples without IPTG induction. (B) ALAD protein levels in fhy3, far1, fhy3 far1 mutants and WT by immunoblotting using ALAD antibody. CBB, Coomassie brilliant blue. 3

4 Supplemental Figure 5. HEMB1 and HEMB2 Expression Patterns in Different Stages and Tissues. Samples were harvested from different tissues at various stages. The expression levels are normalized with an endogenous UBQ control. Mean ± SD from three biological replicates. 4

5 Supplemental Figure 6. Phenotype of HEMB1 Overexpression Plants. (A) Representative images of HEMB1 overexpression (35S:HEMB1) homozygous transgenic plants grown in soil for 3 weeks (16 h light / 8 h dark). Bar, 1 cm. (B) Quantitative RT-PCR showing high expression of HEMB1 in the 35S:HEMB1 transgenic lines. The expression levels are normalized to an UBQ endogenous control. Mean ± SD from three biological replicates. 5

6 Supplemental Figure 7. Identification and Characterization of HEMB1 T-DNA Insertion Mutant. (A) Diagram of the genomic structure of HEMB1 and location of T-DNA insertion. Black boxes indicate exons and arrows denote directions and approximate positions of primers used in (B). (B) PCR genotyping of hemb1-1 heterozygous mutant. M: DNA marker. (C) Comparison of embryo development in the hemb1-1 mutant. a-e, wild type-like embryos; f-j, arrested embryos. a and f, b and g, c and h, d and i, e and j are from the same siliques, respectively. Bars, 100 μm. 6

7 Supplemental Figure 8. Interaction between FHY3 and PIF1. (A) LCI assay between NLuc-fused FHY3 and CLuc-tagged PIF1 in Arabidopsis protoplasts. LUC activities were normalized with GUS controls. Mean ± SD, n=3. (B) HEMB1 expression in seedlings of various genotypes. Seedlings were grown in darkness for 5 d, and expression level was determined by real-time RT-PCR. The relative levels are normalized with an endogenous UBQ control. Mean ± SD from three biological replicates. 7

8 Supplemental Figure 9. FHY3 and HEMB1 Are Induced by Light. (A) Bioluminescence assay showing relative FHY3-LUC level of 4-d-old etiolated FHY3p:FHY3-LUC seedlings after light illumination. RLU, relative luminescence unit. Mean ± SD, n=3. (B) HEMB1 expression in fhy3 far1 mutant and No-0 wild type. 4-d-old dark-grown seedlings were exposed to white light for various time periods. The expression levels are normalized with an endogenous UBQ gene control. Mean ± SD of three biological replicates. (C) ALAD protein level determined by immunoblotting with ALAD antibody after light treatment. Anti-tubulin antibody (Agrisera) is used as loading control. Etiolated seedlings of Col wild type were exposed to light for various periods. 8

9 Supplemental Figure 10. Characterization of the arc5 Mutant Phenotype. (A) Relative Pchlide fluorescence of arc5 mutant and Ler wild type. Seedlings were grown in darkness for 5 d. (B) Greening rate of arc5 mutant and Ler when 5-d-old etiolated seedlings were transferred to light for 2 d. Mean ± SD, n=3. 9

10 Supplemental Table 1. List of Primers Used in This Study. Gene AGI gene code Oligo name Sequence (5'-3') Purpose HEMB1 At1g69740 Chip-a1 CCTTGTTTGTCAAGAAGGCA ChIP Chip-a2 GATGAAACAAGCTGCAATGG chip-b1 TCTATCAAGCCCAGTTCCAA chip-b2 GATGATAACTTTGGATGATCGC Chip-c1 AGGCTGAAGGAGCAGACATT Chip-c2 AACTTGGTATGCAGCAATCG HEMB1WF AATT CAAACTCGTAGATAAAGCGCGTGAACATCAGTCTCTAT Yeast one hybrid HEMB1WR TCGA ATAGAGACTGATGTTCACGCGCTTTATCTACGAGTTTG HEMB1MF AATT CAAACTCGTAGATAAAGCTTGTGAACATCAGTCTCTAT HEMB1MR TCGA ATAGAGACTGATGTTCACAAGCTTTATCTACGAGTTTG HEMB1F CCATGG TCATGGCTACTACACCCATC cdna amplification HEMB1R AGATCT CGCTTCTCGCCGCACAAACAAGTAGCAG HEMB1P1 GAATTC AAGCTT GTCAAGAAGGCAACTTTGCAC Promoter amplification HEMB1P2 GGATCC CTCGAG GAATCTGCAACACACACACAGGAACA HEMB1pm1 AAAGCAAGTGAACATCAGTCTC HEMB1pm2 ATCTACGAGTTTGAGAACTG HEMB1B1 AGAGCTC GCTAGTGAATCCGGCAATGGAC Recombinant protein HEMB1B2 AGTCGAC CCGCTTCTCGCCGCACAAACAAGTAG HEMB1 RealT f AGGCTGAAGGAGCAGACATT Real time PCR 10

11 HEMB1 RealT r AACTTGGTATGCAGCAATCG HEMB1I mir-s GATAACGATACTGTTTACCCCACTCTCTCTTTTGTATTCC amirna and RNAi HEMB1II mir-a GAGTGGGGTAAACAGTATCGTTATCAAAGAGAATCAATGA HEMB1III mir*s GAGTAGGGTAAACAGAATCGTTTTCACAGGTCGTGATATG HEMB1IV mir*a GAAAACGATTCTGTTTACCCTACTCTACATATATATTCCT RS300 A CTGCAAGGCGATTAAGTTGGGTAAC RS300 B GCGGATAACAATTTCACACAGGAAACAG HEMB1RNAif CCACTAGTGGTACCGTCGTAATCTTGTCGTAAGAG HEMB1RNAir AAGAGCTCGGATCCAAAGCCACATCAGTGTAG PIF1 At2g20180 PIF1-F GGTACCGAATTCATGGATCCTCAGCAGCAACCT Recombinant protein PIF1-R GTCGACACCTGTTGTGTGGTTTCCGT HEMA1 At1g58290 HEMA1RealTf GTTGCTGCCAACAAAGAAGA Real time PCR HEMA1RealTr AATCCCTCCATGCTTCAAAC HEMA2 At1g09940 HEMA2 RealTf GCTCCTGATCCAAACCATTT HEMA2 RealTr GATCAGGAATCGAGCAGACA HEMA3 At2g31250 HEMA3RealTf AGAGAAGCAGCATTGTGGTG HEMA3RealTr GTGATTCAAAGCGCACAACT GSA1 At5g63570 GSA1RealTf AATGACTGCAGGGATTCACA GSA1RealTr CCGTTGGTTAGTTCCTTCGT GSA2 At3g48730 GSA2RealTf CGGTTTCGGTAGAGGAGAAG GSA2RealTr TGGTTGTCCACCAACAGATT HEMB2 At1g44318 HEMB2RealTf AGAGAGCTGCCTTCCAAGAG 11

12 HEMB2RealTr GTCTCCAGCCAAGCATGTAA HEMC At5g08280 HEMCRealTf TAGCCTCACTGAACCACGAG HEMCRealTr TGCGTATCCAGCAATAGGAG HEMD At2g26540 HEMDRealTf GGTGCAGAGTGTTGATACGG HEMDRealTr GCAACGTAATTGCTCCATTG HEME1 At3g14930 HEME1RealTf TGAGCGAATGAAAGGAACTG HEME1RealTr CAGGAAGCGGAGAGAATAGG HEME2 At2g40490 HEME2RealTf AGACCGGCTAGGAAGAGACA HEME2RealTr TGAATCCGGCTTGTGATAAA HEMF1 At1g03475 HEMF1RealTf TAGGGCTGCTCAAGACAGTG HEMF1RealTr CTCAAAGACATTCCCGTCCT HEMF2 At4g03205 HEMF2RealTf TTCCGGAAACAGAAAGACCT HEMF2RealTr TTCTTCGACTGCTTCAATGG HEMG2 At5g14220 HEMG2RealTf ATTCCCGTTGTATGACAGCA HEMG2RealTr CCCTCGATGATTACCTGCAT HEMG1 At4g01690 HEMG1RealTf ACCGGAATTCTGTCCAAGTC HEMG1RealTr TGAGGCCATACCCTAACTCC HEMF3 At5g63290 HEMF3RealTf GACGCCTTCTCTTGTTCCTC HEMF3RealTr CCTTCAGCTTCTGACCATCA FC1 At5g26030 FC1RealTf CTCTACAACGGGTTCAAGCA FC1RealTr CTTCTCAATGAGGTCAGCCA FC2 At2g30390 FC2RealTf TTGCATATGTCGAAGAAGCC 12

13 FC2RealTr CTGGTCCAACTCTGCTCTGA HO1 At2g26670 HO1RealTf AAGTCGCCGTCTTTAGTGGT HO1RealTr AGCCACAAACCTCATCTCCT HO2 At2g26550 HO2RealTf CTTTCCCATTCCAAATCTCAA HO2RealTr TCTTCCTCTGTGAGGCCTTT HO3 At1g69720 HO3RealTf AGCCGACTGTGGAAGGATAC HO3RealTr AATCCCGTGTTCTTGAAACC HO4 At1g58300 HO4RealTf AGCAGAAGACTTGACTGCGA HO4RealTr TTTGCTCTTCGGACAAGATG HY2 At3g09150 HY2RealTf AGGCTTTGTTCTCTGCGTTT HY2RealTr TGGCTCTCACATGAGATGGT CHLD At1g08520 CHLDRealTf GTGGTGGTGGTTCTCCTCTT CHLDRealTr TCGGTTATCGCAACAATCAT CHLH At5g13630 CHLHRealTf CAGCCAACATCAGTCTTGCT CHLHRealTr ACCTGCTTCTTCTCAGCCAT CHLI1 At4g18480 CHLI1RealTf GGAATCCAAATAAGGCCAAA CHLI1RealTr AAATGGATAAACCGGTCTCG CHLI2 At5g45930 CHLI2RealTf CATCCTGCTCGGTTTATCCT CHLI2RealTr GCGTCTCTAACCGTCCCTAC CHLM At4g25080 CHLMRealTf GCTATGGTTGCTGAAGCTGA CHLMRealTr GCGGGTAATGTATCAACACG CRD1 At3g56940 CRD1RealTf AGAGTTCGAGGCTCTGCTTC 13

14 CRD1RealTr CAACGAAGATCTGTCGGAGA PORA At5g54190 PORARealTf TCTCCTCTGCTTTCTCTGTC PORARealTr GGATCCAACAATGATGAGACG PORB At4g27440 PORBRealTf CAAAGATGCGAAGTTGAATGC PORBRealTr GATTCGCCTTCGGTGGTACA PORC At1g03630 PORCRealTf ACTGGAGCTTCGTCTGGTTT PORCRealTr AGAAAGTTCCTGCAAGCCATG DVR At5g18660 DVRRealT-F AGCAGCGTTTATAGCGGATT DVRRealT-R CTCCTTGCTCTAATGGCGTT CAO At1g44446 CAORealTf TGAACGAGGGACGTATTCAA CAORealTr GGCCAAATCCAGATCATACC CHLG At3g51820 CHLGRealTf CTTATTATGCGTTGGCGTTG CHLGRealTr TGCCGTTACAAATATTCCGA UBQ At3g52590 UBQf TTCCTTGATGATGCTTGCTC UBQr TTGACAGCTCTTGGGTGAAG 14