Supplementary Information. A chloroplast envelope bound PHD transcription factor mediates. chloroplast signals to the nucleus

Transcription

1 Supplementary Information A chloroplast envelope bound PHD transcription factor mediates chloroplast signals to the nucleus Xuwu Sun, Peiqiang Feng, Xiumei Xu, Hailong Guo, Jinfang Ma, Wei Chi, Rongchen Lin, Congming Lu & Lixin Zhang* Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing , China. Correspondence and requests for materials should be addressed to L.Z. (zhanglixin@ibcas.ac.cn). 1

2 Supplementary Figure S1. The visible phenotypes and identification of the mutants. (a) The wild-type (WT), gun1, abi4, and ptm plants were grown under normal growth conditions. (b) Schematic diagram of T-DNA location in the PTM gene (not to scale). Closed blue boxes represent exons. Green boxes represent introns. Red boxes represent the untranslated regions of the gene. Arrows indicate the location of primers used for PCR analyses; LB: left border primer. FP and RP represent the forward primer and the reverse primer, respectively. PCR analysis of genomic DNA from WT and ptm were used to confirm the homozygosity of the mutants. RT-PCR analysis was performed to analyze PTM gene expression. RT-PCR was performed with primers specific to PTM and actin. (c) Immunoblot analysis of PTM in WT and ptm mutant plants. Total proteins (30 µg) isolated from WT and ptm were separated by SDS-PAGE and immunodetected with anti-ptm. Immunoblot analysis with anti-h3 and anti-dhar2 were performed as controls. The black arrows indicate the full-length PTM protein, and the red arrows indicate the processed form of full-length PTM. (d) Schematic diagram of the T-DNA location in the GUN1 gene (not to scale). PCR analysis of genomic DNA from WT and gun1 were used to confirm the homozygosity of the mutants. RT-PCR analysis was performed to analyze GUN1 gene expression with primers specific to GUN1 and actin. (e) PCR analysis of genomic DNA from WT and mutant plants for the confirmation of the homozygosity of ptm abi4. (f) PCR analysis of genomic DNA from WT and mutant plants for the confirmation of the homozygosity of ptm gun. 2

3 Supplementary Figure S2. qrt-pcr analysis of expression of Lhcb and APX2. (a) Transcript levels of Lhcb in the presence of Lin. WT, ptm, abi4, and gun1 seedlings were grown in the presence of Lin for four days in the dark. After treatments, RNA was extracted, and the levels of Lhcb mrna were analyzed by qrt-pcr. (b) Transcript levels of APX2 in WT, ptm, abi4, and gun1 under HL. Four-week-old WT, abi4, ptm, and gun1 mutant seedlings grown under normal light were exposed to HL intensities for the indicated times. After treatments, RNA was extracted and the levels of APX2 mrna were analyzed by qrt-pcr. (c), Transcript levels of Lhcb in the presence of DMBIB, paraquat, and rose bengal. WT, ptm, gun1 seedlings were grown for 3 weeks, and treated with DMBIB (50 μm), paraquat (25 μm), and rose bengal (50 μm) for 12 h under normal light conditions. After treatments, RNA was extracted and the levels of Lhcb mrna were analyzed by qrt-pcr. (d), Transcript levels of Lhcb in the presence of Lin. The WT, ptm, abi4, gun1, and N-PTM/gun1 seedlings were grown in the presence of Lin, and harvested after 5 days. The samples without Lin treatment were used as controls (Con.). After treatments, RNA was extracted, and the levels of Lhcb mrna were analyzed by qrt-pcr. Values are means of triplicate measurements with error bars representing the standard deviation. 3

4 Supplementary Figure S3. PTM is conserved in Sorghum, Selaginella, Vitis and Oryza. (a) Sequence alignment of PTM with putative Sorghum, Selaginella, Oryza, and Vitis. Multiple alignments were performed using ( and edited with ESPript ( Identical amino acid residues and similar amino acid residues are depicted in white on black background and in black on white background, respectively. The DDT and PHD domains are labeled, and the conserved cysteines and histidines of the C4HC3 motif are indicated by asterisks. For clarity, the less conserved extremities of the alignment were removed. (b) The spatial arrangement of DDT domain, PHD finger, and transmembrane regions in PTM and homologs in Sorghum, Selaginella, Oryza, and Vitis (not to scale). Prediction of transmembrane regions was performed using Tmpred ( 4

5 Supplementary Figure S4. The prediction of PHD domain structure. (a) The Arabidopsis PTM protein contains a DDT domain and a PHD motif in the N-terminal region. Transmembrane helices were predicted by the TMHMM Server v. 2.0 ( and are indicated by. (b) Alignment of the Arabidopsis PHD motifs of PTM with those of Human, Oryza and Vitis PHD-containing proteins. The main PHD regions were color-coded as indicated. Asterisks indicate key cysteine and histidine residues. (c) Superimposition of the predicted PHD domain structure of Vitis (yellow), Arabidopsis (blue), and Oryza (purple) to the NMR solution structure of Human (green) PHD domain (PDB code 1XWH). The core structure possesses a small, two-stranded anti-parallel -sheet. The domain is presumably stabilized by two Zn atoms (purple balls) chelated by seven cysteines and one conserved histidine. The heavy atoms of the conserved cysteines and histidine residues of the C4HC3 motif of these proteins are shown using a stick model. 5

6 Supplementary Figure S5. Immunoblot analysis of PTM. (a) Salt washing of chloroplast outer envelope membrane. The outer envelope sample was treated with 0.5 M NaCl, and 0.1 M Na 2 CO 3, ph 11.5 as indicated. After treatments, the outer envelope sample was pelleted and the pellets were separated by SDS-PAGE followed by immunodetection with anti-ptm. The anti-h3 and anti-toc75 antibodies were used as controls. (b) Immunoblot analysis of PTM with anti-c-ptm. Total proteins (30 µg) or nuclear extract were isolated from the HL treated leaves, separated by SDS-PAGE followed by immunodetection with anti-c-ptm. The anti-h3 antibody was used as a control. (c) Effects of high light on PTM processing in WT and gun1 mutant plants. Total proteins (30 µg) were isolated from the HL treated leaves of WT and gun1 mutant, separated by SDS-PAGE and immunodetected with anti-ptm. The anti-h3 antibody was used as a control. (d) Effects of protease inhibitors on PTM processing. Total proteins (30 µg) were isolated from the HL treated leaves in the presence of pefabloc (+PI1) and pepstatin (+PI2) or absence of protease inhibitors (-PI) for 6 hrs, separated by SDS-PAGE and immunodetected with anti-ptm. The anti-h3 antibody was used as a control. The black arrows indicate the full-length PTM protein, and the red arrows indicate the processed form of full-length PTM. (e) In vitro cleavage of PTM. The intact chloroplasts were treated with Lin in the presence (+PI) or absence (-PI) of protease inhibitor pefabloc for 3, or 6 hrs, separated by SDS-PAGE followed by immunodetection with anti-ptm. The anti-psbo antibody was used as a control. These assays (a-e) were repeated three times, and the results from a representative experiment are shown. 6

7 Supplementary Figure S6. Seed germination phenotypes in the presence of ABA. Germination of WT (dark square), gun1 (red circle), abi4 (blue triangle), and ptm (deep blue inverted triangle) after 7 days by the indicated concentration of ABA. Values are means of triplicate measurements with error bars representing the standard deviation. 7

8 Supplementary Figure S7. Expression of PTMp::Luc/WT and ABI4p::Luc/WT in response to stress treatments. (a) and (b) The LUC activities of transgenic PTMp::Luc/WT and ABI4p::Luc/WT lines after HL treatment. Four-week-old transgenic PTMp::Luc/WT (a) and ABI4p::Luc/WT (b) lines were subjected to HL treatment for various numbers of hours, and the LUC activities were measured. (c) and (d) The LUC activities of transgenic PTMp::Luc/WT and ABI4p::Luc/WT lines after treatments with Lin and NF. The transgenic PTMp::Luc/WT (c) and ABI4p::Luc/WT (d) lines were grown in the ½ LS media in the presence of Lin(white) and NF (gray), and the LUC activities were measured. The samples without treatments (black) were used as controls. (e) and (f) The LUC activities of transgenic ABI4p::Luc/WT and Lhcbp::Luc/WT lines after HL treatment. Four-week-old transgenic ABI4p::Luc/WT (e) and Lhcbp::Luc/WT (f) lines were subjected to HL treatment in the presence of pefabloc (+PI1) and pepstatin (+PI2) or absence of protease inhibitors (-PI) for 6 h, and the LUC activities were measured. The samples without HL treatments were used as controls (Con.). Values are means of triplicate measurements with error bars representing the standard deviation. 8

9 Supplementary Table S1. A list of synthesis peptides used in this study Name Purpose Peptides PTM Anti-body KEKEVTDSSTNESKDLDSRCTNK(KLH) H3 ARTKQTARKSTGGKAPRKQ H3K4Me3 Pull-down ARTK(Me3)QTARKSTGGKAPRK(Bio)Q H3K9Me3 Anti-body ARTKQTARK(Me3)STGGKAPRK(Bio)Q H4K20Me3 SGRGKGGKGLGKGGAKRHRK(Me3)V(Bio)L 9

10 Supplementary Table S2. A list of oligonucleotides used in this study Purpose Gene Name Sequence (5' > 3') Plant transformation psn1301-ptm PTM PTM1301s GAC CCG GGA TGG AAG CGA AGG TTC CTA GAC C PTM1301a GGG AGC TCG GTG TTC CGG CAG ATT AGT ACG T pcam1301- PTMNs GCA TCG ATA TGG AAG CGA AGG TTC CTA GAC PTM N-PTM PTMNa CGG AAT TCG ACA GAT ATG TTA AGC CTG TG GR GR GRs GGC CTC GAG GGA GTC TCA CAA GAC ACT TC GRa CGC ATC GAT TCA TTT TTG ATG AAA CAG AA GAA TTC GAG CTC GGT ACC GTA AAT CGG AAT TTT PTMguss pcam1301- ATT AGA TTC A PTM PTM-GUS CTC AGA TCT ACC ATG GTG TTC TAT GTC CAA AAC PTMgusa ATA CTG CT Transient expression assay N-PTM-GFP PTM PTMgfps CCC ATC GAT ATG GAA GCG AAG GTT CCT AGA CC PTMgfpa CCG GAA TTC GAC AAC TGC ATC TCC ATT GAC AG ABI4p::LUC ABI4 ABI4lucs ACC GAA GCT TAT TTT AGA GGT GAC CAT TTG AAC ABI4luca TCC GGG ATC CGT GTT GAT GTT GGG AAG CTA AAG PTMp::LUC PTM PTMlucs GCA AGC TTG ATC TTA GCA AAG AAG TAA ATC GG PTMluca ATG GAT CCG GGG AAT TTC ATA CTC TAT GGC Lhcbp::LUC Lhcb Lhcblucs GCA AGC TTT GAG CCT GTG AGG TCT AGT GAT TGT Lhcbluca ATG GAT CCA GTG AGA GTG ATT AAA ACT GGT TCG Recombinant protein expression pgst-ptm-phd PHD(D412A) PHD(G421A) PTM PHD(L423A) PHD (C426A,C429A) Yeast transcription activity gstphds CCG GAA TTC AAC GAG TCT AAA GAT TTG GAC gstphda ACG CGT CGA CAG AAG AGT CGC CCA TGT G (D412A)s GAT CTA GAT GGA AAC AGT GCG GAA TGT AGG AT (D412A)a CGC ACT GTT TCC ATC TAG ATC TGA GCT AAC T (G421A)s AGG ATT TGT GGA ATG GAT GCG ACC TTG CTT TGT (G421A)a CGC ATC CAT TCC ACA AAT CCT ACA TTC ATC AC (L423A)s TTG TGG AAT GGA TGC GAC CGC GCT TTG TTG TGA (L423A)a CGC GGT CGC ATC CAT TCC ACA AAT CCT ACA TTC (C429A)s CTT TGT GCT GAT GGA GCT CCT TTA GC (C429A)a GCT CCA TCA GCA CAA AGC AAG GTT CC CAT GGA GGC CGA ATT CAT GGA AGC GAA GGT TCC PTM PTMbds pgbkt7 TAG ACC -PTM GCA GGT CGA CGG ATC CGG TGT TCC GGC AGA TTA PTMbda GTA CG T CAT GGA GGC CGA ATT CAT GGA AGC GAA GGT TCC pgbkt7 PTMNbds TAG ACC -N-PTM PTMNbda GCA GGT CGA CGG ATC CG ACA GAT ATG TTA AGC 10

11 CTG TG N-PTM (C429A)s CTT TGT GCT GAT GGA GCT CCT TTA GC (C426A,C429A) (C429A)a GCT CCA TCA GCA CAA AGC AAG GTT CC N-PTM (C426A)s GAT GGA ACC TTG CTT TGT GCT GAT GGA TGT CCT (H426A) (C426A)a GCA CAA AGC AAG GTT CCA TCC ATT CCA CAA ATC N-PTM (C429A)1s CTT TGT TGT GAT GGA GCT CCT TTA GCA TAC (H429A) (C429A)1a GCT CCA TCA CAA CAA AGC AAG GTT CCA TCC N-PTM (C417A)s GAT GAA GCT AGG ATT GCT GGA ATG GAT (C414A,C417A) (C417A)a GCA ATC CTA GCT TCA TCA CTG TTT CC N-PTM (H434A)s GAT GTC CTT TAG CAT ACG CCT CAA GAT G (H434A) (H434A)a GCG TAT GCT AAA GGA CAT CCA TCA CAA C N-PTM (C437A)s GCA TAC GCC TCA AGA GCC ATT GGT GTG (H434A,C437A) (C437A)a GCT CTT GAG GCG TAT GCT AAA GGA CAT G N-PTM (C455A)s TGG TTT GCT CCA GAG GCC ACT ATT AAC (C452A,C455A) (C455A)a GCC TCT GGA GCA AAC CAT GGC CCA TC Yeast one-hybrid assay GAD-PTM GAD-PTMDDT PTM GAD-PTMPHD GAD- PTM(DDT+PHD) ABI4p-1::LacZ ABI4p-2::LacZ ABI4p-3::LacZ ABI4p-4::LacZ Mutant confirmed PTMgads CCG GAA TTC ATG GAA GCG AAG GTT CCT AG PTMgada CGC CTC GAG GAC AGA TAT GTT AAG CCT GTG PTMddts CCG GAA TTC GTT CCA CCA GTG GAT TTA CCT C PTMddta TAC TCG AGT TTC CTT GTA AGC TGA GGT TTT AG PTMphds CCG GAA TTC AAC GAG TCT AAA GAT TTG GAC PTMphda ACG CCT CGA GAG AAG AGT CGC CCA TGT G PTMddts CCG GAA TTC GTT CCA CCA GTG GAT TTA CCT C PTMphda ACG CCT CGA GAG AAG AGT CGC CCA TGT G ABIas CGG AAT TCT GAT CCT TTT TAG TAT ACT TAT CTG ABIaa CGC TCG AGA ACA AAG AAA ATG ATT TAT GAG TTC ABIbs CGG AAT TCT TTT CTT TGT TAT CTT TGT CTA CTG ABIba CGC TCG AGA AAT TCA CCA AGG TTA GGA ATA TTG ABI4 ABIcs CGG AAT TCA ATA TTC CTA ACC TTG GTG AAT TTG ABIca CGC TCG AGT GTT ACA CAT TGG AAT GTA ATC AAG ABIds CGG AAT TCT ACA TTC CAA TGT GTA ACA AGT AAC ABIda CGC TCG AGA AGA AGA GGA AGA GGA AGT AGA GAG PTM_LP PTMLP TTG ACA ACT GCA TCT CCA TTG PTM PTM_RP PTMRP CTA GCA GAT TTG GTC ATT GGG GUN1_LP GUN1LP ATG TTT AGT AGC CAC GCA TGG GUN1 GUN1_RP GUN1RP TTG ATC GAT GGG TAC TCG AAG SALK_LB SalkLB ATT TTG CCG ATT TCG GAA C SAIL_LB SailLB TAG CAT CTG AAT TTC ATA ACC AAT CTC GAT AC Semi-quantitative RT-PCR GUN1 Gun1rts TCT GCA ACT ATG GAC AGA TCA GCC G 11

12 Gun1rta PTM PTMrts PTMrta ABI4 Abi4rts Abi4rta actin actins actina Real-time RT-PCR GUN1 Gun1rt1s Gun1rt1a PTM Srert1s Srert1a ABI4 Abi4rt1s Abi4rt1a LHCB Lhcbrt1s Lhcbrt1a APX2 Apx2rts Apx2rta actin Actrt1s Actrt1a Northern blotting LHCB Lhcbs Lhcba APX2 APXs APXa Chromatin immuno-precipitation (CHIP) chipas ABI4p-1 chipaa chipbs ABI4p-2 chipba ABI4 chipcs ABI4p-3 chipca chipds ABI4p-4 chipda Actchips actin Actchipa CAC GCA GCC AAG TTG CTA CCA CC GGG CTA GCT GCA TTG ATT GGA GCT TGC GCA AGC TTC TCT GGA CAA AAC CAT GGC GGC GCT AGC CTA ACC CAT AAT AAT CCT CAA TCC CGC AAG CTT ACT AGG GCA TAA CAT AGA GGT CCC AAC TGG GAT GAT ATG GAG AA CCT CCA ATC CAG ACA CTG TA TTG GAG ACG GAG CAC TAA GAC GA GAA GAG GCT GTA AAG CAA ACG AC TCT ACT CCG TTA CCT GCC ACT CC CTC TGC CTC GGG TTT CAC AAT A GCG TTA GGG CAG GAA CAA GG TCC AGA CCC ATA GAA CAT ACC G CGT GAC CAT GCG TCG TAC CGT C CCT CAG GGA ATG TGC ATC CG CAT GGA CAC CAA ACC CGC TCA TTT TCA AGA AGA GCC TTG TCG GTT GGT GGT AAC ATT GTG CTC AGT GGT G CTC GGC CTT GGA GAT CCA CAT C GAG AGT TCC CTG GAG ACT ACG G TTC CGG GAA CAA AGT TGG TG GAT GCC AAC AAT GGT CTT GAT ATT G AGC ATA TTT TTC AAC AAA TGG GAG A ATA CTT ATC TGT TAA TGC TTG TTA ACC A ATC AAG TGA ATA AAA CAG TAG ACA AA AAC GAT GCT TTT AAT TCT TGA ACT C AGA GAA CTG AAT CAA ATT CAC CAA G TAT ATA TGT TGC AAT ATT CCT AAC C GAA ATT TGT GTT GTC ATA AGT TAC T TGT TTT TAT AAT ATC CTT GAT TAC ATT CCA AGA TGA AGA AGA AGA AGA AGA AGA AGA GGA GGT AAC ATT GTG CTC AGT GGT GG AAC GAC CTT AAT CTT CAT GCT GC 12

13 Supplementary Methods Mutant complementation and generation of double mutants To generate PTM/ptm, psn1301-ptm, driven by the 35S promoter, was introduced into lines homozygous for the ptm mutation. To generate N-PTM/gun1, pcambia1301-n-ptm was introduced into homozygous of the gun1 mutation. To generate N-PTM-GR/ptm and N-PTM/ptm, pcambia1301-n-ptm-gr and pcambia1301-n-ptm were introduced, respectively, into lines homozygous for the ptm mutation. To generate the double mutants ptm gun1 and ptm abi4, the gun1 and abi4 mutants, respectively, were crossed with ptm. The ABI4p::Luc/ptm mutant was obtained by crossing the ABI4p::Luc/WT with ptm. The double mutant ptm gun1 homozygous lines were confirmed by PCR using the following gene specific and T-DNA specific primers: PTMLP, PTMRP, and T-DNA LB for PTM and GUN1LP, GUN1RP, and T-DNA LB for GUN1. To screen the double mutants ptm abi4, the M2 seeds were surface-sterilized and plated on ½ Linsmaier and Skoog (½ LS) medium (PhytoTechnology Laboratories, LLC, USA) containing 2% sucrose and 0.8% agar in the presence of 1.5 µm ABA (Sigma Aldrich) and the seedlings grown normally were selected for further confirmation by PCR using the following gene specific and T-DNA specific primers: PTMLP, PTMRP, and T-DNA LB for PTM. Constructs for plant transformation To generate the psn1301-ptm, the full-length cdna of PTM was PCR amplified using PrimeSTAR HS DNA polymerase (Takara) from the Arabidopsis cdna that was obtained by RT-PCR with a PrimeScript II 1st Strand cdna Synthesis Kit and then inserted into the SamI and SacI sites of the binary vector psn1301. To generate the pcambia1301-n-ptm, we first generated the pbsk-35s vector. The 35S promoter and PolyA were PCR amplified using PrimeSTAR HS DNA polymerase (Takara) from the binary vector pub-gfp, and the 35S promoter and the PolyA were subsequently inserted into the KpnI and XhoI and the XbaI and SacI sites of pbluescript_sk+, respectively, to produce the pbsk-35s vector. Then the N-terminus (1-1,560 bp of PTM) that contains the complete DDT and PHD finger domain was 13

14 PCR amplified using PrimeSTAR HS DNA polymerase from a PTM full-length cdna clone and inserted into the ClaI and EcoRI sites of pbsk-35s. Finally, the fragment of 35S::N-PTM was cut by KpnI and SacI and then inserted into the KpnI and SacI sites of pcambia1301. To generate the pcambia1301-n-ptm-gr, the GR steroid-binding domain was PCR amplified using PrimeSTAR HS DNA polymerase and then inserted into the XhoI and ClaI sites of pcambia1301-n-ptm. To construct the transcriptional LUC reporter vectors, the promoter regions of the PTM, Lhcb, and ABI4 were amplified by PCR using gene-specific primers (Table S1). The amplified DNA fragments were digested with HindIII and BamHI and then inserted into the HindIII and BamHI sites of the PUC-35S::Luc vector for the replacement the 35S promoter to produce PUC-PTMp::Luc, PUC-Lhcbp::Luc, and PUC-ABI4p::Luc. These three vectors were subsequently digested with HindIII and SacI and inserted into the HindIII and SacI sites of the binary vector PCAMBIA1301, thus generating the PCAMBIA1301-PTMp::Luc, PCAMBIA1301-Lhcbp::Luc, and PCAMBIA1301-ABI4p::Luc vectors, respectively. To construct the PTM-GUS reporter vectors, the promoter region and the full open reading frame region of PTM were amplified by PCR using the gene-specific primers (Table S1). The amplified DNA fragment was inserted into the KpnI and NcoI sites of the binary vector PCAMBIA1301, and the PCAMBIA1301-PTM-GUS vector was generated using the In-Fusion PCR Cloning Kit (Clontech). Constructs for the transcription activity assay To generate pgbkt7-ptm and pgbkt7-n-ptm, the N-terminal (1-1,560 bp of PTM) and full-length PTM were PCR amplified using PrimeSTAR HS DNA polymerase from Arabidopsis cdna and then inserted into the EcoRI and BamHI sites of the pgbkt7 vector using the In-Fusion PCR Cloning Kit. To generate the N-PTM (C414A), N-PTM (C417A), N-PTM (C426A, C429A), N-PTM(C426A), N-PTM(C429A), N-PTM (H434A), N-PTM (H434A, C437A), and N-PTM (C452A, C455A), the pgbkt7-n-ptm was used as template with the Fast Mutagenesis System (Transgen Biotech) according to the manufacturers instructions. 14

15 Constructs for the yeast one-hybrid assay To generate GAD-N-PTM (1-1,560 bp), GAD-PTMDDT (526-1,132 bp), GAD -PTMPHD (1,156-1,456 bp), and GAD-PTM (DDT+PHD) (526-1,456 bp), the N-termini (1-1,560 bp, 526-1,132 bp, 1,156-1,456 bp, and 526-1,456 bp of PTM) were PCR amplified using PrimeSTAR HS DNA polymerase from Arabidopsis cdna and then inserted into the EcoRI and XhoI sites of the pjg4-5 vector. To generate the ABI4p-1::LacZ, ABI4p-2::LacZ, ABI4p-3::LacZ, and ABI4p-4::LacZ reporter genes, the upstream bp of the ABI4 gene initiation codon, ATG, was split into 4 fragments with an overlap of approximately 250 bp, referred to as ABI4p-1 (-951/-708), ABI4p-2 (-717/-480), ABI4p-3 (-507/-270), and ABI4p-4 (-288/-20). These overlapping fragments were PCR amplified using PrimeSTAR HS DNA polymerase from genomic DNA and then inserted into the EcoRI and XhoI sites of the reporter plasmid placzi (Clontech). Constructs for the transient expression assay in Arabidopsis protoplasts To generate the effector constructs, pbsk-35s::n-ptm and pbsk-35s::n-ptm (C426A, C429A), the N-terminus (1-1,560 bp of PTM), which contains the complete DDT and PHD finger domain, was PCR amplified using PrimeSTAR HS DNA polymerase from a PTM full-length cdna clone and inserted into the ClaI and EcoRI sites of pbsk-35s. To generate the pbsk-35s::n-ptm (C426A, C429A), the pbsk-35s::n-ptm was used as a template with the Fast Mutagenesis System and verified by sequencing. To generate the ABI4p::Luc reporter gene, the sequence from bp upstream of the ABI4 gene initiation codon, ATG, was PCR amplified using PrimeSTAR HS DNA polymerase from genomic DNA and then inserted into the HindIII and BamHI sites of the puc-35s::luc vector. Construct for recombinant protein expression To express the GST-PTM-PHD recombinant fusion protein, a fragment containing the PHD finger (386 aa-485 aa) of PTM was amplified by PCR using PrimeSTAR HS 15

16 DNA polymerase with primers PHDgsts and PHDgsta. The PCR fragments were digested with EcoRI and SalI and then inserted into the EcoRI and SalI sites of the pgex-5x-1 vector (Amersham Biosciences) to generate pgst-ptm-phd. To generate the mutants, pgst-ptm-phd (D412A), pgst-ptm-phd (G421A), pgst-ptm-phd (L423A), and pgst-ptm-phd (C426A, C429A), pgst-ptm-phd was used as a template with the Fast Mutagenesis System and verified by sequencing. Plant transformation The psn1301-ptm, pcambia1301-n-ptm, PCAMBIA1301-ABI4p::Luc, PCAMBIA1301-PTMp::Luc, PCAMBIA1301-Lhcbp::Luc, PCAMBIA1301-PTM-GUS, and pcambia1301-n-ptm-gr constructs were transformed into Agrobacterium tumefaciens strain C58 via electroporation. Then the Agrobacterium tumefaciens that contained the constructs of psn1301-ptm, pcambia1301-n-ptm, and pcambia1301-n-ptm-gr were introduced into the corresponding homozygous ptm line. The Agrobacterium tumefaciens that contained the constructs of pcambia1301-n-ptm was introduced into the homozygous gun1 line. The Agrobacterium tumefaciens that contained the constructs of PCAMBIA1301-ABI4p::Luc, PCAMBIA1301-PTMp::Luc, PCAMBIA1301-Lhcbp::Luc, and PCAMBIA1301-PTM-GUS were introduced into wild-type plants 48. The transformant plants were selected on a medium containing ½ Murashige and Skoog salt mix (PhytoTechnology Laboratories, LLC, USA), 50 μg/ml hygromycin, 2% sucrose, and 0.8% agar. The resistant plants were transferred to soil to grow to maturity, and their transgenic status was further confirmed by PCR analyses. Homozygous transgenic plants were used in all experiments. LUC analysis by CCD imaging The luciferase assay was performed using the substrate luciferin (Promega, Cat. E1602). The seedlings were sprayed with 1 mm luciferin and left in the dark for 10 minutes to take up the luciferin substrate. The seedlings were imaged with the CCD 16

17 imaging system (CHEMIPROHT 1300B/LND, 16 bits; Roper Scientific). Each image was recorded for 5 min and analyzed using the software program WinView/32. Structure modeling of the PTM PHD finger To generate the PTM PHD finger 3D structures, the 3D structure of NMR Structure of the first PHD finger of autoimmune regulator protein of human (AIRE1) (the PDB ID: 1XWH) was used as a template to generate an appropriate 3D structure 49. 3D models for the PTM PHD finger were constructed based on the template structures using MODELLER 50. GFP fusion constructs for transient expression in protoplasts Isolation of the Arabidopsis protoplasts and polyethylene glycol-mediated transfection were performed as described 51. Fusion proteins with sgfp were transiently expressed in protoplasts under the control of the cauliflower mosaic virus 35S promoter, and the GFP signals were obtained by confocal microscopy. The chloroplasts were visualized by chlorophyll autofluorescence. The constructs used for transformation were Chl., signal of chloroplast localization protein, RbcL; Nuc., signal of nuclear localization protein, fibrillarin; Mit., signal of mitochondrial localization protein, FRO1; N-PTM, signals from the N-PTM-GFP fusion protein. The GFP signals were examined using a confocal laser scanning microscope (LSM510; Carl Zeiss). GUS staining and GUS enzyme activity The GUS staining was performed essentially as described 52. The leaves were completely submerged in the GUS staining solution (50 mm sodium phosphate buffer ph 7.0, 10 mm EDTA ph 8.0, 0.1% (v/v) Triton X-100, 0.5 mg/ml X-Gluc) following vacuum infiltration. After incubation at 37 C with agitation for approximately 10 hours, the samples were transferred to 70% EtOH to remove their chlorophyll. For the -glucuronidase (GUS) enzymatic assay, 5 μl of the extract was incubated with 50 μl of a 4-methylumbelliferyl -D-glucuronide assay buffer (50 mm sodium phosphate ph 7.0, 1 mm 4-methylumbelliferyl -D-glucuronide, 10 mm EDTA, 10 mm 17

18 -mercaptoethanol, 0.1% sarkosyl, 0.1% Triton X-100) at 37 C for 15 min, and the reaction was stopped by the addition of 945 μl of 0.2 M Na 2 CO 3. The fluorescence was measured using a Modulus Luminometer/Fluorometer with a UV fluorescence optical kit (Turner Biosystems). Supplementary References 48. Zhang, X., Henriques,R., Lin, S.S., Niu, Q.W. & Chua, N.H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral-dip method. Nat. Protoc. 1, (2006). 49. Bottomley, M.J., Stier, G., Pennacchini, D., Legube, G., Simon, B., Akhtar, A., Sattler, M. & Musco, G. NMR structure of the first PHD finger of autoimmune regulator protein (AIRE1). Insights into autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) disease. J. Biol. Chem. 280, (2005). 50. Sali, A., Potterton, L., Yuan, F., van Vlijmen, H. & Karplus, M. Evaluation of comparative protein modeling by MODELLER. Proteins 23, (1995). 51. Sun, X.W., et al. The stromal chloroplast Deg7 protease participates in the repair of photosystem II after photoinhibition in Arabidopsis. Plant Physiol. 152, (2010). 52. Jefferson, R.A., Kavanagh, T.A. & Bevan, M.W. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, (1987). 18