For all experiments, ... 22°C, and were watered daily with a 1:1500 dilution of Miracle-Gro 20-20-20 fertilizer. .... Fixed tissues were ground in liquid nitrogen and powder was ... EDTA, 1% SDS, 1mM PMSF, 1% Plant Protease Inhibitor cocktail).
Supplemental information for The SAND domain protein ULTRAPETALA1 acts as a trithorax group factor to regulate cell fate in plants
Cristel C. Carles and Jennifer C. Fletcher
This PDF file contains: Supplemental Materials and Methods Supplemental Text Supplemental References Supplemental Table S1 and S2 Supplemental Figures S1 through S6
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Supplemental Materials and Methods Plant material and growth conditions The Arabidopsis thaliana Landsberg erecta (Ler) ecotype, in which the ult1-1 through ult1-3 alleles were previously described (Fletcher 2001; Carles et al. 2005), was used as the wild-type strain. The clf-2 (Goodrich et al. 1997), ag-3 (Bowman et al. 1991), ap3-3 (Jack et al. 1992) and knat2 (Byrne et al. 2002) mutant alleles were also in the Ler background. For all experiments, plants were grown either on Murashige and Skoog (MS) salts medium or on a 1:1:1 mixture of perlite:vermiculite:topsoil under continuous cool-white fluorescent lights (120 μmol m–2 s–1) at 22°C, and were watered daily with a 1:1500 dilution of Miracle-Gro 20-20-20 fertilizer. Transgenic lines were generated by the floral dip method (Clough and Bent 1998). Genetic analyses Double mutants were generated by crossing plants homozygous for each ult1 allele separately to clf-2 homozygous plants. Plants were genotyped using the primers: oult1-1 CAPS RsaI-F and oULT1-R for the ult1-1 EMS allele; oult1-2dCAPS-F and oult1-2dCAPS-R for the ult1-2 EMS allele; oULT1-F, oULT1-R and pROK2LBb1 for the ult1-3 insertion allele; oCLFex1-F, oCLFex2-R and oDs-3’ for the clf-2 insertion allele. Phenotypic analyses were performed on the F2 and F3 generations. For analysis of 35S::ULT1 effects in ag-3, ap3-3 and knat2 mutant backgrounds, crosses were performed between 35S::ULT1 homozygous lines and ag-3/+, ap3-3/+ or knat2 plants. Because class I 35S::ULT1 plants display fertility defects, the 35S::ULT1 construct was transformed into ag-3/+ plants in order to conduct statistical analyses of the effects of high levels of ectopic ULT1 in ag-3/+. The F2, T1 and T2 generations were analyzed for 35S::ULT1 phenotypes. Primer sequences used for genotyping are listed in Supplemental Table S2.
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Constructs for transgenic plants and transient assays 35S::ULT1 construct The full-length ULT1 coding sequence was cloned into the binary vector pCD223 (kindly provided by Chris Day) at the EcoRI site, flanked 5 by a double CaMV 35S promoter and 3 by a nopaline synthase transcription termination signal. The clones were then screened by PCR to obtain the ULT cDNA insert in the sense (S) orientation. Transgenic plants were selected on MS medium containing gentamycin (100 μg/ml). 35S::HA-ULT1 construct The full-length ULT1 coding sequence was cloned into the pENTR/D-TOPO vector for subsequent recombination into the binary vector pEarleyGATE201 (Earley et al. 2006). The pEarleyGATE201 empty vector or the pEarleyGATE-ULT1 construct was introduced into ult1-2 plants and transformants were selected on MS medium containing BASTA (15 μg/ml). 35S::ULT1-(Ala)10-NY and 35S::ULT1-(Ala)10-YC constructs The pEZS-CL vectors carrying the CaMV 35S-MCS-(Ala)10-NY cassette or the CaMV 35SMCS-(Ala)10-YC cassette were kindly provided by Bassem Al Sady and are modified version of the pEZS-CL plasmid generated by David Ehrhardt. The ULT cDNA fragments were cloned into the EcoRI and BamHI sites of the MCS to give the pEZS-CL-ULT1-NY and pEZS-CL-ULT1YC constructs. Primer sequences used for cloning are listed in Supplemental Table S2. RT-PCR and quantitative real-time RT-PCR (RT-qPCR) Total RNA was isolated from 4 day-old-seedlings or rosette leaf tissues using the RNeasy plant kit (Qiagen), treated with RNase-free DNaseI (Roche) for 20 minutes at 37°C, and then purified with phenol/chloroform. First strand cDNA synthesis was performed on 5 μg of total RNA using
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Superscript III RNase H– reverse transcriptase (Gibco BRL, Life Technologies) and an oligo dT primer (18mer), according to the manufacturer’s instructions. From 20μl of the reversetranscription (RT) product, 1μl was used for each PCR reaction. The annealing temperature was 54°C for all primer pairs and 30 cycles of PCR were performed for all genes, except for EF1 (25 cycles) and AGL19 (36 cycles). The specificity of all primers used in this study has been previously reported (Serrano-Cartegena et al. 2000; Katz et al. 2004; Carles et al. 2005; GomezMena et al. 2005; Huang et al. 2006; Schönrock et al. 2006; Schubert et al. 2006; Calonje et al. 2008). For quantification of AG and AP3 cDNAs by real-time PCR, we used SYBR Green PCR master mix (Applied Biosystems) and the ABI 7000 Thermocycler (Applied Biosystems). The specificity of the amplification was determined by performing a dissociation curve analysis. Three technical replicates were done for each sample. Relative quantification (RQ) values were calculated using the 2-Ct method (Livak and Schmittgen 2001). The Ct was calculated using the EF1 gene as the endogenous control. Values given in Figure 3A represent the RQ average of two biological replicates, with the RQ of clf-2 set at 100%. Primer sequences are listed in Supplemental Table S2. Scanning Electron Microscopy (SEM) Scanning electron microscopy was performed as described (Bowman et al. 1989) using a Hitachi 4700 electron microscope with digital imaging capability. RNA in situ hybridization Probes for in situ hybridization were transcribed using a digoxigenin-labeling mix (Roche). The AG antisense probe was generated by T7 RNA polymerase activity from a 1 kb insert cloned into the pBS KS+ vector. The AP3 antisense probe was generated by T7 RNA polymerase activity from the full length AP3 ORF cloned into the pGEM-3Z vector. The specificity of the AG and
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AP3 probes has been previously reported (Yanofsky et al. 1990; Jack et al. 1992). Tissue fixation and in situ hybridization were performed as described (Ambrose et al. 2000), with the modification that seedlings tips were chopped before infiltration to facilitate fixative penetration. GUS staining For analysis of pAG::GUS, AG-I::GUS, pAG-I::GUS (Sieburth and Meyerowitz 1997) and pKNAT2::GUS (Laufs et al. 1998; Lin et al. 2003) reporter activity, the GUS staining reaction, subsequent tissue embedding, and sectioning were performed as described (Sieburth and Meyerowitz 1997) with the modification that GUS localization was visualized after 3 hours of staining with 2mM 5-bromo-4-chloro-3-indolyl--Dglucoronide (X-GLUC, Bioworld). RNA blot hybridization RNA was isolated from 10-day-old-plant rosette leaves using the RNeasy plant kit (Qiagen). Total RNA (20 μg per lane) was resolved on 1% agarose/MOPS-2.2M formaldehyde gels and analyzed as described (Carles et al. 2002). The 32P-labelled DNA probe corresponding to the full-length AG ORF was generated by random primer extension, using the Rediprime kit (Amersham). The specificity of this AG probe (Cheng et al. 2003) under our hybridization conditions was verified using wild-type and ag insertion mutant tissues as controls. Signal detection was quantified by phosphorimager analysis (Storm 640, Molecular Dynamics). Ethidium-bromide-stained ribosomal RNA was used as an internal standard. Chromatin ImmunoPrecipitation (ChIP) ChIP was performed on 20 μg of chromatin isolated from 4-day-old seedlings (for analysis of the histone marks) or from inflorescences (for analysis of the ULT1 DNA-binding sites), following a protocol adapted from (Gendel et al. 2002; Bowler et al. 2004; Schubert et al. 2006; Saleh et al. 2008) and the Pikaard lab protocol, published online at
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http://www.biology.wustl.edu/pikaard/Protocols%20page.html. The tissues used for this analysis were grown together with the tissues used to generate the qPCR expression data. Fresh tissue (24 g) was infiltrated twice in 37 mL of 1% formaldehyde buffer (0.4M sucrose, 10mM TrisHCl pH8, 1mM PMSF, 1mM EDTA), for 2 and 8 minutes under vacuum at 25 psi. The reaction was stopped by adding 0.1M glycine. Fixed tissues were ground in liquid nitrogen and powder was homogenized in 15mL of fresh extraction buffer (0.44M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20mM Hepes KOH pH7.4, 10mM MgCl2, 0.5% Triton-X100, 5mM DTT, 1mM PMSF, 1% Plant Protease Inhibitor cocktail (Sigma)) and filtered through two layers of Miracloth. After 2 cycles of centrifugation at 2000xg for 15 min and washing the pellet in 1mL of extraction buffer, the pellet was resuspended in 500μL of nuclear lysis buffer (50mM Tris-HCl pH8, 10mM EDTA, 1% SDS, 1mM PMSF, 1% Plant Protease Inhibitor cocktail). The chromatin was sonicated using a Bioruptor apparatus (Position High, 8 pulses of 15 sec, with 1 min pauses). After pelleting debris, the DNA of the shred chromatin samples was quantified using a Nanodrop spectrophotometer. 20μg of each chromatin sample was diluted 10 times (generally 500 μL final volume) in ChIP dilution buffer (16.7mM Tris-HCl pH8, 1.2mM EDTA, 1.1% Triton X-100, 167mM NaCl, 1% Plant Protease Inhibitor cocktail) and incubated overnight at 4ºC with 35μL of protein A-agarose / Salmon sperm DNA (Upstate) in binding/washing buffer (150mM NaCl, 20mM Tris-HCl pH8, 2mM EDTA, 1% Triton X-100, 0.1% SDS, 1mM PMSF, 1% Plant Protease Inhibitor cocktail) and 5 μL of antibody, under constant rotation. Antibodies used were anti-H3K27me3 (Upstate), anti-H3K4me3 (Abcam), and anti-HA (Sigma-Aldrich). An aliquot of the original input chromatin (1% of each sample) was retained before the immunoprecipitation reaction and was further processed in parallel with the immunoprecipitated samples for reference during the Real-time PCR quantification. Samples were washed 2 x 5 min and 2 x 15 min with 1
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mL of binding/washing buffer. For reverse cross-linking and elution of immunoprecipitated chromatin, 75μL of Chelex resin (Biorad) was added to the samples and the mix boiled for 10 min. Proteins were degraded by treatment with 20μg of Proteinase K. After protease inactivation, the samples were centrifuged for 2 min at 10,000xg and 2μL of each supernatant was used per PCR reaction. Immunoprecipitated DNA was analyzed by Real-time PCR (Thermocycler ABI 7000) using SYBR Green and absolute quantification settings (Applied Biosystems). The specificity of the amplification was determined by performing a dissociation curve analysis. PCR reactions were performed in triplicate in a final volume of 20μL, using Ex-Taq DNA Polymerase (Takara). Relative enrichments were calculated as the percentage of the obtained values for the immunoprecipitated and input fractions by applying the 2-Ct method (SuperArray ChIP-qPRC user manual, Bioscience Corporation). The average was determined for immunoprecipitates from ChIP assays of two independent experiments, as a percentage of corresponding input DNA, with error bars representing standard error. Primer sequences are listed in Supplemental Table S2. Bimolecular Fluorescence Complementation (BiFC) assay The pEZS-CL-NY, pEZS-CL-YC, pEZS-CL-ULT1-NY, pE-SPYCE-ATX1(Saleh et al. 2007), pUC-SPYNE-bZIP63 (Walter et al. 2004), pUC-SPYCE-bZIP63 and pRecA-RFP constructs were transformed into onion epidermal cells by particle bombardment using a Biolistic PDS1000/He unit (BioRad, Richmond, CA), as described (Sanford et al. 1993). Negative controls with vectors bearing NY or YC, alone or fused to the bZIP63 ORF, were carried out in each experiment to verify the specificity of the interaction. Co-bombardments were processed with a total of 20 μg of DNA used per shot. Ten micrograms of each YFP fusion construct plasmid, with or without 0.5 μg of pRecA-RFP, were used in the following combinations: pEZS-CLULT1-NY / pE-SPYCE –ATX1, pEZS-CL-ULT1-YC/ pE-SPYNE –ATX1, pEZS-CL-ULT1-
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NY / pUC-SPYCE-bZIP63, pEZS-CL-ULT1-YC / pUC-SPYNE-bZIP63, pUC-SPYNE-bZIP63 / pUC-SPYCE-bZIP63, pEZS-CL-ULT1-NY / pEZS-CL-YFP-YC, pE-SPYNE –ATX1 / pEZSCL-YFP-YC. Bombardments were repeated at least three times for each combination. For visualization, epidermal peels were examined 24 and 36 hours after bombardment using a Zeiss Axiophot microscope. Images were acquired with a 12-bit MicroMax cooled CCD camera operated by IPLab software (Scanalytics, Fairfax, VA).
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Supplemental Table S1. Percentage of flowers with a 5th whorl.
Genotype
%
± Std Err
clf-2 ult1-3 ult1-3 clf-2
0 ± 0 16 ± 3 22 ± 3
The percentages represent average values from two biological replicates for which the first ten flowers of 10 plants (n=100 flowers) were examined. The difference between the values of the ult1-3 and ult1-3 clf-2 genotypes is not significant.
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Supplemental Table S2. Primers. Primer
Sequence 5’-3’
Purpose
Cloning and genotyping ATGGCGAACAATGAGGGAGAG oULT1-F TCAAGCTTTGACATTGCTGG oULT1-R CAATGGGTGATCCAAACTGGAAG oult1-1 CAPS RsaI-F oult1-2 dCAPS-F TGACACCAGCTGCATTCGAGAAGAATT oult1-2 dCAPS-R GCAAGCGGCATTCGTCTCTGC GCGTGGACCGCTTGCTGCAACT opROK2LBb1 ATGGCGTCAGAAGCTTCGCC oCLFex1-F CTGGACCTCTCTCCTCCGC oCLFex2-R GTACCGACCGTTACCGACCG oDs-3’ CACCATGGCGAACAATGAGGGAG oULT1-F-Gate GAAGTATTACCCGAATCCGCCCCAAGAA oag-3 dCAPS BslI GTCGATTTCAGAAAATAAGAGCTCA oag-3 dCAPS BslI ACCACCGGAGACAATCAAAG oKn2-Ds-F1 GCTGAATTCCACTATATTGTAG oKn2-Ds-R1 TCCGTTCCGTTTTCGTTTTTTAC oDs5-2a CGATTACCGTATTTATCCCGTTC oDs3-2a RT-PCR and RT-qPCR CAATTGATGGGTGAGACGAT oAG-F CGCGGATGAGTAATGGTGAT oAG-R ATGATAATCATCGCAAGACCG oAGtrans NOS-Ra GCTTGCTCAACCCAATTCTG oAG 3utr-Ra CCACCAGAACCATCACCACTATT oAP3-F AAGAGCGTAAGCACGTGACC oAP3-Rb GCAGCTCCTCAAACATGCTC oSEP1-F CTGAGCTTGATGATGCGCG oSEP1-R CTGCAGCACCTCCAACATGC oSEP2-F CTCTGAGCACACTGGATGGC oSEP2-R GTAGTTCGAGCATGCTTCGG oSEP3-F CACACTTGGTCCTGCTCCC oSEP3-R CAGCCCTAGTGGTATGGCG oSEP4-F GCTACATTGCCTTGTAGAGGC oSEP4-R ATGGTGAGGGGCAAAACGGAG oAGL19-F CCAGATGTTTCGTCTCTCGC oAGL19-R GAAGAGATTCAGCGAGAGAACC oKNAT2-F GAATCGTCCATCATATCAAACGGCATG oKNAT2-R GATGATGTCACCGGAGAGTCTC oKNAT6-F GACTCGACACCAGTACATAGGTTC oKNAT6-R GTGCTCCTGCCTATTCTCTAATG oSTM-F CTATCCTCAGTTGTGGATCTAC oSTM-R GGGTATGGAAGAATACCAGC oBP-F TATGGACCGAGACGATAAG oBP-R TGATGCATACGAGCCTTATCA oPNY-F ACCTACAAAATCATGTAGAAACTG oPNY-R CAGGCTGATTGTGCTGTGTTCTTATCAT oEF1-F
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Cloning, mutant genotyping and RT-PCR dCAPS genotyping of ult1-1 dCAPS genotyping of ult1-2 ult1-3 genotyping clf-2 Ds insertion genotyping and RT-PCR clf-2 Ds insertion genotyping Used with oULT1-F for cloning into pENTR-DdCAPS genotyping of ag-3 knat2 Ds insertion genotyping
for RT-PCR and RT-qPCR Rev. primer for total AG by RT-PCR and RT-qPCR Reverse primer for AG transgene by RT-PCR Reverse primer for AG endogene by RT-PCR for RT-PCR and RT-qPCR on AP3 for RT-PCR on SEP1 for RT-PCR on SEP2 for RT-PCR on SEP3 for RT-PCR on SEP4 for RT-PCR on AGL19 for RT-PCR on KNAT2 for RT-PCR on KNAT6 for RT-PCR on STM for RT-PCR on BP for RT-PCR on PNY for RT-PCR on EF1
oEF1-R qPCR for ChIP oAG-A-F oAG-A-R oAG-real2-F oAG-real2-R oAG-B-F oAG-B-R oAG-real4-F oAG-real4-R oAG-real1-F oAG-real1-R oAG-H-F oAG-H-R oAP3-p-F oAP3-p-R
CTTGTAGACATCCTGAAGTGGAAGA TCAAAACCAATTTGCAGTGG CCACAATCGAAGGTTGCTTT TGAGTGATTGCCCAACTTGA TGGTGGGTAGTTCTTGTGTGG CACAAAAGAAAAGGGAATAGAGCTG TAAGGACACCCCCAAATTGA GGGAAACAAATTGGGGAGAG CAACAATGGAGGATGGATGA TGGGTACTGAGAGGAAAGTGAGA GGATCGTAGAAGGCAGACCA ACCGAATCGAATCCAAACAC CACGGTAACAATGCGTTGAC AAAGCCAACCAAATCCACCTGCAC GGAGCTCCGTTAGCTTCTACTTTG
For AG-p.1 For AG-p.2 For AG-p.2’ For AG-i.1 For AG-i.2 For up-AG For AP3-p
Supplemental Text A total of 466 T1 plants were obtained from the transformation of the 35S::ULT1 construct into the ag-3/+ background. Among the 68 (14.6%) transformants that showed overexpression phenotypes, only 7 (10.3%) were homozygous for the ag-3 mutation, whereas 105 (26%) out of the remaining 398 normal-looking transformants were homozygous for the ag-3 mutation. The 7 35S::ULT1 ag-3 homozygous plants resembled ag-3 homozygous plants in that they produced rosette leaves of relatively normal shape and size, along with indeterminate flowers displaying homeotic conversions of stamens and carpels. The size of the flowers was similar to that of ag-3 flowers. However, these 35S::ULT1 ag-3 plants were still distinguishable from ag-3 plants because they had increased branching and produced flowers with wrinkled and radial petals. In the small T2 progenies of the 35S::ULT1 ag-3/+ overexpressor lines, only petal and branching defects were observed among the 35S::ULT1 ag-3 progeny plants, whereas their 35S::ULT1 ag3/+ siblings displayed strong overexpression phenotypes. Thus the loss of wild-type AG activity severely attenuates the 35S::ULT1 overexpression phenotypes.
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Supplemental References Ambrose, B.A., Lerner, D.R., Ciceri, P., Padilla, C.M., Yanofsky, M.F., and Schmidt, R.J. 2000. Molecular and genetic analyses of the Silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Mol Cell 5: 569-579. Bowler, C., Benvenuto, G., Laflamme, P., Molino, D., Probst, A.V., Tariq, M., and Paszkowski, J. 2004. Chromatin techniques for plant cells. Plant J 39(5): 776-789. Bowman, J.L., Smyth, D.R., and Meyerowitz, E.M. 1989. Genes directing flower development in Arabidopsis. Plant Cell 1: 37-52. -. 1991. Genetic interactions among floral homeotic genes of Arabidopsis. Development 112(1): 1-20. Byrne, M.E., Simorowski, J., and Martienssen, R.A. 2002. ASYMMETRIC LEAVES1 reveals knox gene redundancy in Arabidopsis. Development 129: 1957-1965. Calonje, M., Sanchez, R., Chen, L., and Sung, Z.R. 2008. EMBRYONIC FLOWER1 Participates in Polycomb Group-Mediated AG Gene Silencing in Arabipdopsis. The Plant Cell 20(2): 277-291. Carles, C., Bies-Etheve, N., Aspart, L., Leon-Kloosterziel, K., Koornneef, M., Echeverria, M., and Delseny, M. 2002. Regulation of Arabidopsis thaliana Em genes: role of ABI5. Plant J 30(3): 373-383. Carles, C.C., Choffnes-Inada, D., Reville, K., Lertpiriyapong, K., and Fletcher, J.C. 2005. ULTRAPETALA1 encodes a putative SAND domain transcription factor that controls shoot and floral meristem activity in Arabidopsis. Development 132: 897-911.
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Cheng, Y., Kato, N., Wang, W., Li, J., and Chen, X. 2003. Two RNA binding proteins, HEN4 and HUA1, act in the processing of AGAMOUS pre-mRNA in Arabidopsis thaliana. Dev Cell 4: 53-66. Clough, S.J. and Bent, A.F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743. Earley, K.W., Haag, J.R., Pontes, O., Opper, K., Juehne, T., Song, K., and Pikaard, C.S. 2006. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45(4): 616-629. Fletcher, J.C. 2001. The ULTRAPETALA gene controls shoot and floral meristem size in Arabidopsis. Development 128: 1323-1333. Gendel, A.V., Lippman, Z., Yordan, C., Colot, V., and Martienssen, R.A. 2002. Dependence of heterochromatic histone H3 methylation patterns on the Arabidopsis gene DDM1. Science 297(5588): 1871-1873. Gomez-Mena, C., de Folter, S., Costa, M.M., Angenent, G.C., and Sablowski, R. 2005. Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis. Development 132: 429-438. Goodrich, J., Puangsomlee, P., Martin, M., Long, D., Meyerowitz, E.M., and Coupland, G. 1997. A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386(6620): 44-51. Huang, W., Pi, L., Liang, W., Xu, B., Wang, H., Cai, R., and Huang, H. 2006. The proteolytic function of the Arabidopsis 26S proteasome is required for specifying leaf adaxial identity. Plant Cell 18: 2479-2492.
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Jack, T., Brockman, L.L., and Meyerowitz, E.M. 1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68(4): 683-697. Katz, A., Oliva, M., Mosquna, A., Hakim, O., and Ohad, N. 2004. FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development. Plant J 37: 707-719. Laufs, P., Dockx, J., Kronenberger, J., and Traas, J. 1998. Mgoun1 and mgoun2: two genes required for primordium initiation at the shoot apical and floral meristems in Arabidopsis thaliana. Development 125: 1253-1260. Lin, W., Shuai, B., and Springer, P.S. 2003. The Arabidopsis LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC LEAVES2 functions in the repression of KNOX gene expression and adaxial-abaxial patterning. Plant Cell 15: 2241-2252. Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408. Saleh, A., Al-Abdallat, A., Ndamukong, I., Alvarez-Venegas, R., and Avramova, Z. 2007. The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish 'bivalent chromatin marks' at the silent AGAMOUS locus. Nucl Acids Res 35: 6290-6296. Saleh, A., Alvarez-Venegas, R., and Avramova, Z. 2008. An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3(6): 1018-1025. Sanford, J.C., Smith, F.D., and Russell, J.A. 1993. Optimizing the biolistic process for different biological applications. Methods Enzymol 217: 483-509.
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Schönrock, N., Bouveret, R., Leroy, O., Borghi, L., Kohler, C., Gruissem, W., and Hennig, L. 2006. Polycomb-group proteins repress the floral activator AGL19 in the FLCindependent vernalization pathway. Genes & Dev 20: 1667-1678. Schubert, D., Primavesi, L., Bishopp, A., Roberts, G., Doonan, J., Jenuwein, T., and Goodrich, J. 2006. Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27. EMBO J 25: 4638-4649. Serrano-Cartegena, J., Candela, H., Robles, P., Ponce, M.R., Perez-Perez, J.M., Piqueras, P., and Micol, J.L. 2000. Genetic analysis of incurvata mutants reveals three independent genetic operations at work in Arabidopsis leaf morphogenesis. Genetics 156: 1363-1377. Sieburth, L.E. and Meyerowitz, E.M. 1997. Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9: 355-365. Walter, M., Chaban, C., Schütze, K., Batistic, O., Weckermann, K., Näke, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. 2004. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40: 428-438. Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A., and Meyerowitz, E.M. 1990. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346(6279): 35-39.
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Carles_SupplFigS1
A
C
B
WT
35S::ULT1
D
pSe
Pe
dSe
E
35S::ULT1 Pe
F
WT Pe
G
WT St
H
I
Se
Supplemental Figure S1. Homeotic transformations observed on inflorescences of 35S::ULT1 transgenic plants. (A-B) 35S::ULT1 inflorescence stems, bearing highly carpeloid flowers that terminate in compact and mosaic floral organs. (C) 35S::ULT1 flowers are smaller than wild-type (WT) flowers. (D) Scanning electron micrographs (SEM) of mosaic floral organs, including stigmatic papillae (arrowheads) developing on the distal end of a sepal (dSe), ovules (arrows) forming on the proximal end of a sepal (pSe), and a petal (Pe) with morphological characteristics of a stamen; Se, sepal; Pe, petal. (E) SEM images of mosaic cell types in 35S::ULT1 second whorl organs (35S::ULT1 Pe) that are intermediate between WT petal (WT Pe) and WT stamen (WT St) cell types. (F) inflorescence from an ag-3 mutant plant. (G) inflorescence from a 35S::ULT1 ag-3 plant. (H-I) 35S::ULT1 ag-3 flowers on which sepals and petals have been removed from 4 and 7 whorls, respectively. Petals are short and narrow and display some stamenoid features.
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Carles_SupplFigS2
C
B
A
AG AP3 SEP1
KNAT2 KNAT6
CLF STM
SEP2 SEP3 SEP4
ULT1 ULT2 EF1a
BP PNY
AGL19
Supplemental Figure S2. ULT1 and CLF oppositely regulate a common set of downstream target genes. RT-PCR with RNA from rosette leaves of 10-day-old WT, clf-2 and 35S::ULT1 plants. (A) Expression of floral homeotic MADS box genes and the floral activator AGL19. (B) Expression of class I KNOX genes and the BELL class homeobox gene PNY. (C) Expression of ULT1, ULT2 and CLF, with EF1a shown as the reference gene. gDNA, genomic DNA.
Carles_SupplFigS3
Supplemental Figure S3. The CLF expression pattern is unaffected by ectopic ULT1 activity. RNA in situ hybridization showing CLF expression in inflorescence meristem and young floral tissue shortly after bolting. (A) Wild-type Ler. (B) 35S::ULT1. Scale bar, 50 m.
Carles_SupplFigS4
A
B
clf-2
ult1-3
ult1-3 clf-2
C
D
E
clf-2
ult1-3
ult1-3 clf-2
F
G
clf-2
ult1-3
I
H
ult1-3 clf-2
Supplemental Figure S4. Rescue of the clf-2 phenotypes by the ult1 null mutation. (A) Individual rosette leaves of clf-2, ult1-3 and clf-2 ult1-3 plants. clf-2 ult1-3 leaves are the same size and shape as ult1-3 leaves. (B) Graphs of flowering time measured as mean days to bolting (left) and mean leaf number at bolting (right). clf-2 ult1-3 plants flower at the same time and with the same number of leaves as ult1-3 plants. (C-E) Top view of inflorescences. clf-2 ult1-3 plants produce a similar number of floral buds as ult1-3 plants, but more than clf-2 plants. (F-H) Scanning electron micrographs of individual flowers. clf-2 ult1-3 flowers resemble ult1-3 flowers and lack the carpeloid sepal characteristics (boxes) of clf-2 flowers. (I) Graph of floral organ numbers. clf-2 ult1-3 and ult1-3 flowers have similar mean flower organ numbers. Results shown here were obtained with the ult1-3 null mutant allele. Identical results were obtained in experiments using the independent ult1-1 and ult1-2 mutant backgrounds.
Carles_SupplFigS5
AG AP3 SEP1 SEP2 SEP3 SEP4
EF1a ACT2
Supplemental Figure S5. Expression levels of floral MADS box genes are reduced in ult1 clf double mutant compared to clf single mutant plants. RT-PCR with RNA from rosette leaves of 11-day-old WT, clf-2 and ult1-3 and clf-2 ult1-3 plants. EF1a and ACT2 are shown as the reference genes. gDNA, genomic DNA.
Carles_SupplFigS6
C
B
A ULT1
NY
+
ATX1
YC
bZIP63 NY
+ bZIP63
YC
ULT1
NY
+
bZIP63 YC
Reconstituted YFP
RecA-RFP
Bright field
Test
+ Control
- Control
Supplemental Figure S6. BiFC assay of ULT1-NY and ATX1-YC, co-transformed with the RecA-RFP fusion construct. (A) ULT1-NY/ATX1-YC interaction. The pictures on the right are at higher magnification, to show the sub-nuclear pattern of the interaction. (B) bZIP63-NY/bZIP63-YC interaction was used as a positive control. (C) ULT1-NY/bZIP63-YC co-transformation was a negative control. RFP fusion to the RecA protein (RecA-RFP) was used as an internal marker to identify transformed cells; RecA-RFP localizes in plastids. The green signal indicates physical interaction between ULT1 and ATX1 (A) , or self-interaction of bZIP63 (B) fusion proteins inside the nucleus. Pictures at the bottom show bright-field images of the transformed cells. NY, N-terminal half of the YFP protein; YC, C-terminal half of the YFP protein. Yellow signal corresponds to RFP fluorescence leakage in the YFP channel.
