Supplemental Figure 1. Protein and Gene Structures of DWA1 and DWA2. (A) Protein structures of DWA1 and DWA2. WD40 region was determined based on the NCBI conserved domain databases (B, C) Schematic representation of the T-DNA insertions in DWA1 and DWA2 genes, and genotyping and RT-PCR analysis of wildtype, dwa1 and dwa2. Closed boxes represent exons. First and last lines of each diagram indicate 5 - and 3 -untranslated regions, respectively. Lines between closed boxes of each diagram represent introns. Black arrows indicate the location and direction of the designated primers used for genotyping and RT-PCR analysis. Grey arrows indicate the location and direction of the LBb1 primers used for genotyping analysis. Pink and red boxes represent WD40 region and DWD domain, respectively. RPN6a was used as internal control for the RT-PCR assays.
Supplemental Figure 2. Increased ABA Sensitivity of dwa1 and dwa2 Mutants. (A) ABA-hypersensitivity phenotypes of dwa1 and dwa2 mutants. Wild-type (Col-0), dwa1 and dwa2 seedlings were grown vertically on germination medium (GM) plates with or without 1 μm ABA for 5 days after stratification. (B) Root lengths of wild-type, dwa1 and dwa2 grown on GM with or without 1 μm ABA. Relative root length compared with that of Col-0 grown on GM plates is indicated. Values are means ± SD (n =20). Statistically significant differences between wild-type and dwa mutants (p < 0.01) are indicated by asterisks.
Supplemental Figure 3. Root Growth Inhibition of Wild-Type, dwa1, dwa2, dwa1 dwa2 and cul4cs after ABA Application. Seedlings were grown on MS plates for 5 days and then transferred to control plates (MS plate without ABA) or plates with 5 μm ABA. The lengths of the primary roots were measured at 0 and 5 days after transfer. Growth of each line at 5 days after transfer to control plates was considered to be 100%; growth on the ABA-containing plate was normalized based on this value. Values are means ± SD (n =10).
Supplemental Figure 4. Enhanced Tolerance to Drought in dwa1 dwa2 Mutant. Wild-type, dwa1-1, dwa2-1 and dwa1-1 dwa2-1 were grown in pots for 3 weeks. Watering was withheld for 18 days, then plants received 2 days of re-watering.
Supplemental Figure 5. Expression Levels of Various Proteins Fused with LexA or B42-HA in Yeast Used in Figures 2A and 8A. B42 indicates the transformation of the empty vector, JG4-5, which possesses B42AD- HA as a tag protein, into yeast. Since B42-HA peptide was not detected using B42 antibody in (A), the absence of B42-HA was additionally confirmed using HA antibody (B). The asterisk on the anti-b42 panel indicates non-specific bands that reacted with this antibody.
Supplemental Figure 6. Confirmation of Endogenous ABI5 Protein. Seedlings were grown in the absence or presence of 5 μm ABA for 5 days after stratification. The arrowheads on upper panel indicate the positions of endogenous ABI5. A band (arrow) that cross-reacts with α-abi5, but is unaffected by ABA treatment and the presence/absence of ABI5 protein is indicated as a loading control. Con, a sample without ABA treatment.
Supplemental Figure 7. Level of ABI5 Proteins from the Material in Figure 3C.
Supplemental Figure 8. T7-ABI5 Protein Level in Wild-Type, dwa1-1, dwa2-1, dwa1-1 dwa2-1 and cul4cs Extracts after Treatment with MG132.
Supplemental Figure 9. Self-Interactions of DWA1 and DWA2 Proteins. (A) In vitro interaction between DWA1 proteins detected by pull-down assays. Recombinant GST, GST-DWA1 and MBP-DWA1 proteins were incubated with GST resin at 4 C. The eluates were resolved by SDS-PAGE and blotted. The blot was probed with anti-gst or anti-mbp antibody. The detailed methods are shown in the Supplemental Methods. (B) In vitro interaction between DWA2 proteins detected by pull-down assays. Recombinant MBP, MBP-DWA2 and GST-DWA2 proteins were incubated with amylose resin at 4 C. The eluates were resolved by SDS-PAGE and blotted. The methods are in the Supplemental Methods.
Supplemental Figure 10. Hypocotyl Lengths of Wild-Type, dwa1 and dwa2 after Treatment with ACC. Seedlings were grown on GM plates with or without 1 μm ACC for 3 days in darkness. Relative root lengths compared with that of Col-0 grown on GM plates are indicated. Values are means ± SD (n =20).
Supplemental Table 1. T-DNA Insertion or Mutant Lines of DWD and Additional DCAF Genes in This Study. Gene ID Obtained T-DNA or Insertion status Phenotype of Source of mutant lines site a homozygous line homozygous line At1g04510 SALK_143098 2nd Exon Homo NA ABRC d At1g10580 SALK_068763 1st Exon Homo NA ABRC At1g11160 SALK_078009 15th Exon Homo ND Deng Lab e At1g15440 SALK_037412 5th Exon Homo ND ABRC At1g15470 SALK_075873 6th Exon Homo NA ABRC At1g19485 SALK_091278 2nd Exon Homo NA ABRC At1g19750 SALK_144623 2nd Exon Homo ND Deng Lab At1g20540 SALK_026098 3rd Exon Hetero At1g27840 SALK_030558 4th Intron Homo NA ABRC At1g29260 SALK_005354 5' UTR Hetero At1g29320 SALK_057959 1st Exon Hetero At1g36070 SAIL_346_C09 6th Intron Homo ND Deng Lab At1g49040 SALK_046851 6th Exon Hetero At1g49910 SALK_151687 4th Exon Homo NA ABRC At1g52730 None At1g53090 spa4 (Ref. 1) NA At1g55680 SALK_142665 4th Exon Homo ND ABRC At1g61210 SALK_046984 10th Exon Homo A, N Deng Lab, ABRC SALK_058391 16th Exon Homo A, N ABRC At1g62020 SALK_003425 2nd Exon Homo NA ABRC At1g65030 SALK_140248 2nd Intron Hetero At1g69400 SALK_058038 4th Exon Hetero At1g73720 SALK_123852 11th Exon Hetero At1g76260 SALK_034658 (dwa2-1) 5th Intron Homo A, N ABRC At1g78070 SALK_048461 Promoter Homo NA ABRC At1g80670 SALK_149413 Promoter Homo NA ABRC At1g80710 SALK_001238 1st Exon Homo ND ABRC
Gene ID Obtained T-DNA or Insertion status Phenotype of Source of mutant lines site homozygous line homozygous line At2g01330 None At2g05720 SALK_063822 2nd Exon Homo ND Deng Lab At2g16780 SALK_001214 1st Exon Homo ND Deng Lab At2g19430 SALK_051022 (dwa1-1) 1st Exon Homo A, N Deng Lab, ABRC SALK_021789 (dwa1-2) 5' UTR Homo A, N ABRC At2g19520 SALK_027724 8th Exon Hetero At2g19540 SALK_014373 8th Intron Hetero At2g20330 SALK_140479 5' UTR Homo ND ABRC At2g21390 SALK_103968 2nd Exon Homo NA ABRC At2g22040 SALK_018605 1st Intron Homo NA ABRC At2g25420 SALK_068825 5th Exon Homo NA ABRC At2g32950 cop1 (Ref. 2) NA At2g33340 SALK_144856 5' UTR Homo ND ABRC At2g34260 SALK_063054 10th Intron Homo NA ABRC At2g41500 SALK_070009 3rd Exon Homo NA ABRC At2g43770 None At2g43900 SALK_065920 1st Exon Homo NA ABRC At2g46280 SALK_028797 2nd Exon Hetero At2g46290 None At2g46340 spa1 (Ref. 3) NA At2g46560 SALK_097515 3rd Exon Hetero At2g47410 SALK_017510 7th Exon Homo ND Deng Lab At2g47790 SALK_099078 5'UTR Homo ND Deng Lab, ABRC At2g47990 None At3g05090 SALK_059570 13th Exon Homo ND ABRC At3g08850 SALK_022096 13th Exon Hetero At3g09080 SALK_092404 8th Exon Homo NA ABRC At3g10530 SALK_068610 Promoter Homo NA ABRC At3g13340 None
Gene ID Obtained T-DNA or Insertion status Phenotype of Source of mutant lines site homozygous line homozygous line At3g15354 spa3 (Ref. 1) NA At3g15610 SALK_071771 5' UTR Homo NA ABRC At3g16650 SALK_133885 1st Exon Hetero At3g18060 SALK_026426 5' UTR Homo NA ABRC At3g18140 SALK_002459 4th Exon Homo NA ABRC At3g19590 None At3g20740 SALK_042962 5th Exon Homo NA ABRC At3g27640 SALK_005472 7th Exon Hetero At3g44600 SALK_024686 2nd Exon Homo NA ABRC At3g45620 SALK_129467 1st Exon Homo ND Deng Lab At3g49180 SALK_021729 1st Intron Hetero At3g51930 None At3g52030 SALK_077738 5' UTR Homo NA ABRC At3g56990 None At3g63460 SALK_012544 1st Exon Homo NA ABRC At4g01860 SALK_106172 2nd Exon Hetero At4g02730 SALK_116999 1st Exon Homo NA ABRC At4g03020 None At4g04940 SALK_070800 12th Intron Homo NA ABRC At4g05410 SALK_022234 5' UTR Hetero At4g11110 spa2 (Ref. 4) At4g15900 prl1 (Ref. 5) NA A b,c At4g21130 SALK_122164 5' UTR Homo ND Deng Lab At4g21520 SALK_092432 4th Intron Homo NA ABRC At4g28450 SALK_036854 12th Intron Homo ND Deng Lab At4g29380 SALK_049979 8th Exon Hetero At4g29730 SALK_022118 1st Exon Homo NA ABRC At4g29830 SALK_083364 1st Exon Homo ND Deng Lab At4g29860 SALK_097510 9th Exon Hetero
Gene ID Obtained T-DNA or Insertion status Phenotype of Source of mutant lines site homozygous line homozygous line At4g31160 SALK_065499 3rd Exon Homo NA ABRC At4g34280 SALK_056954 8th Intron Homo ND ABRC At4g34460 SALK_061896 4th Exon Hetero At4g35050 None At4g35560 SALK_045948 1st Intron Homo NA ABRC At4g38480 SALK_051074 1st Intron Homo NA ABRC At5g01770 SALK_072800 2nd Intron Hetero At5g05970 None At5g07590 SALK_093768 4th Exon Homo NA ABRC At5g08390 SAIL_14_E08 5th Exon Hetero At5g10940 SALK_040151 2nd Intron Homo NA ABRC At5g12920 SALK_038176 15th Intron Homo ND ABRC At5g13480 SALK_047002 1st Exon Hetero At5g14050 SAIL_1162_D12 5' UTR Hetero At5g14530 SALK_124562 5' UTR Homo ND Deng Lab At5g15550 None At5g17370 SALK_136079 2nd Intron Homo NA ABRC At5g18525 None At5g19920 SALK_053741 2nd Exon Homo NA ABRC At5g23430 SALK_129667 2nd Exon Homo ND Deng Lab, ABRC At5g23730 SALK_015765 5' UTR Homo ND ABRC At5g27080 SALK_002496 1st Exon Homo NA ABRC At5g27570 SALK_083223 1st Exon Homo ND Deng Lab At5g49430 SALK_093473 3rd Exon Homo A, N ABRC At5g52250 SALK_060638 1st Exon Homo ND Deng Lab, ABRC At5g52820 SALK_032034 7th Intron Hetero At5g54520 SALK_132649 3rd Intron Hetero At5g56130 SALK_093273 5' UTR Homo NA ABRC At5g56190 SALK_019889 10th Exon Homo NA ABRC
Gene ID Obtained T-DNA or Insertion status Phenotype of Source of mutant lines site homozygous line homozygous line At5g58230 None At5g58760 SALK_038892 5 'UTR Homo ND Deng Lab At5g60940 SALK_128902 3rd Intron Homo NA ABRC At5g63010 SALK_115399 2nd Exon Homo ND ABRC At5g64730 None At5g66240 SALK_109069 5 'UTR Hetero At5g67320 None Homo, homozygous line. Hetero, heterozygous line. ND, no difference from wild type. NA, not analyzed. A, ABA-hypersensitive. N, NaCl-hypersensitive. a For Arabidopsis genes with splice variants, the inserton site was determined based on the structure of the representative gene model listed at TAIR. b NaCl hypersensitivity was not checked. c ABA hypersensitivity was checked in our previous paper (Lee et al., 2008). d ABRC, homozygous T-DNA insertion lines were directly obtained from ABRC. e Deng Lab, T-DNA insertion lines were obtained from ABRC and then, homozygous lines were selected by genotyping analysis from Deng lab. Cited references in this table 1. Laubinger, S., and Hoecker, U. (2003). The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light. Plant J. 35: 373-385. 2. Deng, X.W., Caspar, T., and Quail, P.H. (1991). cop1: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis. Genes Dev. 5: 1172-1182. 3. Hoecker, U., Xu, Y., and Quail, P.H. (1998). SPA1: a new genetic locus involved in phytochrome A-specific signal transduction. Plant Cell 10: 19-33. 4. Laubinger, S., Fittinghoff, K., and Hoecker, U. (2004). The SPA quartet: a family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis. Plant Cell 16: 2293-2306. 5. Nemeth, K., Salchert, K., Putnoky, P., Bhalerao, R., Koncz-Kalman, Z.,
Stankovic-Stangeland, B., Bako, L., Mathur, J., Okresz, L., Stabel, S., Geigenberger, P., Stitt, M., Rédei, G.P., Schell, J., and Koncz, C. (1998). Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis. Genes Dev. 12: 3059-3073.
Supplemental Table 2. Oligonucleotide Sequences of the Primer Pairs Used for Genotyping Analysis. Gene ID T-DNA line Forward Primer Reverse Primer genotyping a At1g11160 SALK_078009 acctctctgtcctgctaccc gccgaatatatctgataggg F/LBb1 At1g19750 SALK_144623 gactctccagctctctaatcg agcttagtacctacctgcga R/LBb1 At1g36070 SAIL_346_C09 gtaaatcagcctggggttgc tgcaccgtaataccaagtcc R/LBb1 At1g61210 SALK_046984 gccataaccactgtgtgacc atccaacagccactgactgc R/LBb1 At1g76260 SALK_034658 atgcaaggaggatcatcggg tcttcttggcaggaaaggct F/LBb1 At2g05720 SALK_063822 gtatggtatgtagttggggg tcgggttttgattcgcttgc R/LBb1 At2g16780 SALK_001214 agcccttgatcagctctgtg acgaagaatctccagggatc R/LBb1 At2g19430 SALK_051022 atgtacggagacgctacaaa ttacaatgaactgctacgaa F/LBb1 SALK_021789 aagctagtaatttgatcgac ttccatcccctaactctacc F/LBb1 At2g47410 SALK_017510 ttcagtcccagacaggcgtc ccttgcattgctgtcagagc R/LBb1 At2g47790 SALK_099078 atcactgaccgatcggagcc gtcacaaaaccagagttggg R/LBb1 At3g45620 SALK_129467 acacccgactacgaatcacg ttcacacatccctcatgacc R/LBb1 At4g21130 SALK_122164 tgtttggctttggattcggg gcagggtttactggcgatgg F/LBb1 At4g28450 SALK_036854 gtagaaagttggacgaagcc agattggtgtcatcactgccc F/LBb1 At4g29830 SALK_083364 actggatctcttgacgagacgg ttttccgagcagagtagatgaccc R/LBb1 At5g14530 SALK_124562 cagcagcagcagcaaccgcc ctcggaagctggattccacc F/LBb1 At5g23430 SALK_129667 tgccgtgtttgatctactgc tcaattccacttgagtgccc F/LBb1 At5g27570 SALK_083223 aaggcacaagcttcgtgtcc gtgtctcccaaagcaatggc R/LBb1 At5g52250 SALK_060638 tgttctgctctgagatacccaa tccatgctcgtctctctcgg R/LBb1 At5g58760 SALK_038892 tgaaaattgggccatagccc gatgacgatttctgggtccc F/LBb1 LBb1 gcgtggaccgcttgctgcaact a primer sets for confirmation of homo- or heterozygous lines
Supplemental Table 3. DNA Sequences of the Primer Pairs Used for Generation of Various Constructs. DWA1_SpeI_F DWA1_SpeI_R DWA2_KpnI_F DWA2_KpnI_R DWA1_EcoRI_F DWA1_EcoRI_R DWA2_XhoI_F DWA2_XhoI_R ABI5_EcoRI_F ABI5_EcoRI_R RGA_BamHI_F RGA_BamHI_R MYC_SacI_F MYC_SacI_R DWA2_SpeI_F DWA2_SpeI_R DWA2_SpeI_R-1 DWA1_BiFc_F DWA1_BiFc_R DWA2_BiFc_F DWA2_BiFc_R 5'-GGACTAGTATGTACGGAGACGCTACAAAC-3' 5'-GGACTAGTTTACAATGAACTGCTACGAA-3' 5'-CGGGGTACCATGCAAGGAGGATCATCGGGT-3' 5'-CGGGGTACCCTATCTTCTTGGCAGGAAAGG-3' 5'-CGGAATTCATGTACGGAGACGCTACAAAC-3' 5'-CGGAATTCTTACAATGAACTGCTACGAA-3' 5'-CCGCTCGAGATGCAAGGAGGATCATCGGG-3' 5'-CCGCTCGAGCTATCTTCTTGGCAGGAAAG-3' 5'-CGGAATTCGGATGGTAACTAGAGAAACGAAG-3' 5'-CGGAATTCTTAGAGTGGACAACTCGGGT-3' 5'-CGGGATCCGGATGAAGAGAGATCATCACCA-3' 5'-CGGGATCCTCAGTACGCCGCCGTCGAGA-3' 5'-CGAGCTCATGAGTGGTGAACAAAAGTTG-3' 5'-CGAGCTCTTAGCTAGTGGATCCGTTCAAGTC-3' 5'-GGACTAGTATGCAAGGAGGATCATCGGG-3' 5'-GGACTAGTTCTTCTTGGCAGGAAAGGCT-3' 5'-GGACTAGTCTATCTTCTTGGCAGGAAAGGCT-3' 5'-CACCATGTACGGAGACGCTACAAA-3' 5'-TTACAATGAACTGCTACGAA-3' 5'-CACCATGCAAGGAGGATCATCGGG-3' 5'-CTATCTTCTTGGCAGGAAAG-3'
Supplemental Table 4. DNA Sequences of the Primer Pairs Used in Northern Blot or RT-PCR Experiments. Forward Primer Reverse Primer RD29B 5 -CGGTCCGTTGAAGAGTCTCC-3 5 -ACCCAATCTCTTTTTCACAC-3 RD29A 5 -GGAGGAGAAGAAGAGAAGAA-3 5 -TAAAGCTCCTTCTGCACCGG-3 RD22 5 -CAAAATCGCGGCGGCTGGGG-3 5 -TACAGACCACAAAAGACAGC-3 ABI5 5 -ATGGTAACTAGAGAAACGAAG-3 5 -TTAGAGTGGACAACTCGGGT-3 MYC2 5 -CGGTGAAGATGGGTTTTAGG-3 5 -TACAGACTCAAACATAGAGC-3 RPN6a 5 -AAGGCACGATAGATCTGCAGA-3 5 -TCGAGGGTAGCCGAGTAGAT-3 DWA1 5 -ATGTACGGAGACGCTACAAA-3 5 -TTACAATGAACTGCTACGAA-3 DWA2 5 -ATGCAAGGAGGATCATCGGG-3 5 -CTATCTTCTTGGCAGGAAAG-3 5 -CAGCAGGTTCATTGACTTTG-3
Supplemental Table 5. DNA Sequences of the Primer Pairs Used for Quantitative Real- Time PCR Analysis. Forward Primer Reverse Primer RD29B 5 -ACGGAAACATCGGACTGG-3 5 -TCCGTTGACCACCGAGAT-3 RD22 5 -TTCGGAAGAAGCGGAGAT-3 5 -CAGTGGAAACAGCCCTGA-3 MYC2 5 -CGCTGTTGATGAGGAGGTG-3 5 -TCCCAAACACTCCTCCTT-3 ACTIN2 5 -CAAGGCCGAGTATGATGAGG-3 5 -GAAACGCAGACGTAAGTAAAAAC-3
SUPPLEMENTAL METHODS Preparation of Yeast Protein Extracts Preparation of yeast protein extracts was performed based on Yeast Protocols Handbook (Clontech). Isolated protein extracts were mixed with 2X SDS sample buffer and boiled for 5 minutes. The boiled samples were then resolved by SDS-PAGE and transferred to nitrocellulose membranes. After membrane blocking, the membrane was incubated with B42 antibody (Sigma), HA (Roche) or LexA antibody (Abcam), and additionally incubated for 1 hr with secondary antibody with peroxidase-conjugated anti-igg. In Vitro Pull-Down Assay DWA1 cdna was amplified with two primers, DWA1_EcoRI_F and DWA1_EcoRI_R, that include EcoRI sites at 5' ends (Supplemental Table 5 online). Then, it was introduced into the same site of pmal (NEW ENGLAND BioLabs) and pgex-4t-1 (GE Healthcare) vectors. Amplified DWA2 cdna with DWA2_XhoI_F and DWA2_XhoI_R (that include XhoI sites at 5' ends) was introduced into XhoI site of pgex-4t-1 and into SalI site of pmal (Supplemental Table 5 online). GST-DWA1, GST-DWA2, MBP-DWA1 and MBP-DWA2 were expressed in Escherichia coli strain BL21 (DE3) and purified with Glutathione sepharose 4B (GE Healthcare) or amylase resin (New England Biolabs), respectively. For the in vitro pull-down assays between GST-DWA1 and MBP-DWA1, 2 μg of purified MBP-DWA1 proteins were incubated with 2 μg of either GST or GST-DWA1 in binding buffer [50 mm Tris-HCl (ph 7.5), 100 mm NaCl and 0.1% triton X-100] for 4 hours at 4 C. After 30 µl of GST resins were added, the mixtures were incubated for another hour at 4 C. The samples were washed three times with washing buffer [50 mm Tris-HCl (ph 7.5), 200 mm NaCl and 0.1% triton X-100] and eluted using 2X SDS sample buffer with boiling for 5 minutes. The blot was probed with either anti-gst (GE Healthcare) or anti-mbp antibody (New
England Biolabs). In the case of assays between GST-DWA2 and MBP-DWA2, 2 μg of purified GST-DWA2 proteins were incubated with 2 μg of either MBP or MBP-DWA2 in binding buffer as above mentioned. After 30 µl of amylose resins were added, the mixtures were incubated for another hour at 4 C. The samples were washed three times with washing buffer [50 mm Tris-HCl (ph 7.5), 300 mm NaCl and 0.1% triton X-100] and eluted using 2X SDS sample buffer with boiling for 5 minutes. Genotyping Analysis Genomic DNAs were obtained using the extraction buffer [200 mm Tris-HCl (ph 7.5), 250 mm NaCl, 25 mm EDTA and 0.5% SDS] from rosette leaves of wild-type and various T-DNA insertion lines (0.1-0.2 g). The DNAs were used as templates for PCR analysis with LBb1 and, forward and reverse primers for each gene shown in Supplemental Table 1. Oligonucleotide sequences of the primer pairs used for genotyping analysis were shown in Supplemental Table 2.