MATERIAL S AND METHODS Plant materials and growth conditions Arabidopsis ecotype Columbia (Col0) was used for this study. SALK_072009, SALK_076309, and SALK_027645 were obtained from the Arabidopsis Biological Resource Center, and confirmed by genomic DNA PCR and RT-PCR with specific primers. Seeds were surface-sterilized and plated on MS medium in proper growth conditions as described previously (Zhang et al., 2007). To compare the growth phenotype of the plants, the seeds were collected and stored for the same period before they were used. Gene expression analysis For gene expression analysis, total RNA was isolated using the hot phenol method from 2-week-old seedlings treated with or without ABA or IAA for different durations (Xie et al., 1999). To examine ABI1 and MKK1 expression, 3 RACE PCR, RT-PCR and quantitative RT-PCR were performed using different primer pairs (Supplemental Table 1). The expression levels of Actin1 or 18S were adopted as controls. For the RNA gel blot analysis, 10 µg RNA was applied in each lane, and hybridization was performed as described previously (Zhang et al., 2007). Exon-specific PCR fragments are amplified as probes. ABI1, nucleotides 109-1090; GH3.2, nucleotides 1491-2054; GH3.3, nucleotides 1465-2071; MKK1, nucleotides 755-1373. Construction of plant complementation vector and generation of transgenic plants To create the transgenic construct of the 4.6-kb MKK1 genomic DNA to complement the abi1-3mkk1-2 plants, the DNA was PCR-amplified using the following primers: forward primer: 5 -CCTCTAGATACGTTACCAGTTACCAC-3, reverse primer: 5 -GCCTGCAGCAGATATTACTAGCGAGA3-, and cloned into the XbaI and PstI restriction sites in the vector pcambia1300. This fragment is corresponding to chromosome 4:13221521-13216863, containing 0.934 Kb promoter region and the 1
transcribed region (including exon and intron) plus a 1.8 Kb fragment downstream stop codon. Transformation of Arabidopsis was performed by vacuum infiltration. The transgenic plants were selected on an MS plate containing hygromycin. The MKK1 expression level was checked by quantitative RT-PCR. 2
Supplemental Table 1. Primers used in the paper ABI1sense 5'ATCTTTACCGTTAATGGAGG3' ABI1antisense 5'CTCTCTGCCTCAGTTCAAGG3' MKK1sense 5' ATGAACAGAGGAAGCTTATGC3' MKK1antisense 5'CTAGTTAGCAAGTGGGGGAATC3' Actin1 sense 5' CATCAGGAAGGACTTGTACGG3' Actin1 antisense 5' GATGGACCTGACTCGTCATAC3' LP 5'TCAGGAATGATGGATGGTTTC3' RP 5'GGTTTCGTTACCGGAGACTTC3' LBb1 5'GCGTGGACCGCTTGCTGCAACT3' P1 5'AACATGCTATCTGCCATCTGC3' P2 5'CCACCACTAGCAGAGAAACATAAC3' P3 5'TGTTTTGTCGGTTTGATCTCC3' P4 5'AATGATTCGCCTCTCATGATG3' P5 5 TGACAGCCGGAACACGGCGGCAT3 P6 5 CTCTAGAGGATCCCCGGGTA3 For the qrt-pcr assay 18S Forward primer 5'GTTGATCCTGCCAGTAGT3' 18S Reverse primer 5'ATCCGAGTAGTAGTTACCATC3' MKK1 Forward primer 5'CATTCATCGGGACTTAAAGCCTTC3' MKK1 Reverse primer 5' GCCCACGAAAGAATTAGCAAGAC3' GH3.2 Forward primer 5'AGCAGCAGAAGCATCATTAG3' GH3.2 Reverse primer 5'GTCGCCAACTCTGTAACG3' GH3.3 Forward primer 5'TGGTTTGTGTTAGGATTTGTGAGT3' GH3.3 Reverse primer 5'AAAGGAGGGACAGAGTGGAAA3' Primers for amplifying fragment before T-DNA insertion site of ABI1 Forward primer 5 GCATTAGCCTACCCATTTCCT3' Reverse primer 5 CCTGGATTGTGGGTAATTCGTTAG3' Primers for amplifying fragment cross T-DNA insertion site of ABI1 Forward primer 5 TTCCTTCAATCTTCCTCTGGTT3 Reverse primer 5 CTACAATAGTTCGCTACCTGAGA3' Primers for amplifying fragment after T-DNA insertion site of ABI1 Forward primer 5 GCTGGAGAAGTGGAAGAA3' Reverse primer 5 TTAGCGACGAAGATGTGA3' 3
Supplementary data 1. A. The fragment was amplified with the primers P2 and LBb1 and sequenced. The red letters show the partial fragment of MKK1, and the purple letter show the partial fragment of the vector pbin-prok2. P2 CCACCACTAGCAGAGAAACATAACTGATGATAGTTGTGTTTATAATAGTGAT GTGAAAAGTACACACATTAAATCAAATTTTGTAGACAAGTCTCTTAAGTCAT AACATCTCGTAAATTTATCTTTCATAAAATCTCTTCTAGTGGATATGAAATAA TAACTTGGAGGTTAAAAGATTGTTCAAACTCGGTTCTAGTTAGCAAGTGGG GGAATCAAAGATCCTGCGTCGGTGAAGTAAGCCGAGAGATTTGTATCCGAA TCTTCAAACATCTTTACGAACTTGTGTTCCTATTCACCAAGATGGAACCAGA AAAAAGATCGTTAAAAGTAGTCCAAGACAGTACAAGGTGAAAGACTCAGA GTCTTATATGATTAACGCACCAGAAGCTCCTTTGCTGATTTTCTGTCCCTTG GATCTTTTTGTACACTACACAAGTTAAAGCACCATTTCAAATGCCTACTCGT TATAGCAACAAAAATGACATGACAACTGGTCGAAATCTGTCTCACCATTGC GAGATGAAGGAGCAAAACTCTGGAGAAAAGAGATTGGAAGGTGCACAAG GAGGCGGGTTTTCAACAATGGCGTCCACAAGCTCGTACACGCTACTCCATC CTTTCTTGTGTTCTGGAGGAGTATACGGGAATTTACCCGTTGCACATTCGAG CAAAACCAGTCCCAAGCTCCAAATATCGCTCTTGTTACTGTACAAACTCCC GCTGATTCTCTCTGGCTGCAAAACATGATAACAACACCAGTAAGTCGTATA AAACCACTTGTAACTCTGAAAATTGAGTTTGTATGTATATTGAGTGTTGAGA GGTGAATGGAGGAGACTCACAGACATATAAAGGGTATGTGCCCACGAAAG AATTAGCAAGACTACTTGTGCTTGTCAAGATCTTGCTGACACCAAAGTCTG TGATCTTGACTTCACCTCTATGATTGATTAGCAAGTTTGAAGGCTTTAAGTC CCGATGAGACGGGACTGGGGTGGTGTAAACAAATTGACGCTTAGAAAACT TAATAACACATTGCGGACGTTTTTAATGTACTGGGGTGGTTTTTCTTTTCAC CAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGA GTTGCAGCAAGCGGTCCACGC LBb1 B. The fragment was amplified with the primers P1 and P5 (adjacent to right border in the vector pbin-prok2) and sequenced. The red letters show the partial fragment of MKK1, and the purple letter show the partial fragment of the vector pbin-prok2. P5 (primer adjacent to right border in the vector pbin-prok2) CGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAG CCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCT TGTTCAATCATGCGAAACGATCCAGATCCGGTGCAGATTATTTGGATTGAGA GTGAATATGAGACTCTAATTGGATACCGAGGGGAATTTATGGAACGTCAGT GGAGCATTTTTGACAAGAAATATTTGCTAGCTGATAGTGACCTTAGGCGACT TTTGAACGCGCAATAATGGTTTCTGACGTATGTGCTTAGCTCATTAAACTCC 4
AGAAACCCGCGGCTGAGTGGCTCCTTCAACGTTGCGGTTCTGTCAGTTCCA AACGTAAAACGGCTTGTCCCGCGTCATCGGCGGGGGTCATAACGTGACTCC CTTAATTCTCCGCTCATGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTA TCAGTGTTAATTCGCCTCTCATGATGAATATAACAAAGACCTCGAAGAACCT GCGTTTAGTAGAGAAGAATGAGCTTAGTTATGTGTTTTATTAGAGAAGATAA AAATAGAAAACCGGTAGAGATGTTTAAAGATTTCGCGTAGAGTACTCGCTT GCAGATGGCAGATAGCATGTT P1 C. Sequence of RT-PCR product. RT-PCR with RNA extracted from the abi1-2 mutant. The positions of primers were labelled with underlines. ABI1 sense ATCTTTACCGTTAATGGAGGAAGTATCTCCGGCGATCGCAGGTCCTTTCAG GCCATTCTCCGAAACCCAGATGGATTTCACCGGGATCAGATTGGGTAAAGG TTACTGCAATAACCAATACTCAAATCAAGATTCCGAGAACGGAGATCTAAT GGTTTCGTTACCGGAGACTTCATCATGCTCTGTTTCTGGGTCACATGGTTCT GAATCTAGGAAAGTTTTGATTTCTCGGATCAATTCTCCTAATTTAAACATGA AGGAATCAGCAGCTGCTGATATAGTCGTCGTTGATATCTCCGCCGGAGATGA GATCAACGGCTCAGATATTACTAGCGAGAAGAAGATGATCAGCAGAACAG AGAGTAGGAGTTTGTTTGAATTCAAGAGTGTGCCTTTGTATGGTTTTACTTC GATTTGTGGAAGAAGACCTGAGATGGAAGATGCTGTTTCGACTATACCAAG ATTCCTTCAATCTTCCTCTGGTTCGATGTTAGATGGTCGGTTTGATCCTCAAT CCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGGTTCTCAGGTAG CGAACTATTGTAGAGAGAGGATGCATTTGGCTTTGGCGGAGGAGATAGCTA AGGAGAAACCGATGCTCTGCGATGGTGATACGTGGCTGGAGAAGTGGAAG AAAGCTCTTTTCAACTCGTTCCTGAGAGTTGACTCGGAGATTGAGTCAGTT GCGCCGGAGACGGTTGGGTCAACGTCGGTGGTTGCCGTTGTTTTCCCGTCT CACATCTTCGTCGCTAACTGCGGTGACTCTAGAGCCGTTCTTTGCCGCGGC AAAACTGCACTTCCATTATCCGTTGACCATAAACCGGATAGAGAAGATGAA GCTGCGAGGATTGAAGCCGCAGGAGGGAAAGTGATTCAGTGGAATGGAGC TCGTGTTTTCGGTGTTCTCGCCATGTCGAGATCCATTGGCGATAGATACTTG AAACCATCCATCATTCCTGATCCGGAAGTGACGGCTGTGAAGAGAGTAAAA GAAGATGATTGTCTGATTTTGGCGAGTGACGGGGTTTGGGATGTAATGACG GATGAAGAAGCGTGTGAGATGGCAAGGAAGCGGATTCTCTTGTGGCACAA GAAAAACGCGGTGGCTGGGGATGCATCGTTGCTCGCGGATGAGCGGAGAA AGGAAGGGAAAGATCCTGCGGCGATGTCCGCGGCTGAGTATTTGTCAAAG CTGGCGATACAGAGAGGAAGCAAAGACAACATAAGTGTGGTGGTGGTTGA TTTGAAGCCTCGGAGGAAACTCAAGAGCAAACCCTTGAACTGAGGCAGA GAG ABI1antisense D. Schematic diagram of T-DNA insertion and primers used for RT-PCR. RT-PCR 5
with RNA extracted from the abi1-3mkk1-2 mutant. +RT, with the reverse transcriptase; -RT, without reverse transcriptase. The sizes of PCR product were about 1500 bp. AT4g26080 (ABI1) 550 RT + ABI1 sense ABI1 sense+p6 abi1-3 P6 E. The RT-PCR product was sequenced. The red letters show the partial fragment of ABI1, and the purple letter show the partial fragment of the vector pbin-prok2. The positions of primers were labelled with underlines. ABI1sense ATCTTTACCGTTAATGGAGGAAGTATCTCCGGCGATCGCAGGTCCTTTCAG GCCATTCTCCGAAACCCAGATGGATTTCACCGGGATCAGATTGGGTAAAGG TTACTGCAATAACCAATACTCAAATCAAGATTCCGAGAACGGAGATCTAAT GGTTTCGTTACCGGAGACTTCATCATGCTCTGTTTCTGGGTCACATGGTTCT GAATCTAGGAAAGTTTTGATTTCTCGGATCAATTCTCCTAATTTAAACATGA AGGAATCAGCAGCTGCTGATATAGTCGTCGTTGATATCTCCGCCGGAGATGA GATCAACGGCTCAGATATTACTAGCGAGAAGAAGATGATCAGCAGAACAG AGAGTAGGAGTTTGTTTGAATTCAAGAGTGTGCCTTTGTATGGTTTTACTTC GATTTGTGGAAGAAGACCTGAGATGGAAGATGCTGTTTCGACTATACCAAG ATTCCTTCAATCTTCCTCTGGTTCGATGTTAGATGGTCGGTTTGATCCTCAAT CCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGGTTCTCAGGCAG GATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTG CGGACGTTTTTAATGTACTGGGGTGGTTTTTCTTTTCACCAGTGAGACGGG CAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGC GGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTC CGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATAGGGTT GAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTG AACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAA ATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCG GCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCC ATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCT ATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGT AACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAAT TCCCGATCTAGTAACATAGATGACACCGCGCGCGATAATTTATCCTAGTTTG 6
CGCGCTATATTTTGTTTTCTATCGCGTATTAAATGTATAATTGCGGGACTCTAA TCATAAAAACCCATCTCATAAATAACGTCATGCATTACATGTTAATTATTACA TGCTTAACGTAATTCAACAGAAATTATATGATAATCATCGCAAGACCGGCAA CAGGATTCAATCTTAAGAAACTTTATTGCCAAATGTTTGAACGATCGGGGA AATTCGAGCTCGGTACCCGGGGATCCTCTAGAG P6 REFERENCES Xie Q, Sanz-Burgos AP, Guo H, Garcia JA, Gutierrez C (1999) GRAB proteins, novel members of the NAC domain family, isolated by their interaction with a geminivirus protein. Plant. Mol. Biol. 39: 647-656 Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, Gao T, Guo H, Xie Q (2007) SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis. Plant Cell. 19: 1912-1929 7