Supplementary Figure 1 Transcription assay of nine ABA-responsive PP2C genes. Transcription assay of nine ABA-responsive PP2C genes. Total RNA was isolated from 7 day-old seedlings treated with or without 100 μm ABA for 3 h. Three primer pairs (F1/R1, F2/R2 and F3/R3) were used to detect the PP2C transcripts. The F1/R1 primers was set from ATG to TGA to amplify the full length transcriptions, the F2/R2 and F3/R3 primers were used to amplify the fragments as indicated in the schematic diagram. The UBQ5 was used as the internal control; and three biological replicates were performed and each gave the similar result.
Supplementary Figure 2 Comparison of HAB1.1 and HAB1.2. sequences and the blast result against HsRBM25. (a) Alignment of two isoforms HAB1.1 and HAB1.2 sequences. HAB1.2 lacks the 105 amino acids in the C terminal end. (b) The gene structures of HAB1.1 and HAB1.2 variants predicted in the TAIR
website. (c) AT1G60200 gene is the closest gene of HomoRBM25 by WU-Blast analysis. The cdna (LOCUS: NM_021239) of HomoRBM25 was used to BLAST for the homolog genes in the Arabidopsis genome (WU-BLAST 2.0, http://www.arabidopsis.org/cgi-bin/wublast/wublast). The AT1G60200 sequence produced the highest-scoring segment pairs in TAIR 10 Transcripts (-introns, +UTRs) database.
Supplementary Figure 3 Immunoblot analysis of HAB1.1-GFP and HAB1.2-GFP proteins. 35S::HAB1.1-GFP and 35S::HAB1.2-GFP constructs were transformed into the leaves of N. benthamiana, and western blot was used to detect the expression of HAB1.1-GFP and HAB1.2-GFP proteins. Actin was used as the loading control. The experiment was repeated for three times, and the similar results were observed.
Supplementary Figure 4 The subcellular localization of HAB1.2 in the transgenic line harboring HAB1pro::HAB1.2-GFP. Seven day-old seedlings expressing HAB1pro::HAB1.2-GFP were stained with DAPI for 15 min, and the fluorescence was observed using confocal microscope (scale bar, 20 μm).
Supplementary Figure 5 Expression of HAB1pro::HAB1.1-HA rescued the ABA sensitive phenotypes of rbm25. (a) Phenotypic analysis of WT, rbm25 and WT, rbm25 expressing HAB1pro::HAB1.1 on the media supplemented without or with 0.5 μm ABA. The pictures were taken at 10 days after germination. The wild type plants expressing HAB1pro::HAB1.1 were crossed with rbm25 and the homozygous lines were used for phenotypic analysis. (b) Quantitative analysis of germination and greening rates of WT, rbm25, HAB1pro::HAB1.1 and rbm25/ HAB1pro::HAB1.1 plants. Three biological replicates were performed, and bars represent mean ± SD.
Supplementary Figure 6 Overexpression of RBM25 to the rbm25 mutant rescued ABA sensitivity phenotypes of rbm25. (a) Schematic diagram showing the overexpression construct of RBM25. (b) qrt-pcr analysis of the complementation lines. There are six lines (line 1, 2, 4, 7, 9 and 10) overexpressing RBM25, and two lines (line 3 and 8) having lower RBM25 expression levels than wild type. The RNA was isolated from 7 day-old seedlings, SD of three technical replicates was shown as error bars, and three biological replicates gave similar results..
(c) ABA sensitivity assay of the complementation lines. The seeds were plated on the MS medium supplemented with or without 0.5 μm ABA. The plates were kept at 4 C for two days and then transferred to the constant light. The picture was taken at 8 day after germination. Six transgenic lines overexpressing RBM25 (line 1, 2, 4, 7, 9 and 10) were completely restored for their ABA sensitivity; whereas two lines (line 3 and 8) showing reduced level of RBM25 behaved similarly to the rbm25 mutant.
Supplementary Figure 7 The expression pattern of RBM25 in different tissues and HAB1 in response to ABA. (a) qpcr analysis of RBM25. The bars represent means of three technical replicates ±SD; three biological repeats gave similar results. (b) Microarray assay of HAB1 in response to ABA (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpweb.cgi) (left panel); qrt-pcr analysis for the HAB1 expression without or with 100 µm ABA treatment.
Supplementary Figure 8 Protein structure and phylogenetic analysis of Arabidopsis RBM25. (a) Schematic diagram showing the conserved RRM motif in the N terminal and PWI motif in the C terminal end of RBM25 protein. (b) Alignment of the RRM motif between the human RBM25 and Arabidopsis RBM25. 58% similarity was detected between the two RBM25 proteins (c) Alignment of the PWI motif between the human RBM25 and Arabidopsis RBM25. 74% similarity was detected between the two proteins. (d) Phylogenetic tree of RBM25 proteins in eight species. Pt: Populus trichocarpa; Vv:Vitis vinifera; Rc: Ricinus communis; Mt: Medicago truncatula; Gm: Glycine max; Os: Oryza sativa; Hs: Homo sapiens; At: Arabidopsis thaliana.
Supplementary Figure 9 Sucellular localization of RBM25 in N. benthamiana leaf cells. Subcellular localization of RBM25 following transient transformation of N. benthamiana leaf cells with the 35S::RBM25-GFP construct. The nucleus was indicated by DAPI staining (scale bar, 20 μm).
Supplementary Figure 10 Negative control for the RBM25 and U1-70K interaction. The constructs as indicated in the figure were coexpressed in the leaves of N. benthamiana, and the fluorescence was observed using confocal microscope (scale bar, 20 μm).
Supplementary Figure 11 Binding assay of RBM25 to HAB1 pre-mrna. (a) RBM25 protein can bind to the last intron of HAB1 pre-mrna in EMSA assay. (b) Mutations at the splice sites of HAB1 (from Gg to At) in the third intron decreased the binding ability of RBM25 to the HAB1 pre-mrna.
Supplementary Figure 12 Full size images of all gels and western blots shown in Figure 1, Figure 2 and Figure 3. (1a) Full size image of agarose gel shown in Figure 1a. (1) 1 day after germiantion, (2) 2 day after germination, (3) 3 day after germination, (M) DNA molecular weight ladder. UBQ5 was used as the internal control. (2b) Full size images of agarose gels shown in Figure 2b. (1) wild type, (2) hab1-1, (3) hab1-1hab1.1, (4) hab1-1hab1.2-6, (5) hab1-1hab1.2-8. UBQ5 was used as the internal control. (2c) Full size images of western blotting shown in Figure 2c, *, indicates the non-specific bands, Red arrow indicates the expected proteins. (1) wild type, (2) hab1-1hab1.2-6, (3) hab1-1hab1.2-8. (3d) Full size images of western blotting shown in Figure 3d, Red arrowheads indicate the expected proteins. (3e) Full size images of the gel shown in Figure 3e, Red arrowheads indicate the expected proteins.
Supplementary Figure 13 Full size images of the gels shown Figure 4 (4b) Full size images of agarose gel shown in Figure 4b. (4c) Full size image of agarose gel shown in Figure 4c. (1) 1 day after germination (DAG), (2) 2 day after germination. (M) DNA molecular weight ladder. UBQ5 was used as the internal control.
Supplementary Figure 14 Full size images of the gels in the indicated Figures. (Sup. Fig. 1) Full size images of agarose gels shown in Supplementary Figure 1. MS: without ABA treatments, ABA: with ABA treatment. (Sup. Fig. 3) Three biological repeats of full size images of western blotting shown in Supplementary Figure 3, Rectangles indicate the expected proteins.
Supplementary Figure 15 Biological replicates in qrt-pcr, RNA-ChIP and EMSA Assays in the indicated Figures. (1d) Biological replicates of Figure 1d. (4d) Biological replicates of Figure 4d. (5a) Biological replicates of Figure 5a. (6a) Biological replicates of Figure 6a. (7a) Biological replicates of Figure 7a. (7e) Biological replicates of Figure 7e. (Sup. Fig. 11) Biological replicates of Supplementary Figure 11.
Supplementary Table 1 The list of the oligo sequences in the study. oligo name oligo sequence purpose RBM25GFPF GCGTCGACATGGCCGACGAATCTTCTTC 35S::RBM25-GFP cloning RBM25GFPR GGGGTACCTCAGGCTTTGGATTTTACCG 35S::RBM25-GFP cloning RBM25MYCF GGGTACCCATGGCCGACGAATCTTCTTC 35S::RBM25-myc cloning RBM25MYCR CGAGCTCTCAGGCTTTGGATTTTACCG 35S::RBM25-myc cloning RBM25RTF CTCCGGTATCCTTCTCCATATCC RT-PCR analysis of RBM25 Expression RBM25RTR TGAACAGCACCGATTGTACCA RT-PCR analysis of RBM25 Expression RBM25GWF GGGGACAAGTTTGTACAAAAAAGCAGGCTTC RBM25-BD, RBM25-YFP N ATGGCCGACGAATCTTCTTC cloning Genomic GGACTAGT ATGGCCGACGAATCTTCTTC RBM25genome-GFP RBM25F complementation rescue Genomic RBM25 R GAAGATCT GGCTTTGGATTTTACCG RBM25genome-GFP complementation rescue HAB1F TAATACGACTCACTATAGGGCCAGATAGAGAGGATGA ATATGCA T7 in vivo transcription for EMSA probe HAB1R GTTGTCTTTACTTCCTTTTTGTAGAGC T7 in vivo transcription for EMSA probe RBM25GWF GGGGACAAGTTTGTACAAAAAAGCAGGCTTC RBM25-BD, RBM25-YFP N ATGGCCGACGAATCTTCTTC cloning RBM25GWR GGGGACCACTTTGTACAAGAAAGCTGGGTC RBM25-BD, RBM25-YFP N GGCTTTGGATTTTACCGGG cloning HAB1.1MYC F GGGTACCC ATGGAGGAGATGACTCCCG 35S::HAB1.1-MYC, 35S::HAB1.2pm-MYC cloning HAB1.1MYC R CGGATCCTCAGGTTCTGGTCTTGAACTTTC 35S::HAB1.1-MYC, 35S::HAB1.2pm-MYC cloning HAB1.2MF GTCCATCATtaagcattgcttc HAB1.2 point mutation HAB1.2MR CTAGACATGGCGAGAACACC HAB1.2 point mutation HAB1.1MBP F CGGAATTCATGGAGGAGATGACTCCCG MBP-HAB1.1 and MBP-HAB1.2 cloning HAB1.1MBP CGGGATCCTCAGGTTCTGGTCTTGAACTTTC MBP-HAB1.1 cloning R HAB1.2MBP CGGGATCCTCAAAAGAAGCAATGCTTACCG MBP-HAB1.2 cloning R HAB1.1GWF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAG GAGATGACTCCCG HAB1.1-YFP C,HAB1.2-YFP C and 35S::GFP-HAB1.1 cloning HAB1.1GWR GGGGACCACTTTGTACAAGAAAGCTGGGTCGGTTCTG HAB1.1-YFP C and GTCTTGAACTTTC 35S::GFP-HAB1.2 cloning HAB1.2GWR GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAGAAG CAATGCTTACCG HAB1.2-YFP C cloning
OST1MBPF CGAGCTCGATGGATCGACCAGCAGTGAG MBP-OST1 cloning OST1MBPR GCGTCGACTCACATTGCGTACACAATC MBP-OST1 cloning OST1GWF GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAT OST1-YFP N cloning CGACCAGCAGTGAG OST1GWR GGGGACCACTTTGTACAAGAAAGCTGGGTCCATTGCG TACACAATCTCTCCG OST1-YFP N cloning U1-70KGWF GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGGAGACTCCGGCGat U1-70K-YFP C and U1-70K-AD U1-70KGWR GGGGACCACTTTGTACAAGAAAGCTGGGTC AACATACTCTCGCGATTC U1-70K-YFP C and U1-70K-AD HAB1.1qF CGCACGTGTTTTTGGTGTTC HAB1 and HAB1.2-5 qrt-pcr HAB1.1qR CCGGTTCTGGGATCACATATG HAB1 qrt-pcr HAB1.2-5 TCCTCCTCTGGAACTATGCA HAB1.2-5 qrt-pcr R HAB1.2-3 GCACGTGTTTTTGGTGTTCT HAB1.2-3 qrt-pcr F HAB1.2-3 TCAAGCCACGTTTGTGTGAT HAB1.2-3 qrt-pcr R GAPCF TTGGTGACAACAGGTCAAGCA qrt-pcr GAPCR AAACTTGTCGCTCAATGCAAT qrt-pcr HAB1F ATTGAAGGAAAAATTGGTAGAGCC RT-PCR HAB1R AGGTTCTGGTCTTGAACTTTCTTTG RT-PCR RBM25F CTCCGGTATCCTTCTCCATATCC qrt-pcr RBM25R TGAACAGCACCGATTGTACCA qrt-pcr RBM25TF ATTGAAAAGCACCAAGTGCAC rbm25 homozygous identification RBM25TR AAATCGGAGGAGATTAGCGAC rbm25 homozygous identification LBb1.3 ATTTTGCCGATTTCGGAAC rbm25 homozygous identification ABI1F1 ATGGAGGAAGTATCTCCGGC Splicing variant analysis ABI1R1 TCAGTTCAAGGGTTTGCTCTTG Splicing variant analysis HAB2F1 ATGGAAGAGATTTCACCTGCAG Splicing variant analysis HAB2R1 CAAGATCTGGTCTTGAACTTTCTTTG Splicing variant analysis HAB1F1 TGAAGGAAAAATTGGTAGAGCC Splicing variant analysis HAB1R1 TCAGGTTCTGGTCTTGAACTTTC Splicing variant analysis AHG1F1 ACTGAAATCTACAGAACAATTTCAACC Splicing variant analysis AHG1R1 TGAGAGCTATTCTTGAGATCAATGAC Splicing variant analysis HAI1F1 ATGGCTGAGATTTGTTACGAGAACGAGAC Splicing variant analysis HAI1R1 CTACGTGTCTCGTCGTAGATCAAC Splicing variant analysis HAI2F1 GCGGATATTTGTTATGAAGACGAGA Splicing variant analysis HAI2R1 TCAAGCAACGTGTCTCTTTCTTCT Splicing variant analysis PP2CAF1 ATGGCTGGGATTTGTTGC Splicing variant analysis
PP2CAR1 TTAAGACGACGCTTGATTATTCC Splicing variant analysis HAI3F1 GCCGAGATATGTTACGAAGTAGTGAC Splicing variant analysis HAI3R1 TCTTCTGAGATCAATCACAACGAC Splicing variant analysis ABI2F1 GGACGAAGTTTCTCCTGCAG Splicing variant analysis ABI2R1 TCAATTCAAGGATTTGCTCTTG Splicing variant analysis ABI1F2 ATCCGCTTCCTTGCCATCT Splicing variant analysis ABI1R2 TCAGTTGCGCCGGAGAC Splicing variant analysis ABI1F3 CTCAATCTCCGAGTCAACTCTC Splicing variant analysis ABI1R3 GATGCTGTTTCGACTATACCAAG Splicing variant analysis HAB2F2 GGAGGATGCTGTTAGAGCTTTACC Splicing variant analysis HAB2R2 CCAACAACAGGTCTGTTGATTTT Splicing variant analysis HAB2F3 CGACTGCTGTGGTTGCTTT Splicing variant analysis HAB2R3 CAAAGTCCATCACTGGCCA Splicing variant analysis AHG1F2 CTTCTTGCCAAAAATCGGAAG Splicing variant analysis AHG1R2 CCATCTCATCCATCCTCTTGA Splicing variant analysis AHG1F3 GTTTTGACGCACGATCATATTATTG Splicing variant analysis AHG1R3 AGCTAGCTGACTCGAGAGTACATCC Splicing variant analysis HAI3F2 TCCAAGATACGGTGTTTCTTCG Splicing variant analysis HAI3R2 CGGTGTTTGTAGATCACACTTGC Splicing variant analysis HAI3F3 GCTTGTGATTCCGTCGGA Splicing variant analysis HAI3R3 CAGTCATCGTCTCTCCTGTCC Splicing variant analysis HAI2F2 ATCTGAGGCCGAGATACGG Splicing variant analysis HAI2R2 CACCTACAATTAGCACTCATCACAG Splicing variant analysis HAI2F3 GAGCTTCAAACGCCGGAC Splicing variant analysis HAI2R3 TCATCTTCCTCAGTCCGATCC Splicing variant analysis HAI1F2 CTTCAGTCTGTGGAAGAAGACG Splicing variant analysis HAI1R2 AGTGGATCCCACCGCGT Splicing variant analysis HAI1F3 GATGGTGCGGCAAAATG Splicing variant analysis HAI1R3 AACAACGTCCCAAAGACCG Splicing variant analysis PP2CAF2 ACATGGAAGACGCTGTCTCG Splicing variant analysis PP2CAR2 TCCGAGTAGCACCATTAACAACTAAG Splicing variant analysis PP2CAF3 TGTATCGGTTGTCACGCC Splicing variant analysis PP2CAR3 CCAAGATCAAACACTCATCCTCA Splicing variant analysis ABI2F2 ACTTCGATTTGTGGTAGACGAC Splicing variant analysis ABI2R2 TTCTTCCACTTCTCTTGCCAC Splicing variant analysis ABI2F3 GACTCTAGGGCGGTTTTGTG Splicing variant analysis ABI2R3 GACTCTAGGGCGGTTTTGTG Splicing variant analysis