Supplemental Data. Dai et al. (2013). Plant Cell /tpc Absolute FyPP3. Absolute

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1 A FyPP1 Absolute B FyPP3 Absolute Dry seeds Imbibed 24 hours Dry seeds Imbibed 24 hours C ABI5 Absolute Dry seeds Imbibed 24 hours Supplemental Figure 1. Expression of FyPP1, FyPP3 and ABI5 during seed germination. (A) FyPP1 is expressed in dry seeds, and up-regulated after imbibition. (B) FyPP3 is expressed in dry seeds, and up-regulated after imbibition. (C) ABI5 is strongly expressed in dry seeds, but down-regulated after imbibition. Data was collected from (Winter et al., 2007).

2 Supplemental Figure 2. Germination rates of Col, f1, f3 seeds and seeds from self-pollinated f1 -/+ f3 plants treated with or without ABA at various concentrations. In the absence of ABA (0 µm), the germination rates of Col, f1, f3 seeds and seeds from self-pollinated f1 -/+ f3 plants were comparable. In the presence of ABA (0.5, 1, 2.5 and 5 µm), the germination rates of seeds from self-pollinated f1 -/+ f3 plants were lower than that of Col. while Col, f1 and f3 seeds have comparable germination rates after ABA treatment at various concentrations (0.5, 1, 2.5 and 5 µm). Values are means ± SD (n=100), three replicates.

3 A B 1 cm Supplemental Figure 3. Phenotypes of f1 f3 homozygotes at the seedling stage. (A) 3-days-old Col seedlings. (B) 3-days-old f1 f3 seedlings. Compared to Col seedlings, f1 f3 homozygotes had shorter roots and smaller/fused cotyledons.

4 A B Supplemental Figure 4. Relative root growth of f1 f3 and ethanol-induced F3Ri/f1 mutants after ABA treatment. (A) Increased sensitivity of f1 f3 roots to ABA treatment, as compared to the roots of Col, f1 and f3 mutants. Seedlings were grown on germination medium (GM) plates vertically for 3 days and then transferred onto fresh GM plates with 0 or 1 µm ABA for 5 days before analysis. The percentage of relative root growth was calculated based on control plants grown on unsupplemented media. Values are means ± SD (n=20). Asterisks indicate the levels of statistical significance as determined by Student s t test: * p<0.01 versus Col. (B) Increased sensitivity of F3Ri/f1 roots to ABA treatment, as compared to the roots of Col and f1 mutants. Seedlings were grown on germination medium (GM) plates (with 0.1% ethanol) vertically for 3 days and then transferred onto fresh GM plates (with 0.1% ethanol) with 0 or 1 µm ABA for 5 days before analysis. The percentage of relative root growth was calculated based on control plants grown on unsupplemented media. Values are means ± SD (n=20).

5 Supplemental Figure 5. Expression analysis of FyPP3 in F3Ri/f1 lines. RT-PCR analysis showing the silenced or greatly reduced expression of FyPP3 in F3Ri/f1-3 and -21 transgenic lines after ethanol induction.

6 A B C Supplemental Figure 6. Germination phenotypes of Col, abi5-1 seeds and seeds from self-pollinated f1 -/+ f3, f1 -/+ f3 abi5-1 plants. (A) Germination rates of Col, abi5-1 seeds and seeds from self-pollinated f1 -/+ f3, f1 -/+ f3 abi5-1 plants incubated on GM plates without ABA. Values are means ± SD from three independent experiments (n>100). (B) Germination rates of Col, abi5-1 seeds and seeds from self-pollinated f1 -/+ f3, f1 -/+ f3 abi5-1 plants incubated on GM plates in the presence of ABA (0.5 µm). The germination of seeds from self-pollinated f1 -/+ f3 plants were hyper-sensitive to ABA; while seeds from self-pollinated f1 -/+ f3 abi5-1 plants were insensitive to ABA, similar to the abi5-1 mutants. Values are means ± SD from three independent experiments (n>100). (C) Greening percentages of Col, abi5-1 seeds and seeds from self-pollinated f1 -/ + f3, f1 -/+ f3 abi5-1 plants incubated on GM plates supplemented with 0.5 µm ABA for 5 days. Values are means ± SD from three independent experiments (n>100). Asterisks indicate the levels of statistical significance as determined by Student s t test: *, p<0.01 versus Col.

7 Supplemental Figure 7. Yeast two-hybrid assays between FyPP1/3 and key regulators in ABA signaling. Full-length FyPP1 and FyPP3 were fused with the LexA DNA binding domain (BD) and used in the pairwise interaction with ABI1, ABI2, ABI3, ABI4, ABI5, OST1, Pyr, and Pyl1 fused with the transcriptional activation domain of B42 (AD). 10 µm ABA was added to the color developing plates for the pairwise interactions between FyPP (FyPP1 and FyPP3) proteins and ABA receptors (Pyr and Pyl1). The blue color indicates the protein-protein interaction.

8 A B Supplemental Figure 8. Immunoblot assay of the fusion proteins in the yeast cells for yeast two-hybrid assays. (A) A diagram of parewise combinations of different B42 (AD)- and LexA (BD)-fusions for yeast two-hybrid assays in this work. The combinations are indicated alphabetically. (B) Immunoblots of the target fusion proteins in the yeast two-hybrid assays indicated in (A). The LexA and B42 fusions were detected by anti-lexa and anti-b42 antibodies respectively. A total of 30 µg total protein was loaded for each lane. The loading control was indicated by Ponceau S staining.

9 Supplemental Figure 9. Sequence alignment of the bzip transcription factors. The sequences were aligned with CLUSTALX 2.1 and edited with GeneDoc 3.2. The black shading indicates the conserved amino acids. Based on the sequence identities to ABI5 (indicated in the brackets), the aligned sequences can be classified into two groups (I and II).

10 YFP-FYPP1 YFP-FyPP3 YFP Dark Merge Supplemental Figure 10. Subcellular localization of YFP-FyPP1 and YFP-FyPP3 in Aradopsis root cells. Confical microscopy showing the subcellular localization of YFP- FyPP1 (left panel) and YFP-FyPP3 (right panel) in 4-day old F1OE and F3OE seedling roots. YFP signal was detected in the nucleus (N), the plasma membrane and the cytosol.

11 A B C D E Supplemental Figure 11. Expression analysis of ABI5 mrna. (A) Real-time RT-PCR analysis showing that in the absence of ABA (-ABA), ABI5 showed a similar expression profile in all lines; while in the presence of ABA (+ABA), ABI5 was up-regulated in all lines to various degrees. Values are means ± SD with three replicates (B) RT-PCR analysis showing similar expression pattern of ABI5 in Col, f1 and F3Ri/f1-21 seeds with the same treatment as used in Figure 5B (left panel). At the end of 5 µm ABA treatment (time 0 ), ABI5 was highly expressed; ABI5 expression became undetectable 8, 12, 16 or 20 hours after ABA washout. 26 cycles were used for the RT-PCR reaction. Actin was used as a control. (C) Quantification of ABI5 expression in Col, f1 and F3Ri/f1-21 seeds in response to ABA removal. Values are means ± SD with three replicates. (D) RT-PCR analysis showing reduced expression of ABI5 in Col, F1OE, F3OE, F1DN and F3DN seeds in response to ABA removal. 26 cycles were used for the RT-PCR reaction. Actin was used as a control. (E) Quantification of ABI5 expression in Col, F1OE, F3OE, F1DN and F3DN seeds in response to ABA removal. Values are means ± SD with three replicates.

12 Supplemental Figure 12. Germination rates of Col, F1OE, F3OE, F1DN and F3DN seeds treated with or without ABA. (A) In the absence of ABA (-ABA), the germination rates of all seed types are similar. Values are means ± SD (n=100), three replicates. (B) In the presence of 1 µm ABA (+ABA), the germination rates of F1OE and F3OE seeds were higher, while the germination rates F1DN and F3DN were lower than that of Col seeds. Values are means ± SD (n=100), three replicates. d, Days.

13 Supplemental Tables Supplemental Data. Dai et al. (2013). Plant Cell /tpc Supplemental Table1. List of the primer sequences used in this study. Purpose Plasmid constructing pjg-abi1 peg-abi1 pjg-abi2 peg-abi2 pjg-abi3 peg-abi3 pjg-abi4 peg-abi4 pjg-abi5 peg-abi5 pjg-abi5a4 peg-abi5a4 pjg-abi5d4 peg-abi5d4 pcambia-myc-abi5 pcambia-cluc-abi5 pcambia-abi5-nluc pgex-gst-abi5b pgex-gst-abi5bm py2n-abi5 Genes/ fragment ABI1 ABI2 ABI3 ABI4 ABI5 ABI5A1 ABI5A2 ABI5A3 ABI5A4 ABI5D1 ABI5D2 ABI5D3 ABI5D4 ABI5b/m Name sequence (5'-3') Restriction Enzymes ABI1-F ACGT CAATTG ATGGAGGAAGTATCTCCGGCGATC Mfe I ABI1-R ACGT CTCGAG Xho I GTTCAAGGGTTTGCTCTTGAGTTTC ABI2-F ACGT GAATTC EcoR I ATGGACGAAGTTTCTCCTGCAGTC ABI2-R ACGT CTCGAG Xho I ATTCAAGGATTTGCTCTTGAATTTC ABI3-F ACGT CAATTG Mfe I ATGAAAAGCTTGCATGTGGCGGCCAAC ABI3-R ACGT GTCGAC Sal I ACAGTTTGAGAAGTTGGTGAAGCGACCAC ABI4-F ACGT CAATTG Mfe I ATGGACCCTTTAGCTTCCCAACATC ABI4-R ACGT GTCGAC Sal I ATAGAATTCCCCCAAGATGGGATC ABI5-F1 AGTC GGTACC GAATTC Kpn I, ATGGTAACTAGAGAAACGAAGTTGAC EcoR I ABI5-R1 AGTC CTCGAG GAGTGGACAACTCGGGTTCCT Xho I ABI5-F2 AGTC GGATCCTA BamH I ATGGTAACTAGAGAAACGAAGTTGAC ABI5-R2 AGTC ACTAGT GAGTGGACAACTCGGGTTCCT Spe I ABI5-F3 AGTC GTCGACA Sal I ATGGTAACTAGAGAAACGAAGTTGAC ABI5-R3 AGTC GGATCC GAGTGGACAACTCGGGTTCCT BamH I ABI5A1-F CTTCTTTGGGAAGACAATCCGCTATCTACTCAT TGACCCTTG ABI5A1-R CAAGGGTCAATGAGTAGATAGCGGATTGTCTTC CCAAAGAAG ABI5A2-F CTAGTCTTCCTCGACAAGGCGCTTTGACACTTC CAGCTC ABI5A2-R GAGCTGGAAGTGTCAAAGCGCCTTGTCGAGGA AGACTAG ABI5A3-F GAGACTGCGGCTAGACAACCGGCTTTTGGAGA GATGACACTTG ABI5A3-R CAAGTGTCATCTCTCCAAAAGCCGGTTGTCTAG CCGCAGTCTC ABI5A4-R AGTC CTCGAG Xho I GAGTGGACAAGCCGGGTTCCTCATCAATGTCC ABI5D1-F CTTCTTTGGGAAGACAATCCGATATCTACTCATT GACCCTTG ABI5D1-R CAAGGGTCAATGAGTAGATATCGGATTGTCTTC CCAAAGAAG ABI5D2-F CTAGTCTTCCTCGACAAGGCGATTTGACACTTC CAGCTCC ABI5D2-R GGAGCTGGAAGTGTCAAATCGCCTTGTCGAGG AAGACTAG ABI5D3-F GAGACTGCGGCTAGACAACCGGATTTTGGAGA GATGACACTTGAG ABI5D3-R CTCAAGTGTCATCTCTCCAAAATCCGGTTGTCT AGCCGCAGTCTC ABI5D4-R AGTC CTCGAG Xho I GAGTGGACAATCCGGGTTCCTCATCAATGTCC ABI5b-F GAATTC AGTGGTGGTTCTAGAGGCAAC EcoR I ABI5b-R CTCGAG CTGAGCATTGTTCTGTCCATTAC Xho I

14 peg-ost1 pjg-ost1 peg-pyr pjg-pyr peg-pyl1 pjg-pyl1 pcambia-cluc-abf2 pcambia-abf2-nluc pcambia-cluc- AREB3 pcambia-areb3- nluc pcambia-cluc-eel pcambia-eel-nluc Supplemental Data. Dai et al. (2013). Plant Cell /tpc OST1 Pyr Pyl1 ABF2 ABI5bm-F GTCTTCCTCGACAAGGCGCTTTGACACTTCCAG C ABI5bm-R GCTGGAAGTGTCAAAGCGCCTTGTCGAGGAAG AC OST1-F GAGCT GAATTC ATGGTGGGACTTTTTGTATTTGTAC EcoR I OST1-R AGCT CTCGAG CATTGCGTACACAATCTCTCC Xho I Pyr-F AGAC GAATTC ATGCCTTCGGAGTTAACACC EcoR I Pyr-R AGAC CTCGAG CGTCACCTGAGAACCACTTC Xho I Pyl1-F AGAC CAATTG ATGGCGAATTCAGAGTCCTC EcoR I Pyl1-R AGAC GTCGAC CCTAACCTGAGAAGAGTTGTTG Sal I ABF2-F GGTACC atggatggtagtatgaatttggggaatg Kpn I ABF2-R GTCGAC ccaaggtcccgactctgtcctcctcagc Sal I AREB3-F CAATTG atggattctcagaggggtattg Kpn I AREB3 AREB3-R GTCGAC gaaaggagccgagcttgtcc Sal I EEL Mutant genotyping /expression analysis EEL-F GAATTC atgggttctattagaggaaacattg Kpn I EEL-R GTCGAC gagggaagcagagtttgttcg SALK mutants T-DNA LB1 CGGAACCACCATCAAACAGG ost1 mutant Actin RD29A RD29B ABI5 ost1-f ost1-r Actin-F Actin-R RD29A-F RD29A-R RD29B-F RD29B-R ABI5q-F ABI5q-R CGGGTCTCTGAAAACGTCTC TCGCTCGAAAAGTTCTCCTC CACAATGTTTGGCGGGATTGGTGA TGTACTTCCTTTCCGGTGGAGCAA GCCGACGGGATTTGACG GCCGGAAATTTATCCTCTTCTGA GGCGGGCAAAGCGAG TGCCCGTAAGCAGTAACAGATC TCGACAAGGCTCTTTGACAC ATTACCGCTACCACCACCTC Sal I