Supplemental materials

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1 Supplemental materials Materials and methods for supplemental figures Yeast two-hybrid assays TAP46-PP2Ac interactions I. The TAP46 was used as the bait and the full-length cdnas of the five C subunits of PP2A were used as preys, and an unrelated protein Tom20 was used as a negative prey control. The prey DNA fragments were produced by PCR using the oligonucleotide primers C1-YF1 and C1-YR1 for C1 (At1G59830), C2-YF1 and C2-YR1 for C2 (At1G10430), C3-YF1 and C3-YR1 for C3 (At2G42500), C4-YF1 and C4-YR1 for C4 (At3G58500), C5-YF1 and C5-YR1 for C5 (At1G69960), Tom20-YF1 and Tom20-YR1 for Tom20, cut with the restriction enzymes Eco RI and Xho I, then inserted into the prey vector pjg4-5 (Golemis et al., 1996). Information on PCR primers is listed in Supplemental Table 1. TAP46-PP2Ac interactions II. The TAP46 was used as the prey, the five C subunits of PP2A were used as baits, and Tom20 was used as a negative bait control. The TAP46 prey DNA was produced by PCR using the oligonucleotide primers TAP46-YF1 and TAP46-YR1, cut with the restriction enzymes Eco RI and Xho I, then inserted into the prey vector pjg4-5 (Golemis et al., 1996). The preys were produced by PCR using the oligonucleotide primers C1-YF1 and C1-YR1 for C1 (At1G59830), C2-YF1 and C2-YR1 for C2 (At1G10430), C3-YF1 and C3-YR1 for C3 (At2G42500), C4-YF1 and C4-YR1 for C4 (At3G58500), C5-YF1 and C5-YR1 for C5 (At1G69960), Tom20-YF1 and Tom20-YR1 for Tom20, cut with the restriction enzymes Eco RI and Xho I, then inserted into the prey vector peg202 (Golemis et al., 1996). Protein-protein interactions between TAP46 and other phosphatases. The full-length cdnas of PP4 (PPX1 and PPX2), PP6 (AtFYPP1 and AtFYPP3), and PP1 (TOPP4 and TOPP6) were amplified from a cdna library using oligonucleotide 1

2 primers PPX1-YF1 and PPX1-YR1 for PP4 isoform 1 (At4g26720), PPX2-YF1 and PPX2-YR1 for PP4 isoform 2 (At5g55260), AtFYPP1-YF1 and AtFYPP1-YR1 for PP6 isoform 1 (At1g50370), AtFYPP3-YF1 and AtFYPP3-YR1 for PP6 isoform 2 (At3g19980), TOPP4-YF1 and TOPP4-YR1 for PP1 isoform 1 (At2g39840), TOPP6-YF1 and TOPP6-YR1 for PP1 isoform 2 (At5g43380), digested with restriction enzymes Eco RI and Xho I, then inserted into the bait vector peg202 (Golemis et al., 1996), respectively. Antibody production and quality analysis The full-length cdnas of TAP46 and ABI5 were amplified by PCR from an Arabidopsis cdna library using oligonucleotides TAP46-obF1 and TAP46-obR1, ABI5-obF1 and ABI5-obR1, respectively, digested with restriction enzymes Bam HI and Sac I, then subcloned into the bacterial expression vector pet-30b of Novagen (Madison, Wisconsin, USA). Bacterially expressed ABI5 and TAP46 were purified to homogeneity by following the protocol of the pet bacterial expression system from Novagen (Madison, Wisconsin), then sent to the antibody production company Cedarlane (Burlington, Ontario, Canada) for raising antibodies. Both TAP46 and ABI5 antibodies were tested and confirmed before use. The TAP46 antibody could detect 5 ng purified TAP46 protein and detect a band from 5 µg of cellular protein extracts in Western blot analysis (Supplemental Fig. 7), and so was the ABI5 antibody (Supplemental Fig. 8), indicating that these two antibodies are in good quality. 2

3 Supplemental Table 1. List of oligonucleotide primers used for PCR and cloning experiments Oligonucleotide primers used in the main text ABI5-YF1: ATCGGAATTCATGGTAACTAGAGAAACGAAGTTGACGTC ABI5-YR1: ATCGCTCGAGTTAGAGTGGACAACTCGGGTTCCTC Actin7-F: GGCCGATGGTGAGGATATTCAGCCACTTG Actin7-R: TCGATGGACCTGACTCATCGTACTCACTC Actin8-F: GGAATGGTTAAGGCTGGATTCG Actin8-R: ACGCATCTTTCTGATTCATCCC AtEm1-F1: ATGGCGTCAAAGCAACTGAGC AtEm1-R1: TCACTTGTTGGTGAACTTTGACTC AtEm6-F1: ATGGCGTCTCAACAAGAGAAG AtEm6-R1: TTAGGTCTTGGTCCTGAATTTGG Lba1: TGGTTCACGTAGTGGGCCATCG LeaD34-F1: ATGAGTCAAGAAGAACAACCAAAGC LeaD34-R1: TCATATATCAGCTCTCTCGTTAAGC NCED3-F1: AAAGCCATCGGTGAGCTTCA NCED3-R1: GCAGCTCTGGCGTAGAATAGC RD29A-F1: GCCGACGGGATTTGACG RD29A-R1: GCCGGAAATTTATCCTCTTCTGA RD29B-F1: GGCGGGCAAAGCGAG RD29B-R1: TGCCCGTAAGCAGTAACAGATC tap46-f1: AATGTCAACTCGCAACACAATC tap46-r1: TCAATGCACCTCTTCGTTTTC tap46-f2: AGCATCCCTCTAGCTTTGGTC tap46-r2: CCAATTCAGTAAAAACTCGCG TAP46-OF1: AGCTGGATCCATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-OR1: AGCTGAGCTCTCAGCCACAAGGTGTGAGTTTCTTGTTACC TAP46-Pro-F: AGCTAAGCTTCTTTTGAAGCATCCCTCTAGCTTTGGTCGC TAP46-Pro-R: AGCTTCTAGACACAGGCAACAATTCAAAATTCCTT: TAP46-Com-F: AGCTCCATGGATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-Com-R: ATCGACTAGTGCCACAAGGTGTGAGTTTCTTGTTACC TAP46-GFP-F1: AGCTTCTAGAATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-GFP-R1: AGCTGGATCCGCCACAAGGTGTGAGTTTCTTGTTACC TAP46-qPCR-F1: AAGACAGTTGAAGGATGGAGAG TAP46-qPCR-R1: GGTTTAGAGTACTGTATCCTTGC TAP46-YF1: AGCTGAATTCATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-YR1: AGCTCTCGAGTCAGCCACAAGGTGTGAGTTTCTTGTTACC TAP46-RT-F1: ATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-RT-R1: TCAGCCACAAGGTGTGAGTTTCTTGTTACC Oligonucleotide primers used in the supplemental materials 3

4 ABI5-obF1: AGCTGGATCCGATGGTAACTAGAGAAACGAAGTTGACGTC ABI5-obR1: AGCTGAGCTCTTAGAGTGGACAACTCGGGTTCCTC AtFYPP1-YF1: AGCTGAATTCATGGATTTAGATCAATGGATTTCC AtFYPP1-YR1: AGCTCTCGAGTCACAGGAAATAAGGAACACCT AtFYPP3-YF1: AGCTGAATTCATGGATTTGGATCAATGGATTTCC AtFYPP3-YR1: AGCTCTCGAGTCATAGGAAATACGGAACTCCA C1-YF1: AGCTGAATTCATGCCGTTAAACGGAGATCTCGACCGTC C1-YF1: AGCTGAATTCATGCCGTTAAACGGAGATCTCGACCGTC C1-YR1: AGCTCTCGAGTCACAAAAAATAATCAGGGGTCTTGCGCG C2-YF1: AGCTGAATTCATGCCGTCGAACGGAGATCTGGACC C2-YR1: AGCTCTCGAGTCACAAAAAATAATCAGGGGTCTTCCGAG C3-YF1: AGCTGAATTCATGGGCGCGAATTCTATTCCGACGGAC C3-YR1: AGCTCTCGAGTCACAGGAAATAGTCTGGAGTCCTTCGGG C3-YR1: AGCTCTCGAGTCACAGGAAATAGTCTGGAGTCCTTCGGG C4-YF1: AGCTGAATTCATGGGCGCGAATTCGCTTCCAACGG C4-YR1: AGCTCTCGAGTCAAAGGAAATAGTCAGGTGTCCTTCGGG C5-YF1: AGCTGAATTCATGCCGTTAAACGGAGATCTCGACCGTC C5-YR1: AGCTCTCGAGTTACAAAAAATAATCTGGAGTCTTGCGAGTGGTTTCGG PPX1-YF1: AGCTGAATTCATGTCAGACCTAGATCGGCAAATAGG PPX1-YR1: AGCTGAATTCTTATAGGAAGTAATCAGGGGCCGGC PPX2-YF1: AGCTGAATTCAATGTCAGACCTAGACAAGCAAATAGA PPX2-YR1: AGCTCTCGAGTCACAGGAAATAATCAGGTGCAGGT TAP46-obF1: AGCTGGATCCGATGGGTGGTTTGGCTATGGAGGAAATGCCT TAP46-obR1: AGCTGAGCTCTCAGCCACAAGGTGTGAGTTTCTTGTTACC Tom20-YF1: TAGCGAATTCATGGATATGCAGAATGAAAACG Tom20-YR1: TGACCTCGAGTTACTGCCTTGACACCGGC TOPP4-YF1: AGCTGAATTCATGGCGACGACGACGACGACGC TOPP4-YR1: AGCTCTCGAGTCAAATCTTTGTGGACATCATG TOPP6-YF1: AGCTGAATTCATGGATCCTGGTACTTTAAACTCGG TOPP6-YR1: AGCTCTCGAGTCAGTTGTATTCTTTCGAATTTTGCG

5 Supplemental figures Supp. Fig. 1. Phenotypes of wild-type, TAP46-overexpressing, and tap46 mutants under normal growth conditions for 16 days. Col, wild-type Arabidopsis (Columbia); OE1 and OE2, TAP46-overexpressing line 1 and 2, respectively; tap46-1 and tap46-2, two independent tap46 knock-down mutants. Bar = 1 cm. 5

6 Supp. Fig. 2. Phenotypes of wild-type, TAP46-overexpressing, and tap46-1 mutant plants on MS plate for 14 days. Col, wild-type Arabidopsis (Columbia); OE1 and OE2, TAP46-overexpressing line 1 and 2, respectively. 6

7 Supp. Fig. 3. Transcript analysis of TAP46 in various tissues of Arabidopsis. Real-time PCR was used to analyze the transcript levels of TAP46 in different tissues and at different developmental stages of seed development. The actin gene 8 was used as the reference gene for comparison. Three independent experiments were performed. 7

8 Supp. Fig. 4. Protein-protein interactions between TAP46 and the C subunits of PP2A in the yeast two-hybrid system. A. The C subunits of PP2A were used as baits and the TAP46 was used as prey. Tom20, an unrelated protein used as a negative control. B. The C subunits of PP2A were used as preys and the TAP46 was used as bait. 8

9 Supp. Fig. 5. Expression of the three ABI5 downstream genes RD29A, RD29B, and NCED3 is activated by the PP2A inhibitor cantharidin in 5-day-old seedlings. The transcript levels of these genes were analyzed by real-time PCR technique. The actin gene 8 was used as the reference. Three independent experiments were performed. 9

10 Supp. Fig. 6. TAP46 interacts with PP4 and PP6, in addition to ABI5, in the yeast two-hybrid system. PPX1 and PPX2 are PP4 phosphatases; AtFYPP1 and AtFYPP3 are PP6 phosphatases; TOPP4 and TOPP6 are PP1 phosphatases. Tom20, an unrelated protein was used as a negative control. 10

11 Supp. Fig. 7. Quality analysis of TAP46 antibodies. The TAP46 antibodies could recognize TAP46 in 5 ng of purified TAP46 protein and in 5 µg of total cellular proteins of 5-day-old seedlings in Western blot analysis. 11

12 Supp. Fig. 8. Quality analysis of ABI5 antibodies. A. The ABI5 antibodies can recognize ABI5 in 5 ng of purified ABI5 protein and in 5 µg of total cellular proteins of 5-day-old seedlings in Western blot analysis. B. ABI5 is reduced in abi5-1 mutant and elevated in ABI5-overexpressing plants. O-ABI5-1, O-ABI5-2, and O-ABI5-3, three independent ABI5-overexpressing transgenic lines; WT, wild-type Arabidopsis. 12