SUPPLEMENTAL FILES. Supplemental Figure 1. Expression domains of Arabidopsis HAM orthologs in both shoot meristem and root tissues.

Transcription

1 SUPPLEMENTAL FILES SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure 1. Expression domains of Arabidopsis HAM orthologs in both shoot meristem and root tissues. RT-PCR amplification of AtHAM1, AtHAM2, AtHAM3, along with AtACTIN2 employed as an endogenous control. cdna was prepared from three independent tissue samples of the indicated tissues. Shoot tissue comprises rosette leaf, stem and meristem tissue of plants prior to bolting. Rosette leaf tissue is comprised of expanding rosette leaves. Inflorescence tissue largely excludes mature inflorescence stem. AtHAM ortholog oligonucleotide primer sets used bracket the MIR170/171-binding site, thereby selectively amplifying from full-length transcripts rather than from MIR-mediated cleavage products (Supplemental Table 2). Supplemental Figure 2. Loss-of-function alleles of Arabidopsis homologs of Petunia HAM. (A) Schematic representation showing the location of T-DNA (AtHAMs 1 and 4) and Ds element insertions (AtHAMs 2 and 3) in knockout Atham1-1(FLAG_239), Atham2-1 (SGT11982), Atham3-1 (SGT13186), and Atham4-1 (Salk_110871) alleles. (B) RT-PCR analysis of homozygous Atham2 and Atham3 mutants with primer sets flanking, 5 and 3 of the Ds insertion sites (Supplemental Table 2). The location of primers on their respective target genes, lengths of the amplified fragments, and position relative to the Ds insertions are indicated schematically. Supplemental Figure 3. Reduction in primary root elongation in Atham1,2,3 mutants results from reduced rates of cell division in the root meristem. 1

2 (A) An example of a mature atrichoblast from which cell lengths along the longitudinal cell axis, indicated by the arrow, were measured. (B) Mean atrichoblast lengths of Ler and Atham1,2,3 mutants. Error bars indicate 2xsem. Supplemental Video 1. Arrested inflorescence and flower meristems of an Atham1,2,3 mutant, visualized by digital optical microscopy. A cauline leaf is visible in the background. Cover Art Submission. Arrested inflorescence and flower meristems of an Atham1,2,3 mutant, visualized by digital optical microscopy. 2

3 SUPPLEMENTAL TABLES Supplemental Table 1. DELLA and HAM-subfamily proteins used for phylogenetic analysis in Figure 1. Subfamily Taxon Protein Identifier or Name Sequence Length (aa) N-terminal most 10 aa DELLA Physcomitrella Protein ID MAYQYYPGST Protein ID MAYQYSPGGS Selaginella Protein ID MSNWAMFDSM Protein ID MEDMDMLEAV Oryza Os01g MAMDTFPFQW Os05g MAQFGGFGGW Os03g MKREYQEAGG Arabidopsis At1g14920 (GA-INSENSITIVE) 533 MKRDHHHHHH At2g01570 (RGA) 587 MKRDHHQFQG At1g66350 (RGL1) 511 MKREHNHRES At3g03450 (RGL2) 547 MKRGYGETWD At5g17490 (RGL3) 523 MKRSHQETSV HAM Physcomitrella Protein ID MVITAGSSGG Selaginella Protein ID MEELERMLLS Ceratopteris C. thalictroides HAM 348 ARLNQQVPLK Pinus Pinus HAM 374 IVEQLLKAAE Picea Picea HAM 451 ADIQAAGIHE Allium Alium HAM1 418 VQNPLLYNPT Sorghum Sb10g MSELQDDGAG Sb01g PHAAFPPFVT Sb01g MRGMLFAGSA Sb06g MRAAPFSADG Sb04g MRAALFGAQR Sb04g MRAALFGAQR Oryza Os06g MSSSSCHPHN Os04g MRAAPFSADG Os02g MRAALFGAER Os02g MRAALFGAER Zea Zm2g MTSALPHMSE Zm2g MHMPPLGLGA Zm2g MRGMLFAGSA Zm2g MRFHDGADIK Zm2g MRAAPFSADG 3

4 Zm2g MAKAAEVGNS Zm2g MRAALFGAER Brachypodium Bd1g MSSSSRGNLN Bd1g MPLGDLLLHG Bd3g MPFAGESHAG Bd3g MRAALLGAER Triticum Triticum HAM1 345 PPDGTDIVDP Triticum HAM2 344 SPLGSPSSGR Saccharum Saccharum HAM1 466 RKPPLLGAQP Aquilegia Aquilegia HAM1 673 SLNTISKKKE Aquilegia HAM2 639 SFNPSLAVPS Aquilegia HAM3 323 LVLPIKQEFC Vitis Vitis HAM1 750 MKDMPLPLEL Vitis HAM2 538 MKVPVPAQNN Mimulus Mimulus HAM1 706 MKGMPLPFDF Mimulus HAM2 420 IHPSPLSPPL Solanum Solanum lycopersicum HAM1 536 MKVPFSTNDN Solanum tuberosum HAM1 683 VSGFVPSISS Petunia HAIRY MERISTEM 721 MIAIPQGAK Capsicum Capsicum HAM1 446 EEMGQFHQLQ Euphorbia Euphorbia HAM1 368 QLSFSVGKPQ Carica Carica HAM1 762 MLHASRAENK Carica HAM2 797 MPFQLQGTKG Carica HAM3 534 MRVPVTSSPQ Arabidopsis At4g MKIPASSPQD At2g45160 (AtHAM1) 640 MPLSFERFQG At3g60630 (AtHAM2) 623 MPLPFEQFQG At4g00150 (AtHAM3) 558 MPLPFEEFQG Cucumis Cucumis HAM1 754 MKAMPLPFPF Cucumis HAM2 532 MRVPVSNNQL Cucumis HAM3 508 MRVPVPNHQS Populus Pt0005s MKVPAPSPQN Pt0007s MKVPVPSPQN Pt0003s MPFRLQAKGG Pt0001s MPFHLQAKGG Pt0014s MKAMPLPFEN Pt0014s MKAMPLPSED Pt0002s MKAMPLPFED Pt0002s MPLPFENFQG Malus Malus x domestica 645 RAAAISPDEK Manihot Manihot HAM1 713 MPLPFDDFQG Manihot HAM2 728 MRRSPSPPTS 4

5 Manihot HAM3 498 MKVPVSSPQN Ricinus Ricinus HAM1 733 MPLAFEDFQG Ricinus HAM2 787 MELERGDVDN Ricinus HAM3 539 MKVPVSSSQN Glycine Gm11g MRVPVPSSPQ Gm01g MRVPVPSSPQ Gm02g MRVPSSPQAS Gm16g MRVPSSPQPN Gm01g MPLPFEEFQG Gm01g GYFYMKEALI Gm11g MNEMKAVPLP Gm01g MKAVPLPFQE Medicago Mt5g MKHDMRTFWV Mt8g MRVPPPAVSS Mt4g MSSSHGSGSN 5

6 Supplemental Table 2. Oligonucleotide primer sequences used for RT-PCR amplification of AtHAM gene fragments. AtHAM1 T-DNA flanking primer set* Forward--5ʼ-GTCAACATCAGTTTCAGTTTCCG-3ʼ Reverse--5ʼ-GTTACGGTCACATCCTCTGTCTG-3ʼ AtHAM2 Ds insertion flanking primer set Forward--5ʼ-CAGATCTTCATCAGCTCCGTC-3ʼ Reverse--5ʼ-CGACGACGTTTGGTGAGATTTGC-3ʼ AtHAM2 5ʼ of Ds insertion primer set* Forward--5ʼ-CGCTGTCTTCTTCTCACGGTGGTC-3ʼ Reverse--5ʼ-CTTGAAGGAGAGAGTGAAGAGC-3ʼ AtHAM2 3ʼ of Ds insertion primer set Forward--5ʼ-CGAGTCTCTCGACTCTGGGAATC-3ʼ Reverse--5ʼ-CATTTCCAAGCTGAGACAGTAAC-3ʼ AtHAM3 Ds insertion flanking primer set Forward--5ʼ-CTCAATAATCTCCTTCACAACGTCTCC-3ʼ Reverse--5ʼ-CAAGGGAGATGTTGATCTCAGAGGC-3ʼ AtHAM3 5ʼ of Ds insertion primer set* Forward--5ʼ-GACCACCACTCTGTTCCGCCG-3ʼ Reverse--5ʼ-GGAGACGTTGTGAAGGAGATTATTGAG-3ʼ AtHAM3 3ʼ of Ds insertion primer set Forward--5ʼ-CTCTGAGATCAACATCTCCCTTGAC-3ʼ Reverse--5ʼ-CGGCTGTATAAGAAACCTCTCGATC-3ʼ AtACTIN2 Forward--5ʼ-CTCCGCTTTGAATTGTCTCGTTGTCC-3ʼ Reverse--5ʼ-GGAATAAAGAGGCATCAATTCGATCAC-3ʼ * denotes primer sets that bracket the MIR170/171-binding site, used for analysis in Supplemental Figure 1. 6

7 Supplemental Table 3. Oligonucleotide primer sequences used for PCR genotyping of Atham insertional alleles. Primer Set Product Size AtHAM1 T-DNA flanking primer set Forward--5ʼ-CAGATTCTCAGAAAATCTGGTCC-3ʼ Forward and Reverse--804 bp Reverse--5ʼ-CGGTGTGGTCGCCGTTGTTGTTTC-3ʼ AtHAM2 Ds insertion flanking primer set Forward--5ʼ-GGAGGTCAATGGGCGTCTCTG-3ʼ Forward and Reverse--392 bp Reverse--5ʼ-GGCGCGTCGTTGTTACGGTCG-3ʼ Forward and Ds bp AtHAM3 Ds insertion flanking primer set Forward--5ʼ-CAATCAACAGCTCTCTTCTCC-3ʼ Forward and Reverse--397 bp Reverse--5ʼ-CTTGAGGTTGTCTTGAGTGAAGC-3ʼ Forward and Ds bp Ds Insertion Primer Ds30--5ʼ-AGTTAAATATGAAAATGAAAACGGTAGAGG-3ʼ AtHAM4 T-DNA flanking primer set Forward--5ʼ-CGGAGGAGTGCGCCGTAGAGAC-3ʼ Forward and Reverse--517 bp Reverse--5ʼ-CTTAGCTGTATCGGCCTCATCC-3ʼ Forward and LBb bp Salk T-DNA Insertion Primer LBb1.3--5ʼ-ATTTTGCCGATTTCGGAAC-3ʼ 7