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SUPPLEMENTARY INFORMATION
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ATHB4, a regulator of shade avoidance, modulates hormone response in
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Arabidopsis seedlings.
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Céline Sorin1†, Mercè Salla-Martret1†, Jordi Bou-Torrent1, Irma Roig-Villanova1,
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and Jaime F Martínez-García1,2
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1. Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB), c. Jordi
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Girona, 18-26, 08034-Barcelona, Spain; 2. Institució Catalana de Recerca i
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Estudis Avançats, Passeig Lluís Companys 23, 08010-Barcelona, Spain.
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†These authors contributed equally to this paper
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Supplementary results
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As an initial approach to study the function of ATHB4, transgenic lines
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constitutively overexpressing ATHB4 (P35S:ATHB4) were produced. Adult plants
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segregating from the resulting T1 lines with a single T-DNA insertion displayed
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three phenotypes in terms of rosette size: wild-type-like (azygous), small
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(hemizygous) and dwarf (homozygous) (Figure S1a; data not shown). Because
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homozygous plants were mostly unfertile, the ATHB4-overexpressing lines
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could only be propagated in heterozygosis. Descendent seedlings from
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heterozygous plants showed cotyledons with a range of phenotypes, from wild-
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type to narrow (wt and mut, respectively, in Figure S1) that correlated with the
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absence or presence of the transgene, respectively, estimated by PCR
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analyses (data not shown). Based on this character two groups of seedlings
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were visually selected and different traits were measured. The most obvious
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phenotypes of ATHB4-overexpressing seedlings grown under continuous white
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light (W) were long hypocotyls and narrow cotyledons (Figures S1c, d). In the
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strongest lines, cotyledon longitudinal expansion was also reduced, and in older
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stages primary leaves were shorter than in wild-type plants (data not shown),
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which ultimately resulted in dwarf adult plants.
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Supplementary experimental procedures
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Plant material and growth conditions
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For the identification of homozygous mutant plants obtained from the
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SALK collection, specific oligo combinations were used to genotype them by
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PCR analysis: ATHB4 (JO314 + JO285, and JO291 + JO292), athb4-1 (and
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LBb1 + JO285), athb4-2 (and LBb1 + JO292), HAT1 (CSO23 + CSO24), hat1-1
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(LBb1 + CSO24), hat1-2 (CSO23 + LBb1), HAT2 (JO340 + JO383, and RO3 +
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RO4), hat2-1 (LBb1 + JO383), hat2-2 (LBb1 + RO4), hat2-3 (RO3 + LBb1),
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HAT3 (CSO11 + CSO12) and hat3-1 (LBb1 + CSO12). Homozygous lines were
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selected for further analysis after outcrossing once to Col-0.
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Generation of constructs to overexpress ATHB4
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The whole coding sequence of ATHB4 was PCR-amplified with specific
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primers (JO284 and JO285) using cDNA as a template. The cDNA was
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prepared from total RNA extracted of Col-0 seedlings treated for 1h with
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simulated shade using Moloney Murine Leukemia Virus Reverse Transcriptase
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(Invitrogen, www.invitrogen.com), following manufacturer’s protocol. The PCR
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fragments containing the coding region were cloned into pTZ57T/R (Fermentas,
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www.fermentas.com). The resulting vector pSP22 was sequenced for identity
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confirmation. The plasmid was digested with KpnI and BamHI, and cloned in
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pBinAr (Hofgen and Willmitzer, 1990) digested with the same restriction
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enzymes to give pBF10 (P35S:ATHB4).
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ATHB4 sequence was amplified without the stop codon using specific
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primers (JO284 + CSO7) using cDNA prepared as before as a template. The
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corresponding PCR fragments were subcloned in pCRII-TOPO (Invitrogen) to
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give pCS12. The insert was sequenced and no additional mutations were found
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in the coding region. pCS12 was digested with XbaI and BamHI and cloned in
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frame with the GR domain in the pGREEN0029 vector (Hellens et al., 2000)
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digested with the same restriction enzymes to give pCS13 (ATHB4-GR). The
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EcoRI-HindIII fragment from pBinAr, which contains the 35S promoter and the
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OCS terminator cassette, was subcloned into the same restriction sites of the
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binary vector pCAMBIA1300, which confers hygromycin resistance to transgenic
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plants, to give pCS14. The EcoRI fragment from pCS13 was blunt-ended with
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Klenow and subcloned into SmaI-digested pCS14, generating pCS19
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(P35S:ATHB4-GR).
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RNA Blot Analysis
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Probes for RNA blot analyses were made by amplifying Col-0 genomic
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DNA with specific primers: CSO9 and CSO10 for HAT1; CSO11 and CSO12 for
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HAT3; CSO17 and CSO18 for HAT22; RO1 and RO2 for IAA1; and BO42 and
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BO43 for At5g45670. PCR products were subcloned into pCRII-TOPO or
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pBSK+ to give pCS15, pCS20, pCS17, pIR3 and pJB31 respectively. Inserts
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were sequenced for identity confirmation. Probes for DNA inserts, isolated by
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restriction digestion or by PCR using specific primers, were radioactively labeled
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with [α32P]dCTP by using a random primed DNA-labeling kit (Roche Molecular
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Biochemicals, www.roche-applied-science.com), and purified through Sephadex
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G-50 column (Amersham, www.gehealthcare.com). Images were visualized by
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using a molecular imager FX (Bio-Rad, www.bio-rad.com), and band intensities
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were quantified by using QUANTITY ONE (Bio-Rad) software. Expression levels
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were calculated relative to the “wild-type non treated” value of each set of
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samples after normalization with the 25S rRNA signal.
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Primers used
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The sequence of the primers used for probe generation and plasmid
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construction is: BO42 (5’-TAG-AAA-AAG-ATG-GCG-AGA-ATG-AGT-3’), BO43
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(5’-GAT-CTT-TCT-CTC-TTT-AGA-GAG-ATG-C-3’), CSO7 (5´-GGG-GAT-CCG-
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CGA-CCT-GAT-TTT-TGC-TG-3’), CSO9 (5’-ATC-ATG-ATG-ATG-GGT-AAA-
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GAG-GAT-TTG-3´),
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ATC-3’),
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CSO12 (5’-ACT-CTA-ATG-AGA-ACC-AGC-AGC-AGG-TCG-3’), CSO17 (5’-
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CAA-ATG-GGT-CTT-GAT-GAT-TCA-TGC-AAC-3’), CSO18 (5’-TAA-CTA-ACA-
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TGC-TGC-AGA-AGG-ATT-AG-3’), CSO23 (5’-CCA-CAA-GGA-AAC-AAC-ACA-
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GAT-CC-3’), CSO24 (5’-ATA-CTC-GCA-ATC-CAC-TTC-GGT-C-3’), JO284 (5’-
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AGG-ACA-ATG-GGG-GAA-AGA-GAT-GAT-3’),
CSO11
CSO10
(5’-AAG-TTA-AGA-CCT-AGG-ACG-CAT-CAC-
(5’-AAA-ATG-AGT-GAA-AGA-GAT-GAT-GGA-TTG-3’),
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JO285
(5’-CCT-TCC-CTA-
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GCG-ACC-TGA-TTT-TTG-3’), JO314 (5’-GTG-TGG-TTT-CAG-AAC-CGT-AGG-
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3’),
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JO292
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JO340
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CAC-GTC-TAT-CTT-GCG-AAG-3’), LBb1 (Roig-Villanova et al., 2006), RO1 (5’-
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GTG-AGA-GAA-TAT-GGA-AGT-CAC-C-3’),
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TAA-GGC-AGT-AG-3’), RO3 (5’-AAC-ATG-ATG-ATG-ATG-GGC-AAA-GAA-G-
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3’) and RO4 (5’-AAA-TCA-CGA-TCG-TGG-ACG-CAA-GGC-3’).
JO291
(5’-GGA-AGC-TTA-CTC-TAC-CAT-CCA-CTA-ATG-TTT-TC-3’),
(5’-GTC-GGA-TCC-ACC-ATT-GTC-CTC-AAC-AGA-AAG-AAC-TT-3’),
(5’-GGG-GGA-CAC-AAG-TTG-GAG-ATA-AAG-3’),
RO2
JO383
(5’-GTT-
(5’-GAC-AAT-GGA-TCA-
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The primers used for qPCR were BO33 (5’-GCG-GTC-TAT-GTA-GGA-
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GAG-AAT-GAT-C-3’) and BO34 (5’-CCG-GCA-CCA-CAT-ATC-TCT-TCT-T-3’)
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for SAUR15, BO35 (5’-AAA-CGC-AAA-GAA-GCT-TAT-GAA-GAT-G-3’) and
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BO36 (5’-GCT-GCT-CTT-TGT-TGC-CAT-TTC-3’) for SAUR68, BO59 (5'-GCC-
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TTA-TGA-TCC-ATT-GTC-TCA-AAA-CA-3') and BO60 (5'-TTG-CTT-TTT-CTT-
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TCT-TTA-CAC-CAA-ACT-3') for IAA1, BO68 (5'-AGA-AGT-GAG-AGA-GAA-
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GGA-ATC-TCC-G -3') and BO69 (5'-TCA-TCT-GAG-GTT-CCA-CGT-GAG-TA-
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3') for HAT2, and BO40 (5’-AAA-TCT-CGT-CTC-TGT-TAT-GCT-TAA-GAA-G-
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3’) and BO41 (5’-TTT-TAC-ATG-AAA-CGA-AAC-ATT-GAA-CTT-3’) for UBQ10
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(At4g05320).
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Supplementary References
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Hellens, R., Edwards, E., Leyland, N., Bean, S. and Mullineaux, P. (2000)
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pGreen: a versatile and flexible binary Ti vector for Agrobacterium-
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mediated plant transformation. Plant Mol Biol, 42, 819–832.
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Höfgen, R. and Willmitzer, L. (1990) Biochemical and genetic analysis of
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different patatin isoforms expressed in various organs of potato. Plant Sci, 66,
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221-230.
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