Cell, Volume 138 Supplemental Data mir156-regulated SPL Transcription Factors Define an Endogenous Flowering Pathway in Arabidopsis thaliana Jia-Wei Wang, Benjamin Czech, and Detlef Weigel Table S1. Interaction of mir156/spl with vernalization and GA pathways. Genotype Leaves Average Range Experiment 1 (short day) Wild type 55.9 ± 2.6 53-61 Wild type (+Ver) 38.9 ± 1.4 36-41 p35s:mir156 93.3 ± 3.2 84-99 p35s:mir156 (+Ver) 78.6 ± 3.5 73-83 Experiment 2 (short day) Wild type 55.4 ± 1.2 53-57 Wild type (+GA 3 ) 32.0 ± 1.8 29-35 p35s:mir156 94.2 ± 4.7 86-101 p35s:mir156 (+GA 3 ) 74.1 ± 3.1 70-80 Experiment 3 (long day) FRI FLC 71.2 ± 4.3 63-78 p35s:rspl3 FRI FLC 21.4 ± 1.5 20-24 Experiment 4 (short day) ga1-3 n/f p35s:rspl3 ga1-3 35.4 ± 2.4 30-39 Standard deviation is given. n = 14 for all genotypes. Vernalization treatment was for five weeks. For GA response, plants were sprayed weekly with 100 µm GA 3. n/f: ga1-3 plant never flowered in short days. S1
Table S2. Comparison of flowering times of ft, soc1 and ful mutants. Genotype Leaves Flowering time (days) Average Range Average Range Experiment 1 (long day) Wild type 13.0 ± 0.6 12-14 16.7 ± 0.8 16-18 ful-7 15.1 ± 0.8 14-16 17.9 ± 0.5 17-19 ft-10 38.8 ± 0.7 38-40 35.0 ± 0.9 23-26 soc1-6 22.9 ± 0.5 22-24 24.9 ± 0.9 23-26 ful-7 soc1-6 36.8 ± 0.9 36-38 29. 8 ± 1.1 28-32 ful-7 ft-10 48.1 ± 0.8 47-50 38.5 ± 1.7 36-42 ft-10 soc1-6 63.1 ± 1.5 60-65 47.1 ± 1.6 44-49 Experiment 2 (short day) Wild type 55.3 ± 1.7 53-59 59.6 ± 3.1 54-64 ful-7 63.9 ± 1.1 62-66 61.6 ± 2.6 57-66 ft-10 57.4 ± 1.0 56-59 59.1 ± 2.4 56-64 soc1-6 67.6 ± 1.8 65-70 68.7 ± 2.1 65-72 ful-7 soc1-6 78.4 ± 2.1 74-81 76.5 ± 2.9 72-82 ful-7 ft-10 86.7 ± 1.5 84-90 65.5 ± 2.7 60-70 ft-10 soc1-6 90.1 ± 1.4 88-93 81.9 ± 2.9 77-86 Standard deviation is given. n = 14 for all genotypes. Table S3. Distribution of rosette vs. cauline leaves in long-day grown ft, soc1 and ful mutants. Genotype Rosette leaves Cauline leaves Total leaf number Fraction cauline leaves Wild type 10.7 ± 0.6 2.2 ± 0.5 13.0 ± 0.6 18% ful-7 12.4 ± 0.7 2.7 ± 0.5 15.1 ± 0.8 18% ft-10 30.7 ± 0.7 8.1 ± 0.8 38.8 ± 0.7 21% soc1-6 19.4 ± 0.7 3.6 ± 0.5 22.9 ± 0.5 16% ful-7 soc1-6 25.6 ± 1.1 11.1 ± 0.7 36.8 ± 0.9 30% ful-7 ft-10 34.3 ± 0.9 13.9 ± 0.9 48.1 ± 0.8 29% ft-10 soc1-6 46.0 ± 1.5 17.1 ± 0.7 63.1 ± 1.5 27% Standard deviation is given. n = 14 for all genotypes. S2
Table S4. Oligonucleotide primer sequences. ID Gene Sequence (5-3 ) Purpose Direction G-1936 FUL CTT GAT TCG TTG AGC TTG AAG GAG C T-DNA insertion typing sense G-2187 SOC1 CTT CAG CAT CAC AAA GCA CTG T-DNA insertion typing antisense G-2188 SOC1 CGA AAG CTT CCT CCT GGT TAG G T-DNA insertion typing sense G-15370 FT GCT AGG ACT TGG AAC ATC TGG ATC CAC CAT T-DNA insertion typing antisense G-16384 FT AAA CAA TCA ACA CAG AGA AAC CAC CT T-DNA insertion typing sense G-0654 FUL TTG CAA GAT CAC AAC AAT TCG CTT CTC qrt-pcr sense G-0655 FUL GAG AGT TTG GTT CCG TCA ACG ACG ATG qrt-pcr antisense G-0628 SOC1 ATA GGA ACA TGC TCA ATC GAG GAG CTG qrt-pcr sense G-0629 SOC1 TTT CTT GAA GAA CAA GGT AAC CCA ATG qrt-pcr antisense G-17578 AGL42 TCA TGA AAC CAG CAA TCA CGA CTC A qrt-pcr sense G-17579 AGL42 AGC CTT TCT TTC TCG GAC CTT TCC qrt-pcr antisense G-13301 FT CTA GCA ACC CTC ACC TCC GAG AAT A qrt-pcr sense G-13302 FT CTG CCA AGC TGT CGA AAC AAT ATA A qrt-pcr antisense G-11760 SPL3 AGA GGC TTT GGA GAA GAA GCA GAA AGG qrt-pcr sense G-11761 SPL3 GAA GAC CAG AGA TCC GAA CAT GAG GAG qrt-pcr antisense G-7485 SPL3 AAA CCC GGG ATG AGT ATG AGA AGA AGC AAA GCG GA genomic fragment sense G-8894 SPL3 AAA GAG CTC TTA GTC AGT TGT GCT TTT CCG CCT TCT genomic fragment antisense G-15159 FUL CTG CAG GAT ACT GTA AAC TTT GTT TTC CGA AC promoter GUS fusion sense G-18497 FUL CGG GGA TCC TAT CCT CTC CAT GCT AGA TTT AAG AAA promoter GUS fusion antisense G-16350 FUL GTG TGA TGT GTT AAT CCT ACA AAA A ChIP, region I sense G-16351 FUL GAG AAT CTC AAA CGT ATG ATA CTC TC ChIP, region I antisense G-16348 FUL AAA AAC TTG TCT CCA TGC AAA AAG ChIP, region II sense G-16349 FUL TTG TCG AGT CCT CAT TGG CTA CT ChIP, region II antisense G-16346 FUL AGG TTG CTC TCA TCG TCT TCT CTT ChIP, region III sense S3
G-16347 FUL CCA GTT CTT AGG GAA CAA ACA AAT CT ChIP, region III antisense G-16344 FUL AAA GAA AGG TTG ATT TGA AAC GAT G ChIP, region IV sense G-16345 FUL TGG TGA GTC CAC AAA ACA TCA TAG A ChIP, region IV antisense G-17171 FUL AGA TGG CTT TGT GGA GAG AGT TG ChIP, region V sense G-17172 FUL GAC AAC GGA GTT CCA TCA TAC ATA ChIP, region V antisense G-4065 FUL CGG ATC CCC GAT ATG GGA AGA GGT AGG G genomic fragment sense G-15171 FUL CCC GGG CTA CTC GTT CGT AGT GGT AGG ACG TAA genomic fragment antisense G-16340 FUL GAT AGG ACG TAA CAT CCA AGC GGT CTC TCT TTT GTA TTC C amir-ful G-16341 FUL GAC CGC TTG GAT GTT ACG TCC TAT CAA AGA GAA TCA ATG A amir-ful G-16342 FUL GAC CAC TTG GAT GTT TCG TCC TTT CAC AGG TCG TGA TAT G amir-ful G-16343 FUL GAA AGG ACG AAA CAT CCA AGT GGT CTA CAT ATA TAT TCC T amir-ful G-17804 AGL42 AAA TCC CTT GTT CTT TCT TTC CTC ChIP, region I sense G-17805 AGL42 AAA GAC AAT CGT GGC AGG TTC AAA T ChIP, region I antisense G-17806 AGL42 AGG CTT TAT GAA TTC TCT AGC TCT GA ChIP, region II sense G-17807 AGL42 AAT GAA ATT AGA CTG ATA GGG GAA GAG ChIP, region II antisense G-17808 AGL42 TGA ACA GAA AAC AAC ATC GGA AGT ChIP, region III sense G-17809 AGL42 CTG AAT CAA ACG AGG AAG AGC AAG ChIP, region III antisense G-17810 AGL42 GTT CAA TAA ATG GAC AAA AAA GGG C ChIP, region IV sense G-17811 AGL42 CTG CAT ACT GAT GAT CAA AAA TAG ACA ChIP, region IV antisense G-17812 AGL42 AAG TTA CAT CAA AAG GTG ACC CAT CT ChIP, region V sense G-17813 AGL42 GAA CCA AAT TTT ACC GAA TAG CAC C ChIP, region V antisense G-20474 SOC1 CAA TCG GTA AGA TCT CTT CGT CAT ChIP, region I sense G-20475 SOC1 GAG AAG ACT GAT GAA GTC AAC GAA A ChIP, region I antisense G-20476 SOC1 AAA CTT TAT GAA TTC GCC AGC TC ChIP, region II sense G-20477 SOC1 CCA AGA AAC TCA TTT AAA CAC ACC T ChIP, region II antisense G-20478 SOC1 CTT TGA GAG GTG TGT TTA AAT GAG TT ChIP, region III sense G-20479 SOC1 GCT ACG AAA GAG CAC ATT TTA TTT A ChIP, region III antisense S4
G-20480 SOC1 ACC TAA ATG TTG ATT GTT TTT GAG A ChIP, region IV sense G-20481 SOC1 ATC AAG GAC ATA GCT TGA AAA GTC T ChIP, region IV antisense G-20482 SOC1 TTT TTC TTG GTT ATC TTC AAT CAT CA ChIP, region V sense G-20483 SOC1 GAA GAA CAA GGT AAC CCA ATG AAC ChIP, region V antisense G-20965 AP1 GAA AAC TAT TAC AAC GGA AAT TCA T ChIP, region I sense G-20966 AP1 CAA AAA TCA GTT GAA TAT GTG CTA T ChIP, region I antisense G-20967 AP1 TAT TTT GGT TGG TTC AGA TTT TGT T ChIP, region II sense G-20968 AP1 CTT TAG GAT TTG CGT GTC GAC TT ChIP, region II antisense G-20969 AP1 TTT TTA AGC ATT GTC CTT GTT CTG A ChIP, region III sense G-20970 AP1 TTT ATG CAA TTA AAC CAA GAT TTC C ChIP, region III antisense G-0634 AP1 AGG GAA AAA ATT CTT AGG GCT CAA CAG qrt-pcr sense G-0635 AP1 GCG GCG AAG CAG CCA AGG TTG CAG TTG qrt-pcr antisense G-11965 SPL9 CAA GGT TCA GTT GGT GGA GGA qrt-pcr sense G-11966 SPL9 TGA AGA AGC TCG CCA TGT ATT G qrt-pcr antisense G-7981 SPL9 AAA CCC GGG ATG GAG ATG GGT TCC AAC TCG GGT GR fusion sense G-10093 SPL9 AAA GGA TCC CCG AGA GAC CAG TTG GTA TGG TGA GR fusion antisense G-10087 MIR156a CTC AAG TTC ATT GCC ATT TTT AGG qrt-pcr sense G-10088 MIR156a GAG AGA TTG AGA CAT AGA GAA CGA AGA qrt-pcr antisense G-7536 T-DNA GCG TGG ACC GCT TGC TGC AAC T SALK line genotyping left border G-5578 T-DNA CCC ATT TGG ACG TGA ATG TAG ACA C GABI line genotyping left border N-0078 β-tubulin-2 GAG CCT TAC AAC GCT ACT CTG TCT GTC qrt-pcr sense N-0079 β-tubulin-2 ACA CCA GAC ATA GTA GCA GAA ATC AAG qrt-pcr antisense S5
Figure S1. Expression of mir156-targeted SPL genes and MADS box genes controlling flowering in AtGenExpress. Paralogous SPL genes are shown together. Values refer to absolute expression estimates with Affymetrix ATH1 arrays (Schmid et al., 2005). The rosette series is from short-day grown plants harvested at the indicated age. Transition apex refers to shoot apices that had started to produce S6
flowers, but before bolting. Note rising AGL24 expression in whole rosettes of increasing age, but no dramatic difference between vegetative and transition apex. Figure S2. mir156 expression at the shoot apex after photoperiodic induction. In situ hybridization to detect mir156 (for controls, see Wang et al., 2008) at the shoot apex of 30- day old, short-day grown wild-type plants transferred from short to long days. Scale bars indicate 50 µm. Figure S3. Expression of FD and FT in p35s:mir156 plants. (A) qrt-pcr analysis of FT expression. 14-day old short-day grown plants were shifted to long days for two days, and then returned to short days. Plants were harvested every four hours. (B) In situ hybridization analysis to detect FD in shoot apices of 30-day old, short-day grown plants. Scale bar indicates 50 μm. S7
Figure S4. Identification of SPL targets. (A) Microarray analysis of wild type and plants with reduced (ago1-27, se-1, Wang et al., 2008) or increased mir156 levels (se-1 p35s:mir156). Shoot apices were dissected from 20-day-old, shortday grown plants. (B) qrt-pcr analysis of AGL42 expression in 7-day old long day-grown and 10- day old short-day grown seedlings. (C) Diagram of AGL42 and AP1 genomic regions. Triangles indicate GTAC boxes in promoter and introns. A LFY binding site (Parcy et al., 1998) and an FD response element (Wigge et al., 2005) are indicated. (D, E) ChIP followed by qpcr of AGL42 (D) S8
and AP1 (E) genomic fragments in wild-type and pspl9:gfp-rspl9 seedlings and inflorescences. Relative enrichment of fragments was calculated by comparing samples treated with anti-gfp and anti-myc antibodies. Figure S5. Genetic interaction between FUL and SPLs. (A) A ful mutation suppresses the early flowering phenotype of p35s:rspl3. (B) FUL overexpression suppresses the phenotypic effects of mir156 overexpression. All plants were grown in long days, and photographed after they had begun to flower. Figure S6. Expression of SOC1 and FUL in p35s:fd plants. qrt-pcr analysis of 7-day old long-day grown seedlings. S9
Figure S7. Additional phenotypes of pfd:mir156 fd-2 plants. (A) Long-day grown plants after the inflorescence shoot had started to elongate. (B) Close-up of mature inflorescences. Most of the side shoots in pfd:mir156 fd-2 plants repeated the pattern of the main inflorescence, with many leaves and no signs of flower development. Inset shows tip of an inflorescence that had stopped growing. Only the last few structures were flowers with normal organs, but they were subtended by cauline-leaf like bracts (arrowhead). Scale bars indicate 1 cm. Figure S8. Expression of TFL1 after photoperiod induction. In situ hybridization to detect TFL1 in 30-day old short-day grown wild-type plants shifted to long days. l, leaf primordium. Asterisks indicate shoot apical meristem. Arrows point to expression in secondary shoots. Scale bar indicates 50 µm. S10