Supplemental Data. Jones et al. Plant Cell. (2010) /tpc

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1 IAA synthesis rate Supplemental Data. Jones et al. Plant Cell. ()..5/tpc Supplemental Figure. Root tip specific IAA biosynthesis after induction of the CKX gene. 6 DAG pmdc7:atckx seedling roots were incubated for h in liquid medium containing % HO with or without 5 μm 7-β-estradiol.. The IAA biosynthesis rate was analysed in mm root tips. Error bars indicate S.D. (n=5)... - est + est..

2 Supplemental Figure. Timing in the induction of IAA biosynthesis by cytokinins. Time course experiment showing the difference in the timing of induction of IAA biosynthesis in the root apex between estradiol (+ est) induced AtIPT8/pga roots and roots treated with trans-zeatin (). Data are mean ± s.d. of three independent experiments (n=). P values for +est and +t-z vs -est were determined by two-tailed Student s t-test assuming equal variances (* p <.5, ** p <., *** p <.). IAA synthesis rate..8 - est + est *** *.6 *. **.

3 Supplemental Figure A. qpcr analysis of putative auxin biosynthesis genes after cytokinin treatment. Wild type Columbia seedlings were grown in LD for 7 DAG. The seedlings were then incubated for h, h, h, h or 6h in liquid medium with or without 5 μm t-z. The root tips were harvested, and genes related to auxin biosynthesis were analysed by qpcr. Error bars indicate SE (n=). 5 ASA/WEI PAT/TRP 6.5 IGPS TSA TAA 6 6 AMI 6.5 NIT.5 NIT NIT.5 6 AAO.5 6 6

4 Supplemental Figure B. qpcr analysis of putative auxin biosynthesis genes after cytokinin treatment. Wild type Columbia seedlings were grown in LD for 7 DAG. The seedlings were then incubated for h, h, h, h or 6h in liquid medium with or without 5 μm t-z. The root tips were harvested, and genes related to auxin biosynthesis were analysed by qpcr. Error bars indicate SE (n=)..5.5 YUCCA YUCCA YUCCA YUCCA7 6.5 YUCCA8.5 YUCCA Atg7 At5g At5g.5 6 At5g 6 6

5 Supplemental Figure C. qpcr analysis of genes related to auxin and cytokinin transport and signaling. Wild type Columbia seedlings were grown in LD for 7 DAG. The seedlings were then incubated for h, h, h, h or 6h in liquid medium with or without 5 μm t-z. The root tips were harvested, and genes were analysed by qpcr. Error bars indicate SE (n=). Genes related to auxin signaling Genes related to auxin transport.5.5 TIR PIN IAA PIN5 6 ARF7/NPH PUTPIN 6 Cytokinin related genes CYP75A ARR 6 5

6 Supplemental Table Cytokinin concentration in uninduced and induced AtIPT8/pga seedlings pg/mg fresh weight ipmp ipa ip a 8. ±.6 <..87 ±.8 a + > ± 8 ± 8.7 b ±. ND.9 ±. b + >579 7 ± 95.6 ±.6 c -.75 ±.9 ND.8 ±. c+ >6 ± 5.5 ±. pg/mg fresh weight t-zmp t-zr t-z a. ±,8.9 ±.7.5 ±.5 a + 6 ± 76 ± 68.7 ±.5 b -.88 ±.8.8 ±.6.95 ±.6 b + 56 ± 7 79 ± 5 6. ±.8 c -.7 ±.7.9 ±..85 ±. c+ 75 ± 96 6 ± ± 8. pg/mg fresh weight Z7G Z9G ZOG a 6.86 ±. 6.9 ± ND a +.8 ± ±..6 ±.6 b -.8 ±.66.8 ±.9. ±.5 b ±.6. ±. 5.6 ±. c ± ±..75 ±. c+ 5 ± 7 8 ± 6 5 ± 87 DAG AtIPT8/pga seedlings were incubated for h in liquid medium with (+) and without (-) 5 μm 7--estradiol. After incubation, the seedlings were divided into a) apex and young, developing leaves (leaf and smaller), b) the first two true leaves and c) the root system (according to Figure A), and the cytokinin concentration in these tissues was analysed by LC-MRM-MS. The standard deviation was calculated on three replicates. ipmp, isopentenyladenosine-5 -monophosphate; ipa, isopentenyladenosin; ip, isopentenyladenine; ZMP, zeatinriboside-5 -monophosphate; ZR, zeatinriboside; t-z, trans-zeatin; Z7G, zeatin-7-glucoside; Z9G, zeatin-9-glucoside; ZOG, zeatin-oglucoside 6

7 Supplemental Table Cytokinin concentration in uninduced and induced pmcd7:atckx seedlings pg/mg fresh weight ipmp ipa ip h 6.5 ±.869. ±.8.55 ±.9 h 6.58 ±.. ±.7. ±.5 h +. ±.6.6 ±.9.5 ±.9 6h 8.88 ±.5.88 ±..65 ±. 6h +.57 ±.8.75 ±.8.5 ±. h.6 ±.5. ±.7.58 ±. h +. ±.8. ±.. ±.8 h 8.7 ±..65 ±.9.5 ±. h +.9 ±.5. ±..7 ±. pg/mg fresh weight t-zmp t-zr h.9 ±..6 ±.5 h. ±..69 ±. h +.89 ±.8.96 ±. 6h.9 ±.55. ±.5 6h +.77 ±..9 ±.5 h.77 ±.7.96 ±.6 h +.8±.6.66 ±.8 h.75 ±.9.89 ±.96 h ±.76.6 ±.79 6 DAG pmd7:atckx seedlings were incubated for,, 6, and h in liquid medium with (+) and without (-) 5 μm 7--estradiol. The cytokinin concentration was analysed by LC-MRM-MS, and the standard deviation was calculated on three replicates. ipmp, isopentenyladenosine-5 -monophosphate; ipa, isopentenyladenosin; ip, isopentenyladenine; ZMP, zeatinriboside-5 -monophosphate; ZR, zeatinriboside 7

8 Supplemental Table Oligonucleotide primers for genes analysed by qpcr AGI ID Gene name Sequence 5 to AT5G96 AAO CTG GAG CTT GTC TCC AAG CAT CGG GCG CTA GCG GCA TTG TAC TGT AAA TAA ATG ATG898 AMI CGG ACT TAC TCC AAT GGC TCA G GCT GCT GCA GGA GAA CGC AAC C AT5G689 ATR/MYB CAC CTA CAT GGT GAA GGT GGA TGG CG CGG GTC TTA AGT AAT TAG CCC ATC TC ATG995 CYP79B GGC AAC CCA TTG CTT ACC GCC GAT GAA ATC CAT GGC CCA TTC CAC GGC GTT ATG CYP79B GGC CAG CCT TTG CTT ACC GCT GAT G TCT CCG CAA TGG CCC ATT CCA CGG C ATG5 CYP8B/SUR CCA TCA AAT TCA CTC ACG AAA ATG TC AAG GTA AGT CAT GGC CCA TAC CAC C ATG IGPS TAG CTT GGC TGC CCT TGT TGA GG CTT CGG TTA TTG ATG CCA ACA AGC TCG ATg NIT GGC GTT CAT AAC GAA GAA GGG CGT G TTC CTT CTC TAT GGC TCC CAT TAC C ATg NIT GAG TTC CGC AAG TAC CAT GCT TCT GC GCT GTG CAA TAG AGT GTA TAC CCA TC ATG NIT CTC TAC AGA ACT GCA TTG TAC GCT CCG CAA TGT GAA TCA TCG ATG CTT GCC AT5G NIT GCC TTC TTT GAG AAC CGC AAT GTA TGC C CAA GTG CAA TAT GAGTCATTGATGCTAGCCAAG ATG756 TAA CTC CAA GAT CAC AGG CCA CGC TGG G GAC TCC TTA GAC ACA CCA ATC GAG TTC ATg7 TAA-like CGT CCT CCC AAT CCT CAG TA GAT TAA ACC CAA CGG TCG AA AT5g596 TAA-like AAT AGT CGT CCC CCA AAT CC CCT CAA GGG GAC ACA CAT TT AT5g TAA-like AAG CAT CTC TCG TTT CTT TGC CAC CAT CAA TGG TAA TGG TAC A AT5g TAA-like TGT CGC TCA AGC ATC TAT CG TGT CAA CAA GGG GTT CCC TA ATG IAA CGT GCT GCT GAT TCT GCT TCT CAT GCT GGT TCA TCT CC CCT GTG TGA CCC TAT AGG AGG CCA TCC AAC AAC ATg5 IAA7/AXR GTA TCA ATG GAC GGA GCA CCG TAC TTG AGG GAA GTC TAT CAT TCC TTC TTC TCC TCC ATG 8

9 ATG75 PUTPIN GAA CCG TCG GGC TCT CTT ACG CGT C CTA TGG CTT GAA CTT TCA TTG CGG ACC C ATG698 TIR AAG TGC GAC CAG ATG TTT ACT CT ACA AAC GGA AGC GAG TCA T AT5g799 PAT/TRP TCT CAT CGA TCG GAG CTG TTT C AGC TGC ACT AAA TCC ACC GCT C ATg6 YUCCA CAA AAC CTC ATC CCC GAA G CAG CAA CGG CTA GAC CTG AT AT5g89 YUCCA5 ATT ACC CAA AAT GCC CTT CC CGT ATC GAG CAG ACT GGA CA AT5g56 YUCCA6 GTT CTC GAC GTT GGC ACG CTA G GGG TAC GTT GCT TTT GTA GCC AG ATg YUCCA7 CCA CCG TCG TTG TAT TTCA C TGG AGT GGG CTT ATC TCT TTG ATg87 YUCCA8 TGG TTG GAA AGG AAG GAC AG TCG TGG GTT GTT TTG TTT CA ATg8 YUCCA9 CGC AAA CCG GTT CGA TAT TA CTC CTC TCC GTC TGA GGT TG AT5G579 PIN AGT CGT TAT CCT CGC CGC ACT C ATT CCC ATT ACC AAC GTG TTA G AT5G65 PIN5 CGT GTG GCT CAC ACT CTT GT GGA ACG ACT TCT CTC CCA CA ATG6 IAA CTC TCG ACT ACA TGT TCA ATG C CTC CAA CCA TCA TCC AGT CAC C AT5G7 ARF7/NPH GTT GAG TGA TCA TGA AAG CTC C GGA GGC AAC GAA ATC AAT GGC C ATG6857 ARR TTC ATA TGC CTG ACA TGG ACG G AAC CGC ACC GTG CGT TAC TCC C ATG IAA/SHY GAT TGG ATG CTC ATT GGT GAT G CTC ATA CAC CAC AGC CTA AAC C ATG67 CYP75A CTT CAA CGT CTT TGT GCTC AAG CTA CTC CGA CCG ATC TCT ACA C AT5G57 ASA/WEI CGA ACG TTA TTC CCA TGT TAT GC GCA CCA CTA ACT GTT CCC ACT GG ATG56 TSA CTC GCT GAT ACT TTCA CAC AGC GGG TCA GAG TAA GGA ACA CCC ATG55 IAA/SLR GAT GTA CAC CAG CTA CAA GGA TC GAA ATC TAT CAT CCC TTG TGC TCC 9

10 ATG7 TIP-like GCT CAT CGG TAC GCT CTT TT TCC ATC AGT CAG AGG CTT CC AT5G5697 CKX CCCACCGGATAACTGGAGAT CACGATGTCGTTTGACCATT

11 Supplemental Methods Microarray analysis RNA hybridisation and analysis of auxin and cytokinin related transcripts Total RNA was isolated with the RNeasy Plant Mini Kit (Qiagen). RNA samples were sent to Nottingham Arabidopsis Stock Centre (NASC, for amplification and hybridization. For each treatment and control samples, three independent replicates were collected for RNA isolation and amplification. Unfortunately, the method of amplification was not sufficient to produce enough material for hybridisation of all samples, but all treatments and control samples were analysed in at least duplicates, as seen in the table below. Tissues samples Control/Treatment Time point No. of replicates Control h Control h Estradiol h Estradiol h Zeatin h Zeatin h Transcript profiling was performed at NSAC according to the GeneChip Expression Analysis Technical Manual ( using the two-cycle cdna synthesis protocol, with a starting amount of 5ng of total RNA. Affymetrix ATH GeneChip arrays were stained with streptavidin phycoerythrin solution and scanned with an Affymetrix 7 scanner. Following scanning, non-scaled RNA signal intensity files (.cel) were generated using the GeneChip Operating System software (GCOS version 5, Affymetrix). The NASCArrays experiment reference number is NASCARRAYS-6, and the data has been donated to GEO ( with the GEO accession number GSE. The.cel files were then analysed using the Genespring GX software (Agilent Technologies, USA). Raw.cel files were loaded into the Genespring GX analysis software and pre-normalised to produce a signal value for each gene using the MAS5 algorithm (Hubbell et al., ), using the GCOS software with the following settings; alpha=., alpha=.6, tau=.5, TGT=. The gene signals were normalised to the median signal of that gene in all samples. Selected genes with functions related to auxin and cytokinin biosynthesis and signal transduction were analyzed in a two step process; i) the fold change calculated between treated and control at each time-point and ii) p-values generated using a T-test with a Benjamini and Hochberg multiple testing correction. Genes that passed the filtering steps (flags and low expression) and were significantly differentially regulated between controls and treated samples were highlighted in pink if up regulated and blue if down regulated. Light pink = passed t-test analysis, dark pink = passed T-test with a Benjamini and Hochberg multiple testing correction. Light blue = passed t-test analysis, dark blue = passed T-test with a Benjamini and Hochberg multiple testing correction.

12 GO analysis Genes were selected that had a fold-change >.5 and a p-value <.5 using a T-test at each time point. These were analysed for over-represented GO terms using the GO analysis function in Genespring GX. The significance of the over-representation was calculated using a hypergeometric test with a Benjamini-Yekutieli multiple testing correction. Hubbell, E., Liu, W.M. and Mei, R. (). Robust estimators for expression analysis. Bioinformatics 8:

13 qpcr analysis Wild type Arabidopsis (Columbia) seedlings were grown on vertical plates in LD for 7 days. The seedlings were harvested ( h) or incubated on plates for h, h, h, h and 6h in liquid medium ( x MS, % sucrose, ph 5.7) with or without 5 μm t-z. Root tips (around 5 mm long, mg root tissue/sample collected in triplicates) were harvested by cutting the roots with a scalpel on the nylon mesh. Each sample was immediately frozen in liquid nitrogen and stored at -8 C until RNA extraction. The samples were disrupted with tungsten carbide beads using a MM vibration mill at a frequency of Hz for min (Retsch GmbH, Total RNA was isolated using the Plant RNA Isolation Aid and a RNAqueous-Micro Kit (Ambion, followed by a DNA-free kit (Ambion) treatment to ensure removal of genomic DNA. Thereafter,5 μg of total RNA was reverse transcribed using the iscripttm cdna biosynthesis Kit (Bio-Rad). Primers (Supplemental Table S) were designed using PRIMER ( and synthesized by Invitrogen ( The best reference gene to use in our conditions (TIP-like-Atg7) has been selected among the following genes by using GeNorm (Vandesompele et al., ): ef-at5g69, APT-Atg75, UBQ-Atg5 and TIP-like-Atg7. PCR reactions were performed in triplicate in 96-well plates with a Light Cycler 8 (Roche). Each PCR reaction mixture contained 5 μl of x diluted cdna sample, μl iq TM SYBR Green Supermix (BioRad),.5 μl of. μm forward and reverse primers in a total volume of μl. Thermal cycling conditions were 95 C for 5 min, followed by cycles of s at 95 C, 5 sat 6 C or 6 C (in function of the Tm of the primers) and 5 s at 7 C, followed by a melting curve analysis from 6 C to 95 C with C per step to check the presence of a single product. The Cq values for each sample were acquired by the Light Cycler 8 software release.5..sp (Roche). Three biological and three technical replicates where performed for each sample. Relative expression levels were calculated as follow: the difference in the expression ess level of a target (T) gene between two samples, and, is calculated from E Cq( ) T. The normalization consists of using the Cq( Cq and the efficiency measured for the reference (R) gene to calculate the E T ) /E Cq( ) R ratio, which gives the normalized efficient-corrected relative quantification of the target gene expression in sample compared to sample. The results presented on the graph are normalized to the wild type non treated samples value, which was set at. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. : RESEARCH. Epub Jun 8.