enzymatic families regulated in response to a short-term cytokinin treatment in root apices

Transkriptio

1 A B C D Supplemental tlfigure 1. 1Mtbli Metabolic processes, hormonal pathways and dlarge enzymatic families regulated in response to a short-term cytokinin treatment in root apices The 297 transcripts regulated by cytokinins (BAP 10-7 M, 1h) in Medicago root apices ( Fold Change (FC) >1.5; Adjusted p-value <0.001) were loaded into Mapman software (Tellström et al., 2007). Transcripts absent are shown in grey whereas transcripts that change by less than a given threshold are shown with the colour code scale bar (based on log2 ratios) shown next to the scheme (blue: induction, and red: repression). A. Mapman Metabolism Overview pathway. B. Mapman Secondary Metabolism pathway. C. Mapman hormonal Regulation Overview pathway. D. Mapman Large Enzymatic Families pathway. 1

2 A fter P (cm) ngth a n BAP oot len sfer on Ro trans 2 1,6 pfrn GUS RNAI 1,2 0,8 RNAi Mt RR1-MYB myb RR1domain RNAi B fter P (cm) ngth a n BAP oot len sfer on Ro trans 3,0 2,5 35S 35S:GUS 2,0 35S 35S:Mt MtRR1 15 1,5 0,4 0,5 1,0 0 0,0 control 0 BAP 10-8M -8 M BAP 10-7M 10-7 M Control control BAP M BAP M C WT 35S:Mt RR1.1 35S:Mt RR1.2 D Control BAP 10-8 M Roo t lengt th (cm m) 25 2,5 2,0 1,5 1,0 0,5 * * WT RR1-1 35S:Mt 1 RR1.1 1 RR1-2 35S:Mt RR1.2 BAP 10-7 M 0,0 control Control BAP 10-8M -8 BAP M BAP 10-7M -7 BAP M Supplemental Figure 2. Mt RR1 overexpression in Arabidopsis thaliana affects root sensitivity to cytokinins A. Length of M. truncatula roots expressing a RNAi construct targeting the conserved Mt RR1 MYB domain or a GUS RNAi construct (as control), four days after transfer tobap 10-7 M or 10-8 M under in vitro conditions ( i medium). Error bars represent confidence interval ( =0.05; 005 n>35 >35independent d transgenic roots / construct). One biological replicate out of two is shown. B. Length of M. truncatula roots expressing Mt RR1 under a 35S-CaMV promoter or the GUS reporter (as control), six days after transfer on BAP 10-7 M or 10-8 M under in vitro conditions ( i medium). Error bars represent confidence interval ( =0.05; n>25 independent transgenic roots / construct). One biological replicate out of two is shown. C. Representative A. thaliana Columbia-0 plants expressing Mt RR1 under a 35S-CaMV promoter (two id independent d ttransgenic lines), seven days after transfer on BAP010 0, -7 M or 10-8 M under in vitro conditions (MS [Murashige and Skoog] /2 medium). D. Length of A. thaliana roots expressing Mt RR1 under a 35S-CaMV promoter, as described in C. Error bars represent confidence interval ( =0.05; n>12 plants / genotype). A Kruskal and Wallis test was performed to assess significant differences for each BAP concentration ( <0.05). 2

3 Clone N : 1 to AATGAGACTAGT TAAGATTGTTAT TAGATTGATATG -----AATAAGATCACA--- TTATAATAAAGA CAAAGACAAATAC- -AGGCAATGAGAC ATGGAGAGTGCT AATAAGACTAGT--- --AATCCCAAGACT TATACCAGAATC GGAGATGCCGGA- --ATACCTAAGATT CGAAGATTGCCG ACGAGATACCCC-- ---CTCCCTAGAATC AGTAAGATCAAT GAGATTGTTAT AAGATCCGGGGC AAGACCATACAT GAGACTGGCTGC Supplemental Figure 3. Raw sequences cloned after seven rounds of binding site selection using SELEX Sequences are shown for 38 randomly picked clones (numbers of clones are indicated on the left). 3

4 GST-RR1 BD SELEX* Box SELEX + SELEXmut + Supplemental Figure 4. Competition EMSA comparing the in vitro binding preference of GST-RR1 Binding Domain (BD) for alternative RRBS The major SELEX consensus was labeled l with [ - 32 P]dATP (as indicated d by an asterisk) and competed by the following 20-fold unlabeled probes: the major SELEX consensus, box 1 to 4 from the RR4 promoter, box 5 and 6 from the NSP2 promoter, and a version of the SELEX consensus where the central GAGA/TCTC core was replaced by a ACTG/CAGT sequence (SELEX mut.). Addition of fresh DTT to the binding reaction abolishes upper bands seen in Figure 2B (due to the oligomerization of GST) without changing binding specificity. i 4

5 A I II III B Supplemental Figure 5. Expression pattern of CKX1 in nodules In situ hybridization on mature M. truncatula nodules d l (21 ddays after ft S. S meliloti lil ti inoculation), i l ti ) using an Mt CKX1 specific antisense (A) or sense (B) probe (see Methods for primers used). Nodulation zones are defined according to Vasse et al., 1990: I, meristem; II, infection by Rhizobium; III, III nitrogen fixation zone. zone Bar = 100 µm. Vasse, J., de Billy, F., Camut, S. and Truchet, G. (1990). Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J. Bacteriol., 172,

6 A g of ro oot No odule numbe er / m dry weight 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 * GUS RNAi B g of ro oot 3 2,5 bhlh057 bhlh476 RNAi construct 1 * er / m weight numb dry w No odule ,5 14 dai 14 dai 1 0,5 0 A17 WT bhlh-1 bhlh476-1 Supplemental Figure 6. Nodule density of bhlh476 RNAi and mutant roots Quantification of the number of nodules per root dry weight of (A) roots expressing an RNAi construct targeting bhlh476 or GUS (as control) and (B) bhlh476 mutant roots. Nodulation was dt determined din vitro 14 days after inoculation (dai) with S. meliloti. Error bars represent confidence interval ( =0.05; n>30 independent transgenic roots), and a Mann and Whitney test was performed to assess significant differences ( <0.05). 6

7 A ATGGAAAAATTAAACTCATCTTCAAACTCATCATCAACTAGCTGGTTATCTGATCTGGTAATAAGAAATGTTTTTTTTCTTCATATATTAATCTAGTGACTAT AGTTCTAATTATATGCACAAATGAAATGGATTTAATTATATGAATGATTTTTATTCATTATAGTTTTGTGTTTTGTTTCAGCTATTGGAAGAAATGGAAATAG AGGGTTGTGATTTGTTTCAACAATGTGAACAAAACTTGTTTGGTGAAAAAGAGTTTCTTTCAAATGACATAGCAAGTATTTTTCAACAAGAGGAAAATTTCCA ACATCAACCTTTGTTATCTTCTGAAAGCTATTCTTCTTCTTATCCTTTAGAAAAGAATTTCGAAAGACCCAACAAAAAATTGAAAAAAAACACTTCTTCCCCT TCTTCTACCACTTCTCAGATTCTGTCTTTTGACTGTACCTTAAATACAAAAAAGAACAAGGTTGTTCCATTGTCACAAACAGAATTACCTCAAAATAGAAAAG GGTCATTGCAAAAACAAAACATTGTTGAAACTATAAAGCCACAAGGACAAGGAACTAAGAGGTCTGTGGCTCATAATCAGGACCATATCATAGCAGAGAGAAA GAGAAGAGAGAAACTCAGTCAGTGCTTGATTGCTCTAGCTGCTCTTATTCCTGGCTTGAAGAAGGTATGGTACTCTAATGTCTCACTATGAACTAATTTATTA AAAGTGATCAGATGGACATATTAATTTCATGTTTGGTTATAAACTCCAAAAATAAATTTTGGTTATAAACAGACTTTTTTTTTTTTTTTTGACAAGGGGTATA AATAGAATAAAGAGCAATGCTATATATCAAGAAAGATTTTTTCAAGAAAATTTTAAGAATGACATGGCAGAATAATCACCCTTAGATTTTATCAAGAAATAGT CCTGAAATTAATGTTAACCAATAAAATCTCAGCCTGGCCGGAAATCATGGGGTGGTGGGTGTTATGTATCTTCTTTTAATGAAAGATTAATAAATGAGACTGA AATCTAAAGGTGATTATTGTGCCATGTCATTCTTGAATTTTTCTTATAAAATCATTCTTGATATCTAGCATTTTCCTAGAATAAATGATGAGTTAAAAAAATG TAATATTTTTTCTATTTATGGATATTTTTGTGTGTGAAGTTTGTAGTATTTATTATGTAGGGCCGCTTTGTTTAAGAATCATAAAAATATGTTTTTTTGTTTT GTACGGAAATTTTTATTTATTTATTGTTTTATTTATGGTTATAGTTAAACTATTTCGTTCTCGTGAGTTTAACTCGATTGATAATCACAATACATATGATATG CAAGATCCTCTATGTGAGTTATGTATTCTTTAAACGCAATCTAGATACTAATTCCTTAAAGCATGTAAAACTTCAATGATAACACCACTTTTACTCAAGCAAA TTGAAAAGTTAAAAGCTAGTTGAATTAACTTTTAAGGAAAATCATTTTTAATAACAGGAAGAAATACAAAGTATCTTTCTATAATTATAACTTTAAACAAACA TAAAATAACATACTTTTACTTTTCTTAAAAATCTCCGACAAGTTCTATCTAATTGATTTATATCAAAAATGATTTTATAAAAAAGTTTAAACAGAAACACAAC TAAAACCAGAACATCAAATGTTGTGTGGGTTAAAAGTTTTTCACATGGATTATTTTATAAAACAAAATTATTTTTCCAATATAAAGGGAAAACAATTTCACCC TAAATAATTTTGGATAAATAAACCATCTTAGTGTATGTCTAGATTGACAATGAGATAGTAAAAATTAGAATGAATCAACACACTTTTGGCAAAAAGTACCACT CTATGTAGTTTCAACAAATTATATGTTTGGATTTACAGTGATACATGTTGAATTTTAGTAAATTAATTTTGTTAGATTGATTTTATTTAAAAGTGACATGAAT GAATGTAAAGTGATTTATGTTTTTAATACATTTACATAAATTGATTTGAACAACAAATTTAAGTGAAAAAAAGATCACATTTAGACTCAAAAGCTATAAATCC TAACCTCTATTTAAAATCAATTTTAGATGCAAAATCAATTCCATTGAAGAACAAACAAACTTATCAAACTCTAGTTGCTCTAAACATGAAACCAAACCTAAAT AGTCATGTTAGCAATTCATATTCATATGATGTGAATTAATTGGTGCAAGGTTGTTACAACTACTATATAGGTAGCTATTAATTATAGGAATTTTAGCATCTAA GTAAACTTTATTATGTTGTAAAAAAATCTACAGATGGACAAGGCTTCAGTCCTAGGAGATGCAATCAAGTACGTGAAAGAGCTCCAAGAACGTTTGAGGGTAT TAGAGGAACAAAACAAAAATAGTCATGTTCAGTCTGTGGTGACGGTTGATGAACAACAACTAAGTTACGACTCATCAAACAGTGATGACTCTGAAGTTGCTAG TGGTAATAATGAAACTCTCCCACATGTAGAAGCCAAAGTTTTGGACAAGGATGTGCTCATAAGGATCCATTGCCAAAAGCAAAAGGGACTTTTGCTCAAAATA TTGGTCGAGATTCAAAAGCTTCATCTCTTTGTTGTTAATAACAGTGTCCTACCTTTTGGAGATTCTATCCTTGATATAACCATCGTTGCTCAGGTAAAGAAAA TATCAACTTTTTTAATTTAAAAATAGAGCAAATCTCCTTTTTAATCATTTAAAGTATTAACTAAGTCTAAAACAATTTTATAGTGAAACTTTATAAAATAAAT AAAAAACAGTATATTAGACTTTCACTTGTTGAAACTAAGGTTCGAAAATGGACTCAGGAGTATGTTAGACTTTCACTTCTTCAAAAAGAAGAAGAATATATTA GACTTTTATCTTGATTAACTAAGTTGTGCAAATATCTATAACTAATATGTCATGTTATATATTTATAATTAAATTATATTTATTTGAGGTCTTTTTTTTTTTT TGTCCTAAATTTCGAGGCTTAATTGAAACCTTAATTTAGACTGCTTGGGGCATTTATTCAATGGTAAAAAAAAGTCATAGTTTTTGTTTTATTTTAAAAACAA AGATACTGTCAAATTTACAAAATTTATCATTAGTGATAAGAAAAAGCTAAATTCCAAGAATTAAAAAATGGAAGTAATATTAACACTTCATCATATAAAATTA GACAATTCTACCAAAAAAAATATAAAATTAGACAAGATATTTTTTGATAAAATTCAGCTTTAAATTTTTTTGTCTTTTCTGTGTTTGTCCCTTTCAATCTTCA TAAATGTATCCTTGTTCTCCTTTTCACAAACAGATTTTAAATTGATATGTGAAATTAATTAAGGAGGGGTTTTTATTTTTTATTTGGAAACAAAGAGGGCTTT CGCTAAGAAAAGAGAGAATTACAAAGAAATTAATCGAGGAGTAGCAATTCCAAAAACATCATATAAATAACCACTTGAATGATTTGAGTGGACTTTCTGTTGT TAAGATGTTAAAGATTACGTAACATTTTAATAGTATGGTTAAATTGATGAGTATAATATAACTTGTAACTTAAAACTAAATTGAATAGAGATTAACAGTTTAA TTGGGTACTAAAATGATGATTTTTTCTCCTTCTTGTGTCCTTTTCAATATTTAAAAATGTACTTTGTGTCCTTTTCAATATTATAAATGTATAGTTGTTTTCC TTTTCACAACCAAATTTTGAATTGATTTGAGAAATTAATTAAGGCATGTACTATTTCCCTTGAATTATTTGAGAAATGAAAAACTTACACGAATTAGTCCAAA CGATTTTTTTTTTGCAGATGGGAATAGGGTACAACTTGACCAGAAACGATCTAGTGAAAAAACTACGTGTGGCTGCATTGAGAGCCATGTCATAA B MEKLNSSSNSSSTSWLSDLLLEEMEIEGCDLFQQCEQNLFGEKEFLSNDIASIFQQEENF QHQPLLSSESYSSSYPLEKNFERPNKKLKKNTSSPSSTTSQILSFDCTLNTKKNKVVPLS CWE* QTELPQNRKGSLQKQNIVETIKPQGQGTKRSVAHNQDHIIAERKRREKLSQCLIALAALI Q Q Q Q Q Q Q PGLKKMDKASVLGDAIKYVKELQERLRVLEEQNKNSHVQSVVTVDEQQLSYDSSNSDDSE VASGNNETLPHVEAKVLDKDVLIRIHCQKQKGLLLKILVEIQKLHLFVVNNSVLPFGDSI LDITIVAQMGIGYNLTRNDLVKKLRVAALRAMS Supplemental Figure 7. Nucleotidic and protein sequence of the bhlh476 transcription factor and location of the Tnt1 insertion A. Nucleotidic sequence of the bhlh476 genomic region (from initial iti ATG to stop codons). B. Amino acid sequence of the bhlh476 protein, including the Helix-Loop-Helix DNA binding domain (italics). In both cases, the Tnt1 insertion of the bhlh-1 allele is indicated by an arrowhead. 7

8 A17 bhlh476 Supplemental Figure 8. Root hair and Rhizobium infection phenotype of bhlh476 mutants One week-old wild-type yp and bhlh476 mutant pplants were inoculated with a S. meliloti 2011 strain expressing a ProHemA:LACZ reporter to follow infections. infections Infection phenotype was analyzed five days after inoculation. No significant root hair and infection phenotype was d detected d in i the h mutant roots. Bar B = 100 µm. 8

9 RRBS-like RRBS-like Box 6 ATG Pro Mt ERN Box 1 RRBS-like RRBS-like ATG Pro Mt NIN Supplemental Figure 9. Schematic diagrams of promoter regions of ERN1 and NIN cytokinin nodulation-related genes RRBS boxes are indicated, d based on 8 bp cores defined d in the bioinformatic i screen, in promoters of selected nodulation-related genes: Mt ERN1 and Mt NIN. RRBS-like refers to boxes similar to the SELEX consensus in at least 7 bases. 9

10 MDDFSDRFPIGMRVLAVDDDPTSLLLLETLLRSCQYHVTTTSEAITALTMLQENIDMFDL VIAEVHMPDMDGLKLLELVGLEMDLPVIMLSAHGETELVMKAISHGARDFLLKPVRLEEL RNIWQHVIRNKESQFVWSVELHRKFLETVNQLGVDKAVPKKIFDLMNVENITREDVATHL QKYRLFLKRMDSSGHFHNNTIRSFSPAGIVGNLNTPAGLNGQFGHNSR Supplemental Figure 10. Protein sequence of the Mt RR1 Response Regulator Amino acid sequence of the Mt RR1 protein, including the phosphoreceiver p domain (underlined) and the DNA binding domain (italics). For each domain, conserved residues are highlighted in green and yellow, respectively (based on Hwang et al., 2002). Hwang, I., Chen, H., and Sheen, J. (2002). Two-Component Signal Transduction Pathways in Arabidopsis. Plant Physiol 129,

11 MapMan BIN Name General Functional Category Fisher's exact test pvalue gene number in BIN number of differentially regulated genes Microarray probe ID secondary metabolism.flavonoids.anthocyanins flavonoids 1.38E MT000247, MT001199, MT008218, MT013036, MT014034, MT014304, MT014305, MT014306, MT misc.udp glucosyl and glucoronyl transferases UDP GT 299E E MT000038, MT001070, MT001924, MT008640, MT009155, MT009525, MT015597, MT015712, MT015750, MT hormone metabolism.gibberelin.synthesis- synthesis degradation GA 4.13E MT003894, MT004863, MT007614, MT015706, MT transport.peptides and oligopeptides peptides MT002125, MT002790, MT007686, MT007687, MT MT000596, MT001635, MT002182, MT004863, misc.cytochrome P450 Cyt P MT006682, MT007068, MT007069, MT007426, MT RNA.regulation of transcription.bhlh,basic Helix-Loop-Helix family TF MT001777, MT012710, MT013227, MT013695, MT013931, MT secondary metabolism.flavonoids.dihydroflavonols flavonoids MT001070, MT003018, MT008640, MT RNA.regulation of transcription.ap2/erebp, APETALA2/Ethylene-responsive element TF MT002030, MT003761, MT005007, MT binding protein family misc.peroxidases MT007657, MT007930, MT008878, MT Supplemental Table 1. Differentially expressed functional categories in response to cytokinins (BAP 10-7 M, 1h) in root apices based on Mapman BINs A Fisher s exact test was used to determine which Mapman functional categories ( BINs ) were enriched after a BAP treatment in root apices ( Fold Change (FC) >1.5; Adjusted p-value <0.001). In each case is indicated: the Mapman BIN name, the Mapman General Functional Category, the Fisher'ss exact test p value, the total number of genes ( elements ) in the BIN, the number of differentially regulated genes in the BIN, and the 16K + array ID for differentially regulated genes. UDP GT: UDP Glucosyl Transferases; GA: Gibberellins; Cyt P450: Cytochrome P450; TF: Transcription Factors 11