PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL7 PYL8 PYL9 PYL10 PYL11/12
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1 Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Supplemental Figure 1. Gene expression levels of the PYR/PYL/RCAR ABA receptors in the Arabidopsis transcriptome genomic express database and Arabidopsis whole-genome tiling array (At-TAX). (A-C) Gene expression data were obtained from the Arabidopsis transcriptome genomic express database ( The figure shows the gene expression picture for each PYR/PYL gene using whole-genome microarrays (A) Salk transcriptome (Yamada et al., 23), (B) Exosome (Chekanova et al., 27) and (C) At-TAX (Laubinger et al., 28), Arabidopsis wholegenome tiling array ( (D)Arabidopsis thaliana Tiling Array Express (At-TAX) expression data of the PYR/PYL/RCAR gene family. Gene expression data were obtained from the Weigel World webpage ( microarray/at-tax) using the TileViz web resource ( Data correspond to 9 different tissues at various stages of plant development from Col- wild-type. Root, seedling and vegetative apex materials were obtained from 7-d-old plants; young and old leaf materials were obtained from 17 and 35 day-old plants respectively. Remaining plant material was obtained from plants grown for 21 or more days. Y axis represents the intensity of the gene expression in arbitrary units. (A) Salk transcriptome (Yamada et al., 23) (B) Exosome (Chekanova et al., 27) (C) At-TAX (Laubinger et al., 28) 1/12 1
2 (D)Arabidopsis thaliana Tiling Array Express (At-TAX) expression data of the PYR/PYL/RCAR gene family Root Seedling Leaf young Leaf old Stem Veg. apex Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Flower Fruit Whole Inf
3 Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Pro:GUS Pro:GUS Pro:GUS Pro:GUS Pro:GUS Pro:GUS Supplemental Figure 2. Photographs showing GUS expression driven by Pro, Pro, Pro, Pro, Pro and Pro:GUS genes in roots of 5-d-old seedlings. Upper arrows point to mature root, while lower arrows point the root differentiation zone. Mature roots from Pro:GUS and Pro:GUS lines show cortex expression while expression of the other receptors is located only in vascular tissue. 3
4 Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Pro:GUS Pro:GUS Pro:GUS MS ABA MS ABA MS ABA MS ABA MS ABA Pro:GUS Pro:GUS Pro:GUS Supplemental Figure 3. ABA treatment inhibits or attenuates GUS expression driven by Pro, Pro, Pro, Pro, Pro and Pro:GUS genes. Photographs show 5-d-old seedlings that were either mock or 1 µm ABA -treated for 1 h. MS ABA 4
5 Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Experiment I Experiment II Gene expression (au) without ABA with ABA Gene expression (au) without ABA with ABA Supplemental Figure 4. Expression of PYR/PYL genes in guard cells mock or 1 μm ABA-treated. Data were obtained from experiments reported by Yang et al., (28). Five-six weeks old plants were sprayed with 1 μm ABA or water for 4h before cell isolation. Next, guard cell protoplast isolation and RNA extraction were performed as previously described by Leonhardt et al., (24). Experiment I included the addition of 33 mg/l actinomycin D and 1 mg/l cordycepin during protoplast isolation to inhibit modulation of gene expression in response to stress during guard cell protoplast preparation. Experiment II lacked these compounds. 5
6 Experiment A Experiment B Experiment C +ABA +ABA +ABA +ABA Experiment D Col A 2.2/2.3/2.6 A A Col B 2.2/2.3/2.6 B B Col C 2.2/2.3/2.6 C C Col D 2.2/2.3/2.6 D D Mix Col /2.6 A A /2.6 B B /2.6 C C /2.6 D D A /2.6 C Suppl. Figure 5. Scheme of the transcriptomic experiment. ABA-response of wt, and /2.6 mutants was compared using Agilent s Arabidopsis 44k oligonucleotide microarrays. See also methods. /2.6 B C /2.6 D D /2.6 A B Agilent v4 4x44 Agilent v4 4x44 6
7 Supplemental Data. Gonzalez-Guzman et al. Plant Cell. (212) /tpc Supplemental references Chekanova, J. A., Gregory, B. D., Reverdatto, S. V., Chen, H., Kumar, R., Hooker, T., Yazaki, J., Li, P., Skiba, N., Peng, Q., Alonso, J., Brukhin, V., Grossniklaus, U., Ecker, J. R., and Belostotsky, D. A. (27). Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome. Cell 131, Laubinger, S., Zeller, G., Henz, S. R., Sachsenberg, T., Widmer, C. K., Naouar, N., Vuylsteke, M., Scholkopf, B., Ratsch, G., and Weigel, D. (28). At-TAX: a whole genome tiling array resource for developmental expression analysis and transcript identification in Arabidopsis thaliana. Genome Biol. 9, R112. Leonhardt, N., Kwak, J. M., Robert, N., Waner, D., Leonhardt, G., and Schroeder, J. I. (24). Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16, Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, C. J., Southwick, A. M., Wu, H. C., Kim, C., Nguyen, M., Pham, P., Cheuk, R., Karlin-Newmann, G., Liu, S. X., Lam, B., Sakano, H., Wu, T., Yu, G., Miranda, M., Quach, H. L., Tripp, M., Chang, C. H., Lee, J. M., Toriumi, M., Chan, M. M., Tang, C. C., Onodera, C. S., Deng, J. M., Akiyama, K., Ansari, Y., Arakawa, T., Banh, J., Banno, F., Bowser, L., Brooks, S., Carninci, P., Chao, Q., Choy, N., Enju, A., Goldsmith, A. D., Gurjal, M., Hansen, N. F., Hayashizaki, Y., Johnson- Hopson, C., Hsuan, V. W., Iida, K., Karnes, M., Khan, S., Koesema, E., Ishida, J., Jiang, P. X., Jones, T., Kawai, J., Kamiya, A., Meyers, C., Nakajima, M., Narusaka, M., Seki, M., Sakurai, T., Satou, M., Tamse, R., Vaysberg, M., Wallender, E. K., Wong, C., Yamamura, Y., Yuan, S., Shinozaki, K., Davis, R. W., Theologis, A., and Ecker, J. R. (23). Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 32, Yang, Y., Costa, A., Leonhardt, N., Siegel, R. S., and Schroeder, J. I. (28). Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool. Plant Methods 4, 6. 7
8 Supplemental Data. Gonzalez Guzman et al. Plant Cell. (212) /tpc Supplemental Table 1. List of oligonucleotides used in this work. For amplification of the promoters: Fprom: ACT AAA CTA ATC ATT CTC TAC Rprom: TCGTTCTTCTGGTGTTAACTCCG Fprom: AGCAATATAATTTTACACTCTGGA Rprm: GGAGGAGGACTCTGAATTCGCCAT Fprom: AAACTTATCGAACTGGAATTG Rprm: TAGGCCTTTCACGGCCGGGGATG Fprom: TGTGTCCATGTCCGGAGTTGT Rprom: GGCGGAAGAAGGACGGTGAACG Fprom: GGTGTGATGGTCGAATATTT Rprom: GCCGTGTTGGAGTTGCACCGGT Fprom: ACTGTGAAGCAAACCCTATATAT Rprom: CGTCAAGTTCTCAATCCCGTTAG For genotyping of the mutants: FNc4g1787: ACCATGGCTTCGGAGTTAACACCA R4g1787: TCACGTCAC CTGAGAACCACT F: ATGGCGAATTCAGAGTCCTCC R: TTACCTAACCTGAGAAGAGTT FwNco: ACCATGGGCTCATCCCCGGCCGTGA RvStop: TTATTCATCATCATGCATAGGTG F: ACCATGGTTGCCGTTCACCGTCCTT R: TCACAGAGACATCTTCTTCTTGC F: ATGAGGTCACCGGTGCAACT R: TTATTGCCGGTTGGTACTTCGA FwMEANpyl8: ATGGAAGCTAACGGGATTGAG RvESRVpyl8 : TTAGACTCTCGATTCTGTCGT prok : GCCGATTTCGGAACCACCATC Spm3: ACCGTCGACTACCTTTTTTCTTGTAGTG LB3SAIL: TAGCATCTGAATTTCATAACCAATCTCGATACAC DS5: GTTCGAAATCGATCGGGATAAAAC 8
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Intensity Intensity Intensity Intensity Intensity Intensity 150 50 150 0 10 20 50 C 150 0 10 20 50 D 0 10 20 Distance (μm) 50 20 40 E 50 F 0 10 20 50 0 15 30 Distance (μm) Supplemental Figure 1: Co-localization
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