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1 1 Stomatal Patterning and Differentiation by Synergistic Interactions of Receptor Kinases Elena D. Shpak, Jessica Messmer McAbee, Lynn Jo Pillitteri, and Keiko U. Torii Supporting Online Material Material and Methods Supplementary References and Notes Figs. S1 to S5 Tables. S1 Material and Methods Plant materials and double~quadruple mutant construction Arabidopsis Columbia (Col) plants were used as wild type. Complete loss-of-function alleles of ER (At2g26330), ERL1 (At5g62230), and ERL2 (At5g07180), namely er-105, erl1-2, and erl2-1, respectively, were used for analyses. tmm-1 was acquired from the ABRC (Ohio State Univ). To generate tmm er, tmm er erl1, tmm er erl2, tmm erl1 erl2, and tmm er erl1 erl2 quadruple mutant, tmm-1 plants were crossed with plants of the genotype er/er erl1/+ erl2/erl2. Plants of the correct genotype were isolated from the F3 or F4 populations. The presence of er, erl1 and erl2 mutations were determined by PCR as described previously (1, 2). The presence of the tmm-1 mutation was determined by

2 2 sequencing the PCR-amplified TMM coding region with primer TMM-743 (5 - CACTCACAAGCTGTGGATCGTT-3 ). Reporter constructs and histology The ER::GUS, ERL1::GUS and ERL2::GUS constructs were described previously (1). The 0.5 kb 5 upstream sequence of TMM (3) was amplified by primers TMMp5 (5 - CGAATTCCTTTCGTAGTTACTTCATTATTCA-3 ) and TMMp3 (5 - CGAATTCGGTTTAGGTTCGAAATTGTCAGTGT-3 ) and cloned into EcoRI-digested pesh224 (1) to generate TMM::GUS (pkut574). GUS staining, light microscopy and scanning electron microscopy were performed as described previously (1). For DIC microscopy, tissues were treated in 9:1 ethanol:acetic acid solution, rehydrated through an ethanol series, cleared in 8:1:1 chloral hydrate:water:glycerol, and viewed under Nikon Optishot equipped with a Sony DXC-960MD CCD video camera. The DIC microscopic images in Figure 4 are taken under Olympus BX51 equipped with an Olympus DP million-pixel digital camera. The line drawings of the DIC images were false colored using Adobe Photoshop 7. Quantitative analysis The methods of Geisler et al. (1998)(4) were used as a guide for data collection. For the quantitative analysis, one three-week-old cotyledon, one fully expanded leaf, one mature stem segment, and one fully expanded silique were collected from each of five plants for each genotype grown in the same conditions. This material was cleared, and then stored and viewed in chloral hydrate solution. Epidermal peels were taken from fresh pedicels

3 3 and viewed at 40X. For each organ, five random DIC images were taken at 20X. The numbers of stomata, stomatal lineage ground cells, and pavement cells were counted in each image. Stomatal indices were calculated as the number of stomata divided by number of stomatal cells plus non-stomatal epidermal cells, then multiplied by 100. Supplementary References and Notes 1. E. D. Shpak, C. T. Berthiaume, E. J. Hill, K. U. Torii, Development 131, 1491 (2004). 2. K. U. Torii, L. A. Hanson, C. A. B. Josefsson, E. D. Shpak, in Morphogenesis and patterning in biological systems T. Sekimura, Ed. (Springer-Verlag, Tokyo, 2003) pp J. A. Nadeau, F. D. Sack, Science 296, 1697 (2002). 4. M. Geisler, M. Yang, F. D. Sack, Planta 205, 522 (1998).

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9 Table S1 Stomatal index (SI) for five organs in eight genotypes. SI = (# stomata/ # stomatal cells + # non-stomatal epidermal cells) x100. (±SE, n=5 for each organ) Cotyledon, Leaf, abaxial Stem Pedicel Silique abaxial WT (Col) 20.7±0.6 # 21.0± ± ± ±0.3 er 17.9±0.3 b 21.6± ±0.6 12±0.5 c 16.0±0.2 erl1 22.3± ±0.4 a 15.3±0.5 b 14.5±0.5 c 17.7±0.3 b erl2 20.0± ±0.9 c 14.7±0.6 c 13.0± ±0.5 erl1 erl2 24.2± ±0.7 b 16.3±0.8 b 16.3±0.6 b 19.7±0.3 a er erl1 25.3±1.0 b 26.0±0.4 a 15.1±0.9 c 19.1±0.9 a 18.3±0.3 a er erl2 20.5± ± ± ± ±0.4 er erl 1erl2 37.0±0.4 a 33.7±1.0 a 34.0±2.0 a 39.7±0.6 a 36.3±0.9 a a Significantly different from the WT (p<0.001) b Significantly different from the WT (p<0.01) c Significantly different from the WT (p<0.05) # The Student s T-test was performed for statistic significance. Numbers in black are not significantly different from wild type. Numbers in blue are significantly higher than wild type. Numbers in red are significantly lower than wild type.