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Figure S1. An alignment of C-terminal sequences of ACS isozymes from tomato and Arabidopsis. Box 7 is the 7th conserved domain of the ACSs (partially indicated). Putative CDPK phosphorylation sites are indicated with an asterisk and its conserved flanking sequences are underlined. Putative MAPK phosphorylation sites are indicated with arrowheads and the following Pro residues are underlined. Amino acid changes that result from the eto2 (AtACS5) and eto3 (AtACS9) mutations are highlighted in gray.

Figure S2. Ethylene production in wounded tomato fruit tissue treated with protein kinase/phosphatase inhibitors. Tomato fruit disks were prepared and treated with 0.5 μm K252a or okadaic acid for the indicated times. Ethylene production from tissue was measured.

Figure S3. The phylogenetic tree of CDPKs from Arabidopsis and tomato. The tree was made using the entire CDPK protein sequences using the ClustalW program at GenomeNet in the Kyoto University Bioinformatics Center. The tree includes CDPKs from Arabidopsis and tomato that have been reported so far. CDPKs from Arabidopsis are denoted with only numbers. Roman figures of subgroups are named for the classification by Cheng et al. (2002).

Figure S4. Time-course analyses of CDPK and MAPK activity induced by wounding, and inhibitor effects on CDPK, MAPK and LeACS2 mRNA expression. (a) Relative LeACS2 mRNA expression in wounded fruit tissue treated with DMSO, 100 μm U0126, or 100 μm CMZ. LeACS2 expression was analyzed with real-time PCR. Real-time PCR was performed in triplicate, and values shown are means ± SD (= 3). (b) 40 pmol of Trx-LeACS2 was incubated with 0.05 μg of a crude kinase fraction prepared from wounded tomato fruit tissue in the presence of [γ-32P] ATP. 1 mm EGTA, 100 μm CMZ, or 100 μm U0126 was added to the reaction mixture to characterize the kinase fraction (60 min after wounding). Proteins were subjected to SDS-PAGE and radioactivity was detected (top). Coomassie Brilliant Blue staining shows equal amount of Trx-LeACS2 as substrates (bottom). (c) Proteins prepared from wounded tomato fruit tissue were subjected to in-gel kinase assay using myelin basic protein. After the reaction, radioactivity was detected. To confirm the effects of U0126 and CMZ, proteins from the tissue (60 min after wounding) treated with DMSO, 100 μm U0126, or 100 μm CMZ were analyzed.

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