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Figure S1. The split-ubiquitin yeast two-hybrid assay suggests that SLAC1 interacts with OST1 kinase. (a) Construct design of SLAC1 in pBT3-STE bait vector and OST1 in pPR3-STE prey vector; CYC P: Cyc1 promoter; Cub: C-terminal (amino acids 34–76) of ubiquitin; LexA: LexA DNA binding domain; VP16: VP16 transactivation domain; CYC T: Cyc1 terminator; ADH P: Adh1 promoter; NubG: N-terminal (amino acids 1–33) of ubiquitin with the Ile3rdàGly3rd mutation; (b) Split-ubiquitin membrane yeast two-hybrid assay showed interaction between SLAC1 and OST1. Growth of yeast co-transformed with the constructs indicated were tested on SD-Leu-Trp permissive media (for the presence of both bait and prey constructs; left panel) or the selective SD-Leu-Trp-His media (for bait-prey interaction; middle panel); β-galactosidase activities of transformants were visualized by filter lift-off assay (right panel). As controls, yeast was co-transformed with SLAC1-Cub+Alg5-NubI (positive control) to verify proper expression of SLAC1 in yeast, and with SLAC1-Cub+Alg5-NubG (negative control) to ensure that SLAC1 was not autoactivating the reporter genes.

Figure S2. Mass spectrometry analysis suggests that OST1 phosphorylates at least four serines in the SLAC1–186 fragment. SLAC1–186 fragment phosphorylated by OST1 was analyzed with nanoLC-MS/MS system. MS/MS spectra derived from four phospho-peptides with m/z 1528.73, 1833.80, 843.41 and 1233.68, representing peptides were S59 (a), S86 (b), S113 (c) and S120 (d) appeared to be phosphorylated, respectively. Also peptide with m/z, 2035.89 containing S146 (e), which was not phosphorylated, is shown. The results were confirmed through several replications.

Table S1. List of mutants tested for ozone-triggered rapid transient decrease (RTD) in stomatal conductance. The mutants are grouped into functional categories based on the mutated protein (see separate file).

Table S2. Reactive Oxygen Species (ROS) production in Arabidopsis guardcells. Both genotype and time of ozone exposure were significant factors affecting ROS production (P = 0.000; analysis of variance, GLM procedure). Comparisons between individual means were done using Tukey HSD test. Genotypes with the same letter are not significantly different at P < 0.05.

Appendix S1. Supplementary experimental procedures.

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