1. Arabidopsis OST1 Protein Kinase Mediates the Regulation of Stomatal Aperture by Abscisic Acid and Acts Upstream of Reactive Oxygen Species Production by Anna-Chiara Mustilli, Sylvain Merlot, Alain Vavasseur, Francesca Fenzi, and Jérôme Giraudat Plantcell Volume 14(12):3089-3099 December 1, 2002 ©2002 by American Society of Plant Biologists

2. Wild Type seeds (Ler) Mutant collection Screening Mutagenesis (EMS) Isolation of ost1 and ost2 mutant Stress sécheresse

3. ost1 and ost2 : the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds Isolation of ost1 and ost2 mutant Upon drought ost1, ost2 and abi1 have lower leaf temperature ost1, ost2 and abi1 showed reduced stomatal closure in response to ABA abi1 is insensitive to the inhibition of germination in response to ABA

4. Wild type ost1-2 ost1-1 Physiological characterization of ost1 mutation A- Preopened ost1 and ost2 stomata failed to close in response to ABA B- Normal darkness-induced stomatal closure C- ost1 has no effect on the ability of preclosed stomata to open in response to light ost1 and ost2 are impaired in the ABA inhibition of light induced stomatal opening D- ost1 has no effect on the ability of preclosed stomata to open in response to Cos-free air

5. -OST1 encodes a 362–amino acid protein with a predicted molecular mass of 41 kDa -OST1 is a putative Ser/Thr kinase Cloning of OST1

6. Localization of OST1 expression RT-PCR pOST1:GUS GCP Guard cell protoplast MCP Mesophylle Cell protoplast OST1 expression is not regulate by ABA OST1 expression is confined to guard cells and vascular tissus

7. OST1 is an ABA-activated Protein Kinase In both root and GCP a 42 kD band with kinase activity was enhanced in response to ABA

8. OST1 Acts Upstream of ROS Production A- ABA did not increase ROS levels in ost1 guard cells B- ost1 mutation disrupts ABA signaling upstream of ROS production D- OST1 kinase acts downstream of ABI1

9. Mustilli A et al. Plantcell 2002;14:3089-3099 ©2002 by American Society of Plant Biologists Model for the Position of OST1 in the ABA Signaling Cascade