Higher Order Calcium-Dependent Protein Kinases (CPKs) Mutant Lines Elucidate Roles of CPKs within Abscisic Acid Signal Transduction in Arabidopsis and In Vivo Interactions of Calcium-Dependent Protein Kinases, CPK6 and CPK23, with PP2C Protein Phosphatases within Abscisic Acid Signaling /

Stomata, present in the aerial epidermis of land plants, provide gateways for regulating carbon dioxide and water exchange between plants and the atmosphere. The stress- induced phytohormone abscisic acid (ABA) reduces transpirational water loss crucial for the fitness of plants by inducing stomatal closure. Cytosolic Ca²⁺ has been reported to play a major role in ABA-induced stomatal closure through Ca²⁺ sensors, such as Calcium Dependent Protein Kinases (CPKs). Plants lacking transcripts of CPK3 and CPK6 (cpk3cpk6) have shown impaired ABA- and Ca²⁺- induced stomatal closing. Double knockout mutants of CPK4 and CPK11 (cpk4cpk11) exhibit similar ABA-hyposensitive phenotypes in ABA-induced stomatal closing response as well as a reduced ABA response in seed germination and seedling growth. Single mutant cpk23, however, demonstrated increased sensitivity to ABA, indicating a possible negative regulatory role in ABA signaling for CPK23. To improve the understanding of these proteins and their functions, two quadruple mutant plant lines, cpk3cpk4cpk6cpk11 and cpk5cpk6cpk11cpk23, were established. In Chapter 1, the quadruple mutant lines are phenotypically characterized by examining a variety of ABA -dependent biological responses. Other proteins important in ABA-dependent stomatal responses are Protein Phosphatases 2C (PP2Cs). Group A PP2Cs are known negative regulators within the Ca²⁺-independent ABA signaling pathway; however, experimental evidence suggests other regulatory roles exist for PP2Cs within the Ca²⁺-dependent branch of this signaling network which includes CPKs. In Chapter 2, yeast two-hybrid system is used to evaluate whether PP2Cs interact with CPKs. Observed interactions can provide additional insight to these proteins' functions within the Ca²⁺-dependent ABA signalin