Abscisic acid (ABA) is a central stress hormone in Arabidopsis that mediates rapid cellular responses, down- regulates cell proliferation and causes cell cycle arrest. Early signal transduction networks that down-regulate cell proliferation are of key importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. The long-term goal of this research is to achieve a new and quantitative understanding of the network of events that mediate early abscisic acid signaling in the potent Arabidopsis guard cell system. We will characterize newly identified key cellular signaling mechanisms hypothesized to control the recently revealed early ABA receptor signaling core consisting of ABA receptors, PP2C protein phosphatases and SnRK2 &calcium (Ca2+)-dependent protein kinases, which mediate downstream ABA signaling.
Specific Aims : I. Regulatory proteins of eukaryotic PP2Cs and ABA receptor interactors are not well-understood. We will characterize the functions of a newly identified proposed early receptor-PP2C control loop that can counteract monomeric ABA receptor-induced ligand-free """"""""leaky"""""""" PP2C signaling. This includes the PP2C interacting and regulating ROP10 &11, ABA receptor-interacting GDP/GTP exchange factor 1 and the OST1 protein kinase. II. How the universal second messenger Ca2+ mediates specific responses in eukaryotic cells is a key question in cell signaling research and disease. Our recent results point to a """"""""Ca2+ sensitivity priming"""""""" mechanism, in which ABA primes Ca2+ sensors switching them from an inactivated Ca2+-insensitive state to a Ca2+-responsive """"""""primed"""""""" state. Priming enables a specific Ca2+ response and this novel mechanism may be used by diverse eukaryotic Ca2+-signaling pathways. We will investigate the hypothesis that the PP2Cs directly inactivate the Ca2+-dependent kinase CPK6. In addition, cross-regulation of CPK6 and the OST1 protein kinase in ABA activation of SLAC1 anion channels will be investigated. We will identify the ABA-induced Ca2+ specificity signaling mechanisms using biochemical and dynamic in vivo cell signaling analyses and via parallel functional analyses of the reconstituted multi-component ABA signalosome in Xenopus oocytes. III. In a new chemical genetics screen of 9600 compounds, we have identified a small molecule """"""""DFPM"""""""" that down-regulates ABA signaling. DFPM activates intracellular effector-triggered signaling and thereby rapidly (<3 min) deactivates ABA responses at the level of Ca2+ signaling in guard cells. New chemical genetic mutants showing strong insensitivity to DFPM-regulated ABA/Ca2+ signaling have been isolated. Selected mutants and the underlying mechanisms will be characterized to elucidate new elements and mechanisms mediating DFPM-induced rapid interference with ABA/Ca2+ signaling. This research will result in a new understanding of PP2C regulation and Ca2+ specificity signaling mechanisms, which are fundamental to numerous cell signaling processes and disease states, and will reveal novel mechanisms by which newly identified ABA receptors and regulation mechanisms control ABA signaling.

Public Health Relevance

A fundamental question in cell signaling research is how the universal second messenger Ca2+ mediates specific responses in eukaryotic cells. Furthermore, type 2C protein phosphatases (PP2Cs) play central roles in human disease, but PP2C interactors that regulate catalytic PP2C activity are largely unknown. This research project will make use of a well-developed model system to substantially advance the understanding of the mechanisms mediating specificity in Ca2+ signaling and PP2C regulation, processes that are fundamental to numerous cell signaling pathways and are misregulated in human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Chin, Jean
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University of California San Diego
Other Domestic Higher Education
La Jolla
United States
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Wang, Cun; Zhang, Jingbo; Schroeder, Julian I (2017) Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes: Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor. Bio Protoc 7:
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Li, Zixing; Waadt, Rainer; Schroeder, Julian I (2016) Release of GTP Exchange Factor Mediated Down-Regulation of Abscisic Acid Signal Transduction through ABA-Induced Rapid Degradation of RopGEFs. PLoS Biol 14:e1002461
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