Synthesis and characterization of abscisic acid receptor modulators

The apocarotenoid phytohormone abscisic acid (ABA) regulates several aspects of plant development and stress responses. ABA is synthesized in response to multiple stressors and indirectly activates subfamily 2 Snf1-related kinases (SnRK2s) by receptor-mediated inhibition of clade A type IIC protein phosphatases (PP2Cs), which normally repress SnRK2 activity. The binding of ABA to its receptors triggers a change in receptor conformation that directs the formation of a receptor-ligand-PP2C complex that inhibits the activity of PP2C; this core mechanism can be harnessed for phosphatase activity-based measurements of receptor activation. In this chapter, we describe general methods for determining the effects of small molecules on ABA receptor function and supplement these with methods describing the synthesis of the high-affinity ligands opabactin (OP), which activates subfamily III and II ABA receptors, and the pan-receptor antagonist antabactin (ANT), and TAMRA-ANT fluorescent derivative of ANT. We present simple methods for quantifying receptor-ligand interactions using both PP2C inhibition and fluorescence polarization (FP) assays. Controls for determining the quality of recombinant receptors are provided, which are required for both quantitative analyses and for experimental consistency. Collectively, these methods will facilitate consistent biochemical measurements of the effects of ligand binding on ABA receptor function in vitro. Although the chapter describes ABA-specific methods, they illustrate and address a common need across receptor systems-reproducible assays that enable high throughput discovery and subsequent optimization of receptor modulators.