Abstract

Abscisic acid (ABA) is the main phytohormone involved in abiotic stress response and its adaptation, and is a candidate agrichemical. Consequently, several agonists of ABA have been developed using the yeast two-hybrid system. Here, we describe a novel cell-free-based drug screening approach for the development and validation of ABA receptor agonists. Biochemical validation of this approach between 14 ABA receptors (PYR/PYL/RCARs) and 7 type 2C-A protein phosphatases (PP2CAs) revealed the same interactions as those of previous proteome data, except for nine new interactions. By chemical screening using this approach, we identified two novel ABA receptor agonists, JFA1 (julolidine and fluorine containing ABA receptor activator 1) and JFA2 as its analog. The results of biochemical validation for this approach and biological analysis suggested that JFA1 and JFA2 inhibit seed germination and cotyledon greening of seedlings by activating PYR1 and PYL1, and that JFA2 enhanced drought tolerance without inhibiting root growth by activating not only PYR1 and PYL1 but also PYL5. Thus, our approach was useful for the development of ABA receptor agonists and their validation.

Highlights

  • Abscisic acid (ABA) is an important phytohormone for the regulation of complex networks to cope with abiotic stress in plants[1]

  • Previous studies in Arabidopsis have shown that all 14 ABA receptors (PYR1 and PYL1 to PYL13) interact with specific PP2CAs in an ABA-dependent or -independent manner and that all ABA receptors are involved in the regulation of ABA signals[14,18,21,22,23]

  • Interaction analysis within the ABA receptor family revealed 20 new interactions. By applying this method of chemical compound screening based on the interactions between PYR1 and ABI1, we identified one new unique ABA receptor agonist compound JFA1 from a chemical library consisting of 9,600 compounds

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Summary

Introduction

Abscisic acid (ABA) is an important phytohormone for the regulation of complex networks to cope with abiotic stress in plants[1]. By using a combination of the wheat cell-free system and the “AlphaScreen” luminescence system, we developed a high sensitivity and specificity as well as high-throughput method to analyze ABA-dependent and -independent interactions and to screen ABA receptor agonists. JFA1 and JFA2 suppressed seed germination to the same level; JFA2 induced the expression of ABA-inducible genes and enhanced drought tolerance in Arabidopsis plants compared to JFA1 These results suggested that our method is useful for the biochemical analysis of ABA receptors and development of an ABA receptor agonist against each receptor, allowing large-scale screening of agonist or antagonist compounds for plant hormones

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