Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system

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.


Construction of the in vitro transcription templates
The genes of PYR1, PYL1, PYL2, PYL4-6, PYL8, and PYL11 were amplified by PCR from the RIKEN Arabidopsis full-length (RAFL) cDNA library. The genes of PYL3, PYL7, PYL9, PYL10, PYL12, and PYL13 were amplified by PCR using total cDNA prepared from mRNA of Col-0 plant as a template (described below).
The DNA fragments of the open-reading frame (ORF) were cloned in a pDONR221 vector using the gateway cloning system (Thermo Fisher Scientific). After confirming sequences, we generated these expression vectors by LR Clonase recombination with pEU-His-bls-GW vectors for in vitro transcription. Then, these regions of the gene containing ORF and tag sequence were amplified by PCR and used as transcription templates.
For preparation of in vitro transcription templates for FLAG-PP2CAs and biotinylated SnRK1.1, the genes of ABI1, ABI2, HAB1, HAI1, HAI2, AHG1, AHG3, and SnRK1.1 were amplified by the split-primer PCR method 1 using the RAFL cDNA library as the template. For preparation of in vitro transcription templates for AGIA-tagged ABA receptors, gene regions of ABA receptors were amplified by the split-primer PCR method 1 using the genes sub-cloned into pDONR 221 as templates.
All primer sequences and plasmids used in this study are listed in supplementary Table   2 and Table 3.

Chemical library screening
For the AlphaScreen-based chemical library screening, we used a diverse set     Four-week old plants were sprayed with chemical solutions containing with 25 µM chemical, ABA, JFA1, or JFA2, and 0.02% Silwet L-77. Then plants were incubated in a growth cabinet at 22 C under a light condition. After incubation for 3, 6 and 24 hr, leaves were detached and treated with water containing with 1 µM rhodamine 6G (Sigma) for 2 min. Imaging of stomata was performed with a fluorescence microscope IX-73 (Olympus). Image analysis was performed using ImageJ software (https://imagej.nih.gov/ij/). The width and the length of the stomatal aperture were measured, and the stomatal aperture index was calculated by division of the aperture width through the length. Relative stomatal aperture index was expressed as a relative value with the DMSOtreatment as one. Stomatal aperture indexes are calculated from three independent experiments with 30 stomata per treatment. Error bars represent standard error. Statistically significant changes compared with mock control (DMSO) are indicated (*P < 0.05, **P < 0.01, two-tailed Student's ttest)