A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration

Abstract

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.

Recent Advances in Plant Chemical Biology of Jasmonates

“…However, there are few successful examples of 'biased' plant hormone receptor agonists. Cutler's group successfully developed the ABA receptor agonists [39] pyrabactin [40,41], quinabactin [42], cyanabactin [43], and opabactin [44], which were identified by random screening of a large-scale chemical library, and exhibited remarkable selectivity among 15 ABA receptor subtypes. The same method also resulted in the identification of SPL7, a femtomolar agonist selective for a strigolactone receptor ShHTL7 involved in the seed germination of parasitic plant Striga hermonthica [45].…”

Synthetic Switches and Regulatory Circuits in Plants

“…Another goal is to improve plant stress tolerance to environmental hardships by manipulating phytohormone signaling pathways or introducing orthogonal networks, targeting key plant stress responses. First steps toward this were recently reported based on engineering the receptor for the phytohormone ABA and developing chemical agonists thereof to control the responses to drought (Park et al, 2015;Vaidya et al, 2017). A next step would be to design hybrid circuitry to overcome limitations and bypass endogenous regulation of plant signaling networks to improve the efficiency of existing cascades.…”

“…Recent advances have been made in manipulating different aspects of ABA signaling (e.g. receptor engineering and developing an ABA agonist; Park et al, 2015;Vaidya et al, 2017; Table 1). Cyanabactin is a potent, selective agonist for one distinct ABA receptor family, namely, the subfamily of IIIA receptors.…”

The fungal sesquiterpenoid pyrenophoric acid B uses the plant ABA biosynthetic pathway to inhibit seed germination

“…This reporter 229 line is highly activated by ABA (Fig. 2C,D) (Vaidya et al, 2017). P-Acid B was able to 230 activate the reporter line since pMAPKKK18-LUC + seedlings treated with 200 µM P-231 Acid B during 6 hours showed around 4.5 fold increase on LUC activity due to the 232 activation of the ABA responsive promoter pMAPKKK18 (Fig.…”

“…After ABA binding, allosteric changes induced in ABA receptors facilitates 245 their interaction with PP2Cs inhibiting phosphatase activity. Therefore, the binding of 246 small molecules to ABA receptors can be followed by in vitro PP2C phosphatase assays 247 as reported elsewhere Okamoto et al, 2013;Vaidya et al, 2017). We 248 performed in vitro PP2C phosphatase assays using recombinant PYR1, PYL1, PYL2, 249 PYL4, PYL5, PYL6, PYL8 and PYL10.…”