Dynamic control of plant water use using designed ABA receptor agonists

Abstract

Plant thirst quenched without water Drought causes many billions of dollars of annual losses to farmers worldwide. Central to a plant s water use efficiency are signaling pathways regulated by the hormone abscisic acid and its receptors. Vaidya et al. screened a pool of candidate small molecules and used structure-guided design to optimize the function of an abscisic acid receptor agonist (see the Perspective by Phillips and Sussman). Application of the agonist protected Arabidopsis, wheat, and tomato from underwatering. Science, this issue p. eaaw8848; see also p. 416 Chemical design informed by structural analysis leads to a more effective inhibitor of plant abscisic acid receptors for drought tolerance. INTRODUCTION Climate extremes create a need to mitigate the effects of drought on agriculture. The contributions of water to crop yield vary over a growing season but peak during reproductive development. Water banking strategies, which save soil water early in a growing season, reserve water for flowering and can increase yield under modest drought. Antitranspirants based on mimics of the phytohormone abscisic acid (ABA), which controls stomatal aperture, are sought as next-generation agrochemicals for controlling water use and increasing yield during drought. RATIONALE Information on the structure and function of ABA receptors has created opportunities for agrochemical development. Current lead molecules have low and short-lived bioactivity in some relevant crop species, including wheat, the world’s most widely grown staple crop. This liability is likely a consequence of incomplete activation of different ABA receptor subclasses. We reasoned that the idiosyncratic activity of these molecules was due, in part, to a lack of interaction between the agonist and a conserved lysine in ABA receptors that forms a salt bridge to ABA’s carboxylate. We performed virtual screening to identify candidate agonists that interact with this lysine. RESULTS Two ABA receptor structures were used to screen against the ZINC database, a collection of commercially available ligands, using Glide docking protocols, requiring that hits interact with the conserved lysine. Candidate agonists were obtained and tested for receptor activation using in vitro assays. A family of substituted phenyl acetamido-cyclohexane carboxylic acid ABA receptor panagonists was identified. Scaffold merging was used to improve binding: We grafted an optimized headgroup of an existing sulfonamide onto our phenyl acetamido-cyclohexane carboxylic acid scaffold to yield a chimeric ligand (3CB) that displayed an improvement toward target sites of up to three orders of magnitude. Analysis of a 3CB-PYL10 crystal structure suggested that addition of appropriately situated hydrophobes to 3CB might improve activity. A 3CB derivative was synthesized, yielding an agonist that we have named opabactin (OP) for overpowered ABA receptor activation. Thermodynamic characterization of 3CB or OP receptor binding reactions indicates that the newly generated scaffold’s improvements are enthalpically driven relative to sulfonamides, consistent with the salt bridge observed in our crystal structure. Biological studies show that OP is ~10-fold more active in inhibiting seed germination (a response driven by ABA) than ABA itself. Experiments in wheat, tomato, barley, Arabidopsis, and Commelina demonstrate bioactivity in vegetative tissues across diverse species. Time course experiments in wheat and tomato using thermal imaging show that OP induces a more sustained antitranspirant response than ABA, and they document poor activity of sulfonamide agonists in those two species. To understand which receptors are necessary for OP action, we used Arabidopsis mutant strains to show that OP requires the subfamily III receptors PYR1, PYL1, and PYL2 for maximal activity. Thus, our virtual screening experiments yielded an ABA receptor agonist that functions as an antitranspirant. CONCLUSION A newly generated ABA agonist scaffold was identified and optimized through a structure-guided approach. The chemical biology of ABA receptor agonists can be broadened by designing ligands that engage a conserved lysine residue in the binding pocket. This pharmacophoric feature results in a favorable enthalpic binding profile and lower dissociation constants than existing sulfonamide-based ligands. Our ABA agonist, opabactin, has activity in multiple monocots and eudicots and addresses the limitations of existing sulfonamide molecules being explored as tools for mitigating the effects of drought on crop yields. Water savings and drought protection activity in crops. Virtual screening yielded a scaffold 3B4 whose potency was improved by ~1600-fold by scaffold merging, yielding 3CB. Structure-based optimization gave opabactin (OP). Thermodynamic profiling shows that OP binding to its targets PYR1 and HAB1 is enthalpically driven with about one-tenth the dissociation constant Kd of ABA. OP exhibits more potent and longer-lasting antitranspirant effects than existing sulfonamide-based ligands. Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.