Dynamic control of plant water use using designed ABA receptor agonists

First author: Aditya S. Vaidya; Affiliations: University of California, Riverside (加州大学河滨分校): Riverside, USA

Corresponding author: Sean R. Cutler

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.

引言    极端气候的出现使得我们必须缓和干旱对于农业的影响。水分对于作物产量的影响虽然在不同生长季节各有变化，但是在作物生殖发育时对于作物的产量影响最大。在植物生长季节节约用水，而在开花时供给充足水量的“水库策略”可以在适度干旱的环境条件下增加作物的产量。控制植物气孔开度的植物激素脱落酸的模拟物“止汗剂”被认为是下一代控制干旱条件下作物的水分利用以及增加产量的农用化学品。

原理    有关ABA受体的结构和功能信息为化学农业的发展创造了机会。 当前的前沿分子在某些相关农作物种中的生物活性低且寿命短，包括全球生长最广泛的主粮小麦。 该现象可能是由于不同ABA受体亚类的不完全激活所导致的。 作者认为，这些分子的特定活性至少部分是由于缺乏激动剂与ABA受体中保守赖氨酸之间的互用，而这种互作形成了一个指向ABA羧酸盐的盐桥。 作者进行了虚拟筛选，以鉴定能够与该赖氨酸互作的激动剂候选物。

结果    通过Glide docking方案，即要求靶标能够与保守的赖氨酸互作，作者使用两个ABA受体结构来筛选一个包含所有商业可获取配体的ZINC数据库。作者通过体外试验进一步对候选激动剂进行了受体激活测试。通过这种方法，作者鉴定到了一类取代的苯基乙酰氨基-环己烷羧酸ABA受体泛激动剂家族。作者进一步通过Scaffold merging的方法来改善结合，具体来说，作者将现有的磺酰胺的最优化头基接到苯基乙酰氨基-环己烷羧酸支架上，以产生一个嵌合配体3CB，该配体对目标位点的结合能力提高了约三个数量级。对3CB-PYL10的晶体结构分析表明，向3CB中添加适当位置的疏水基可能会提高活性。因此，作者合成了一种3CB衍生物，产生了一种名为OPabactin的新的激动剂，用于抑制ABA受体活化。 3CB或OP受体结合反应的热力学特征表明，新生成的支架改进相比于磺酰胺是由焓驱动的，这与晶体结构中观察到的盐桥一致。生物学研究表明，OP在由ABA驱动的种子发芽抑制时的活性比ABA要高出10倍左右。在小麦、番茄、大麦、拟南芥和鸭跖草中的研究证明了在不同物种的营养组织中的生物活性。使用热成像在小麦和番茄中进行的时序试验表明，OP比ABA诱导更加持久的抗蒸腾反应，并且同时证明了这两个物种中磺酰胺激动剂的活性较差。为了知道哪些受体是OP发挥作用所必需的，作者通过拟南芥突变体显示OP需要亚家族III的PYR1、PYL1和PYL2受体才能发挥最大活性。因此，作者的虚拟筛选试验产生了一个作为抗蒸腾剂的ABA受体激动剂。

通讯：Sean R. Cutler (http://www.thecutlerlab.org/2008/05/sean-cutler.html)

个人简介：1991年，多伦多大学，学士；1995年，多伦多大学，硕士；2001年，斯坦福大学，博士。

研究方向：1. 利用化学遗传学鉴定能够调控植物细胞扩张的新的化学物质；2. 利用小分子解析自然变异。

doi: 10.1126/science.aaw8848

Journal: Science

Published date: October 25, 2019