Research Article

Dynamic control of plant water use using designed ABA receptor agonists

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Science  25 Oct 2019:
Vol. 366, Issue 6464, eaaw8848
DOI: 10.1126/science.aaw8848
  • 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.

  • Fig. 1 Virtual screening and scaffold merging enabled the design of overpowered ABA receptor agonists.

    (A) Structures of existing ABA receptor agonists; the gray box highlights the 4-cyano-3-cyclopropylphenyl head group used in our scaffold merging strategy. tABA, tetralone ABA; CB, cyanabactin; QB, quinabactin; R, radical. (B) Structures of representative hits (IC50 ≤ 25 μM) belonging to the different scaffolds identified; the yellow box highlights the amide scaffold that was merged with the cyanabactin headgroup. The complete list of hits identified is provided in fig. S1 and table S1. (C) Binding modes of ABA and hit 3B4, as originally predicted by docking to PYR1; the figure illustrates that 3B4 lacks a hydrogen bond acceptor for the Trp-lock water with thin red lines representing hydrogen bonds from 2.7 to 3.1 Å. (D) Hit discovery and optimization. The ZINC collection of 18 million purchasable drug-like compounds was screened against PYR1 and PYL9 to identify candidate binders that interact with K59. Of the top 10,000 hits, 1700 were obtained and tested for activity in vitro. The best hit obtained, 3B4, lacked an H-bond acceptor for the Trp lock. We therefore installed the cyanabactin head group on the 3B4 amide scaffold. A subsequent structure of 3CB-PYL10 suggested that bioactivity could be improved through the addition of an additional hydrophobe to interact with the receptors’ 3′ tunnels, which yielded the agonist opabactin (OP). IC50 values shown on the right (in red) are for the compounds tested against subfamily III receptor PYL2 (see Fig. 2 and fig. S2 for complete IC50 data). Synthetic schemes for the synthesis of 3CB and OP are provided in fig. S2.

  • Fig. 2 3CB and OP are best-in-class ABA receptor agonists.

    (A) Agonist potency against different Arabidopsis receptors, as measured using agonist/receptor-mediated inhibition of ∆N-HAB1 phosphatase activity (n = 3 replicates). A dash indicates that PP2C activity was >75% control at 50 μM test chemical (the highest concentration tested). The colored boxes indicate the subfamily membership of the receptors assayed. (B) Agonist potency against different wheat receptors, using indicated receptors and TaPP2C. A dash indicates that PP2C activity was >75% control at 10 μM test chemical (the highest concentration tested). We note that TaPYL9 is a pseudoreceptor with an atypical binding pocket. (C) OP shows an ~10-fold increase in in vivo potency relative to ABA. The potency of different agonists on Arabidopsis seed germination and their corresponding IC50 values (concentrations required to inhibit germination by 50%). Photographs were taken 4 days post stratification (n = 6 replicates). The full-dose response curves used to infer IC50 values for all three figure panels are shown in figs. S3 and S4 and tables S2 and S3.

  • Fig. 3 Structure of 3CB bound to PYL10.

    (A) Cocrystal structure of 3CB bound to PYL10, dashes representing hydrogen bonds from 2.6 to 3.3 Å. (B) Differences in orientation of the cyclopropyl group present in the 3CB:PYL10 structure versus the CB:PYR1 structure (PDB ID 5UR5). (C) Positioning of H111 and L159 in PYL10 relative to 3CB’s 3-cyclopropyl-4-cyanobenzyl headgroup. This illustrates the site of predicted steric clash that would occur between L159 and OP’s C5 cyclopropyl substituent when bound to Arabidopsis subfamily I receptors. (D) Sequence variation at the selectivity-determining residue (homologous to PYL10 L159), which is the only residue lining the 3′ tunnel that is variable between ABA receptors). (E) Genetic evidence that sequence variation at the selectivity-determining residue (L159 in PYL10) determines OP selectivity. V163 in PYR1 is homologous to L159 in PYL10. To test the role of this residue in OP selectivity, wild-type PYR1, V163I, and V163L were tested for interactions with HAB1 using an established yeast two-hybrid assay (chemicals tested at 25 μM); the bulkier leucine substitution corresponding to that present in Arabidopsis subfamily I receptors blocks OP action. Single-letter abbreviations for the amino acid residues are as follows: E, Glu; F, Phe; H, His; I, Ile; K, Lys; L, Leu; N, Asn; V, Val.

  • Fig. 4 The high binding affinities of 3CB and OP are enthalpy driven, in contrast to the entropy-driven binding of sulfonamides.

    (A) Measured thermodynamic parameters of binding of agonists to PYR1 in the presence or absence of HAB1; the top two rows are previously published observations, and all the rest were determined in this study. The bar graphs summarize the ITC data and illustrate that sulfonamide agonist binding is driven primarily by entropy, whereas the 3CB and OP binding are more enthalpically driven. (B) Representative thermogram and binding isotherm. ITC experiments were conducted by repeated injections of 2.5 μl of OP (60 μM) into a 1:1 mixture of PYR1:∆N-HAB1 (10 μM). Binding isotherms were fitted to an independent one-site binding model using NanoAnalyze software (TA Instruments, USA); n = 3 replicate experiments, standard deviations shown. The red trace is a control of buffer injections (rather than buffer + ligand) into PYR1–∆N-HAB1. The data are consistent with high-affinity binding of OP to the receptor complex; however, precise Kd values in the low nanomolar range are challenging to deduce.

  • Fig. 5 OP has bioactivity and long-lasting effects in tomato and wheat, whereas sulfonamide agonists do not.

    (A) Infrared images of 6-week-old tomato seedlings (UC82) treated with 100 μM of different chemical solutions composed of 0.5% dimethyl sulfoxide (DMSO) and 0.05% Silwet-77. Graphs depict the variation in difference in temperature levels of treated plants relative to mock-treated plants up to 5 days after application (n = 18 replicates for mocks and n = 6 for chemical treatment). (B) Infrared images of 4-week-old wheat seedlings (WB-9229) sprayed with 11 μM of different chemicals solution composed of 0.5% DMSO and 0.05% Silwet-77. Graphs depict the variation in difference in temperature levels of treated plants, relative to mock-treated plants up to 5 days after application. Statistical analyses were conducted in R using a one-way analysis of variance (ANOVA) and post-hoc Dunnett tests to obtain multiplicity adjusted P values for treatment effects relative to mock-treated controls (n = 10 replicates for mocks and n = 5 for chemical treatment). 95% confidence intervals are shown.

  • Fig. 6 OP enhances drought tolerance and hyperactivates ABA transcriptional responses in wheat.

    (A) Effects of ABA receptor agonists in wheat. Two-week-old plants were sprayed with chemical solution containing 0.05% Tween-20 and 50 μM compounds. After 3 days of water deprivation, plants were rewatered, and stress recovery images were acquired after 12 hours. (B) Chlorophyll content of aerial parts of plants after water stress treatments was measured as described in the methods. (C to E) Comparison of transcript levels for TaPP2C6, TaLea, and TaAOS induced by individual ligands in 7-day-old wheat seedlings after a treatment of 50 μM for 12 hours. Statistics were done using a one-way ANOVA and post-hoc Dunnett tests to obtain multiplicity adjusted P values for treatment effects relative to mock-treated controls, with n = 5 replicates for chlorophyll measurements and n = 4 replicates for qRT-PCR studies. Error bars indicate standard error of mean, individual P values are shown on the graph. n.s., nonsignificant.

  • Fig. 7 OP acts primarily through subfamily III receptors and demonstrates that PYL2 contributes to OP’s antitranspirant activity.

    (A) Effects of different ABA receptors agonists on seed germination in wild type and different receptor mutant lines. Chemical effects were tested at concentrations approximately two times their IC50 values (Fig. 2C) to normalize for differences in agonist potency (ABA, 1200 nM; QB, 1800 nM; AMF4, 1600 nM; 3CB, 440 nM; and OP, 120 nM). Photographs were taken after 4-day incubation under constant illumination. (B) Adult Col-0 and different mutant lines were sprayed with 50 μM compounds and imaged by thermography 24 hours after treatment and (C) average leaf temperatures for each treatment were quantified. Statistics done using a one-way ANOVA and post-hoc Dunnett tests to obtain multiplicity adjusted P values for treatment effects relative to mock-treated controls (n = 8 replicates). Error bars indicate standard error of mean. Individual P values for comparisons to mock-treated controls are shown on the graph.

Supplementary Materials

  • Dynamic control of plant water use using designed ABA receptor agonists

    Aditya S. Vaidya, Jonathan D. M. Helander, Francis C. Peterson, Dezi Elzinga, Wim Dejonghe, Amita Kaundal, Sang-Youl Park, Zenan Xing, Ryousuke Mega, Jun Takeuchi, Bardia Khanderahoo, Steven Bishay, Brian F. Volkman, Yasushi Todoroki, Masanori Okamoto, Sean R. Cutler

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S9
    • Tables S1 to S6
    • NMR Spectra
    • References 
     
    Correction (9 December 2019): In fig. S7A, the labels on the color scale bar were inadvertently switched. The quantification shown is not affected. This transposition has been corrected.
    The original version is accessible here.

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