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

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 <i>Pyrabactin Resistance 1</i> (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 <i>Arabidopsis</i> whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its <i>in vitro</i> potency translates to ABA-like activity <i>in vivo</i>. 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

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

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 <i>Pyrabactin Resistance 1</i> (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 <i>Arabidopsis</i> whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its <i>in vitro</i> potency translates to ABA-like activity <i>in vivo</i>. 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

Plant development is affected in several <i>crk</i> mutants.

<p><b>(A)</b> Representative pictures of 17-day old seedlings of Col-0 wild type and <i>crk2</i>. Complementation of <i>crk2</i> with 35S::<i>CRK2-CDS</i>:YFP rescued the growth defect of the mutant. Plants were grown under the following conditions: 250 μmol m^-2 s^-1 light intensity under 12 h-day length (day: 23°C, 70% relative humidity; night: 18°C, 90% relative humidity). Bar = 1 cm. Pictures are representative of three independent experiments. <b>(B)</b> A selection of <i>crk</i> mutant lines showing earlier senescence compared to Col-0 wild type. Results are means ± SE (<i>n</i> = 8). <b>(C)</b> Several <i>crk</i> mutants flowered earlier compared to wild type while <i>crk2</i> flowered later. Results are means ± SE (<i>n</i> = 8). <b>(D)</b> Time course analysis of endosperm rupture showed delayed germination in several <i>crk</i> mutants compared to wild type. Results represent means from three independent biological experiments (<i>n</i> = 30). Testa and endosperm rupture were assessed every 5 hours up to 51 hours of imbibition. A seed was considered as germinated when the radicle protruded through both envelopes. <b>(E)</b> Several <i>crk</i> mutants exhibit a lower pavement cell density (number of pavement cells / mm²) in cotyledons. Results are means ± SE (<i>n</i> = 15). <b>(F)</b> Three <i>crks</i> showed slightly longer roots compared to wild type (measured eight days after stratification). Results are means ± SE (<i>n</i> = 16). (<b>B-F)</b> Differences between mutants and Col-0 wild type were compared and analysed using one-way-ANOVA (<i>post hoc</i> Dunnett, asterisks indicate statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001) for <b>(B, C, E</b>) and linear model with single step p-value adjustment (<b>F</b>). All experiments were repeated three times with similar results.</p

Immunity to bacterial pathogens is impaired in <i>crk</i> mutants.

<p><b>(A)</b> ROS production was enhanced in several <i>crks</i> compared to Col-0 wild type after elicitation with 100 nM flagellin (flg22) in 4 week-old leaves. Data show the percentage of the mean of the total RLU (relative light units) to Col-0 ± SE (<i>n</i> = 24). Asterisks indicate differences between <i>crks</i> and Col-0, statistical significance *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (one-way ANOVA <i>post hoc</i> Dunnett). <b>(B)</b> A subset of <i>crks</i> was more susceptible to <i>Pto</i> DC3000 (spray infection of 2-week old seedlings at 10⁸ cfu ml^-1) compared to Col-0. Disease symptoms were scored 3 days post inoculation: 0, no symptom; 1, one symptomatic cotyledon; 2, two symptomatic cotyledons; 3, dead seedling. Results are means ± SE (<i>n</i> = 48). Asterisks indicate differences between <i>crks</i> and Col-0, statistical significance *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (Mann-Whitney test, Benjamini-Hochberg correction for multiple comparisons). <b>(C)</b> Some <i>crk</i> mutants are impaired in stomatal closure after 2 h treatment with 10 mM flg22. Results are presented as mean of stomatal aperture ratio (width/length) after treatment compared to pre-treatment values in percentages ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control treatment at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (linear model, single-step p-value adjustment). <b>(D)</b> Stomatal apertures were measured 2 h after chitin treatment. Stomatal closure is impaired in several <i>crk</i> mutants after treatment with chitin (1 g l^-1) for two hours (five selected mutants are shown). Results are means of % stomatal aperture ratio (width/length) after treatment ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control treatment at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (linear model, single-step p-value adjustment). All experiments were repeated three times with similar results.</p

Phylogenetic clustering of the <i>Arabidopsis thaliana</i> CRK group of RLKs and summary of the <i>crk</i> T-DNA insertion collection.

<p><b>(A)</b> The coding region of the CRKs of <i>Arabidopsis thaliana</i> (including the truncated <i>CRK9 At4g23170</i> and the putative pseudogene <i>CRK35 At4g11500</i>) was aligned using Muscle. The maximum-likelihood phylogenetic tree was estimated in MEGA6 using all sites (no gap penalty). The initial guide tree was constructed using maximum parsimony. Values at branch nodes represent bootstrap values (1000 replicates). CRK43 (At1g70740), CRK44 (At4g00960) and CRK45 (At4g11890) lack signal peptide, CRK ectodomain (ED) and transmembrane domain. <b>(B)</b> Information on T-DNA insertion lines for corresponding <i>crk</i> mutants is summarized: location of the T-DNA insertion in the gene (detailed information in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s003" target="_blank">S3 Fig</a>), number of T-DNA insertions per line (determined by quantitative PCR; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>) and transcript level of the corresponding <i>crk</i> mutant (according to semi-quantitative RT-PCR and qPCR; detailed information in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>). For two additional <i>crk10</i> alleles (<i>crk10-1</i> and <i>crk10-3</i>) information can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>.</p

Scatter plots of stomatal regulation in <i>crk</i> mutants.

<p>The <i>crk5</i> was insensitive to all studied stimuli, whereas <i>crk31</i> was particularly insensitive to O₃. The lines <i>crk19-1</i> and <i>crk22</i> were more sensitive to the analysed stimuli. <b>(A)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to O₃ (x-axis) and CO₂ (y-axis). (<b>B</b>) Scatter plot of stomatal responses of <i>crk</i> mutants to O₃ (x-axis) and darkness (y-axis) (<b>C</b>) Scatter plot of stomatal responses of <i>crk</i> mutants to CO₂ (x-axis) and darkness (y-axis). Dashed lines indicate the cut-off for reduced, normal, and high response with respect to Col-0. Grey dashed lines show regression fit with correlation (R), coefficient of determination (R²) and significance reported in lower right corner in each plot. Reduced or increased responses were statistically significant in the majority of mutants (see respective barplots in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s017" target="_blank">S17</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s018" target="_blank">S18</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s019" target="_blank">S19</a> Figs).</p

Integrated cluster analysis of <i>crk</i> mutant phenotypes.

<p>An age-matched collection of T-DNA insertion lines in <i>CRK</i> genes was analyzed for developmental and stress-related phenotypes. <b>(A)</b> Analysis of developmental phenotypes of <i>crk</i> mutant lines: senescence, germination (endosperm rupture), epidermal cell segmentation, bolting, flowering, and root length. <b>(B)</b> Analysis of abiotic stresses phenotypes of <i>crk</i> lines: germination of <i>crk</i> lines on medium containing NaCl, cell death (measured by electrolyte leakage) in response to Xanthine-Xanthine Oxidase (X+XO), ultraviolet light (UV-AB), ozone (O₃), or light stress. <b>(C)</b> Analysis of photosynthesis responses upon treatment with DCMU or methyl viologen (MV). <b>(D)</b> Pathogen phenotypes. ROS production in response to treatment with the bacterial elicitor flagellin (flg22). Stomatal aperture ratio in response to flg22 and chitin treatments and <i>crk</i> susceptibility to the hemibiotrophic bacterial pathogen <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (<i>Pto</i> infection) or the biotrophic fungal pathogens <i>Golovinomyces orontii</i> (<i>Go</i>) (virulent on <i>Arabidopsis</i>) or <i>Blumeria graminis</i> f.sp. <i>hordei</i> (<i>Bgh</i>, a barley pathogen, non-pathogenic on <i>Arabidopsis</i>). <b>(E)</b> Analysis of stomatal parameters: fresh weight (for determination of water loss), density, length, aperture, stomatal aperture in response to ABA treatment, steady state stomatal conductance, stomatal closure in response to elevated CO₂, O₃, and darkness. Experiments were made comparable by bootstrap sampling to <i>n</i> = 15 followed by averaging over bootstrap estimates. Red and blue indicate statistically significant increase or decrease in response compared to Col-0 wild type, respectively, while white indicates a response that is similar to wild type Col-0. The intensity of color is proportional to the Benjamini-Hochberg false discovery rate (FDR) adjusted Z statistic which takes the estimated means and their variation into account. As a rough guideline, |Z|>1.67 corresponds to a FDR<10% (shown with light hue), and |Z|<2.6 to a strong FDR<1% (intense color). White: non-significant response; grey: not measured. A corresponding plot displaying the adjusted Z statistics without thresholding is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s025" target="_blank">S25 Fig</a>.</p

Scatter plots for stomatal regulation in <i>crk</i> mutants.

<p><b>(A)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to ABA (x-axis) and flagellin (flg22; y-axis). <b>(B)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to ABA (x-axis) and chitin (y-axis). <b>(C)</b> Scatter plot of stomatal responses of <i>crk</i> mutants to chitin (x-axis) and flg22 (y-axis). Black dashed lines indicate the cut-off for reduced, normal, and high response with respect to Col-0. Grey dashed lines show regression fit with correlation (R), coefficient of determination (R²) and significance reported in lower right corner in each plot. Reduced or increased responses were statistically significant in the majority of mutants (see respective barplots in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s015" target="_blank">S15A</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s023" target="_blank">S23</a> Figs).</p

Stomatal development and responses are impaired in specific <i>crks</i>.

<p><b>(A)</b> A subset of the <i>crk</i> mutants showed altered water loss (shown as decrease of fresh weight) compared to Col-0 wild type plants after detachment of shoots from roots as evaluated from rosette weight. Complementation of the <i>crk2</i><b>(B)</b>, <i>crk5</i><b>(C)</b> or <i>crk45</i><b>(D)</b> mutants restored a wild type-like water loss phenotype as interpreted from decrease of fresh weight of excised rosettes. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results. <b>(E)</b> Stomatal apertures were measured 2 h after abscisic acid (ABA) treatment. Some <i>crk</i> mutants are impaired in stomatal closure 2 h after treatment with 10 μM ABA. Results are means of % stomatal aperture ratio (width/length) ± SE (average number of stomata measured = 250). Asterisks indicate statistical significance between control and ABA treatment at *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 (linear model, single-step p-value adjustment). Lowercase letters indicate statistical significance between wild type Col-0 and <i>crk</i> mutant at <i>P</i><0.05 (a), <i>P</i><0.01 (b) and <i>P</i><0.001 (c) according to one-way ANOVA with <i>post hoc</i> Dunnett’s test. <b>(F)</b> Stomatal density (number of stomata/mm²) is correlated with stomatal length (μm). Most of the <i>crks</i> exhibit a smaller stomata density which correlates with longer stomata (Pearson correlation -0.69, p-value = 0.04). Results are means (average number of stomata measured = 500). <b>(G-I)</b> Time courses of stomatal conductance (relative units) in response to a 3 min pulse of 500–600 ppb of O₃ (<b>G</b>), darkness (<b>H</b>) and elevation of CO₂ from 400 ppm to 800 ppm <b>(I)</b> in a subset of <i>crk</i> mutants and Col-0. Stimuli were applied at 0 time point, which is indicated by an arrow; pre-treatment stomatal conductance was used for normalization. Graph shows the mean of two experiments (<i>n</i> = 6). (<b>J</b>) Overexpression of CRK5 led to lower stomatal conductance compared to Col-0 wild type. (<b>K</b>) Complementation of the <i>crk5</i> mutant restored wild type-like phenotype in the response to a 3-min pulse of 500–600 ppb of O₃, darkness, and elevating CO₂ from 400 to 800 ppm. Asterisks indicate differences between <i>crk</i> mutants or complementation lines and Col-0 with statistical significance at *<i>P</i><0.05 according to one-way ANOVA with <i>post hoc</i> Tukey HSD. The experiment was repeated three times with similar results.</p

Phenotypic analysis of the <i>Arabidopsis thaliana</i> CRK protein family.

<p>A T-DNA insertion collection for the CRK family was compiled and subjected to phenotyping addressing aspects of plant development, biotic and abiotic stress responses, photosynthesis as well as stomatal regulation. Length of red and blue bars in the five phenotyping sections is representative of the number of <i>crk</i> lines found to have phenotypes in the thematic area. Information about the sections in the pie chart is displayed in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005373#pgen.1005373.s026" target="_blank">S1 Table</a>. The red outline in the pie chart highlights the lines included in the analyses and figures throughout the manuscript. The gray scale bar serves as a reference for comparison. The length of the scale bar corresponds to ten lines.</p