a novel chemical inhibitor of aba signaling targets all

A Novel Chemical Inhibitor of ABA Signaling Targets AllABA Receptors1

Yajin Ye2 Lijuan Zhou2 Xue Liu Hao Liu Deqiang Li Minjie Cao Haifeng Chen Lin XuJian-kang Zhu and Yang Zhao

Institute of Plant Physiology and Ecology (Y-JY L-JZ D-QL LX YZ) and Shanghai Center for PlantStress Biology (XL M-JC J-KZ) Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China University of the Chinese Academy of Sciences Beijing 100000 China (Y-JY L-JZD-QL) Faculty of Life Science and Technology Kunming University of Science and Technology Kunming650000 China (YZ) Department of Horticulture and Landscape Architecture Purdue University WestLafayette Indiana 47907 (J-KZ) and State Key Laboratory of Microbial Metabolism Department ofBioinformatics and Biostatistics College of Life Sciences and Biotechnology Shanghai Jiaotong UniversityShanghai 200032 China (XL H-FC)

ORCID IDs 0000-0003-0107-5648 (L-JZ) 0000-0003-4718-1286 (LX) 0000-0001-8697-5148 (YZ)

Abscisic acid (ABA) the most important stress-induced phytohormone regulates seed dormancy germination plant senescenceand the abiotic stress response ABA signaling is repressed by group A type 2C protein phosphatases (PP2Cs) and then ABAbinds to its receptor of the ACTIN RESISTANCE1 (PYR1) PYR1-LIKE (PYL) and REGULATORY COMPONENTS OF ABARECEPTORS (RCAR) family which in turn inhibits PP2Cs and activates downstream ABA signaling The agonistantagonistof ABA receptors have the potential to reveal the ABA signaling machinery and to become lead compounds for agrochemicalshowever until now no broad-spectrum antagonists of ABA receptors blocking all PYRPYL-PP2C interactions have beenidentified Here using chemical genetics screenings we identified ABA ANTAGONIST1 (AA1) the first broad-spectrumantagonist of ABA receptors in Arabidopsis (Arabidopsis thaliana) Physiological analyses revealed that AA1 is sufficientlyactive to block ABA signaling AA1 interfered with all the PYRPYL-HAB1 interactions and the diminished PYRPYL-HAB1 interactions in turn restored the activity of HAB1 AA1 binds to all 13 members Molecular dockings the non-AA1-bound PYL2 variant and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2Using AA1 we tested the genetic relationships of ABA receptors with other core components of ABA signaling demonstratingthat AA1 is a powerful tool with which to sidestep this genetic redundancy of PYRPYLs In addition the application of AA1delays leaf senescence Thus our study developed an efficient broad-spectrum antagonist of ABA receptors and demonstratedthat plant senescence can be chemically controlled through AA1 with a simple and easy-to-synthesize structure allowing itsavailability and utility as a chemical probe synthesized in large quantities indicating its potential application in agriculture

The phytohormone abscisic acid (ABA) controlsmany vital plant physiological processes such as seeddevelopment seed dormancy and germination adap-tive responses toward environmental stresses such asdrought and salinity fruit ripening and leaf senescence

(Fujita et al 2006 Cutler et al 2010) The downstreamfactors of ABA signaling have been establishedincluding group A type 2C protein phosphatases(PP2Cs) SNF1-related kinases2 (SnRK2s) and down-stream effector proteins including transcription factorssuch as ABI3 ABI4 and ABI5 (Zhu 2002) In the ab-sence of ABA PP2Cs bind and physically block theactivity of SnRK2s through dephosphorylation keep-ing downstream effector proteins inactive ABA re-ceptors have been obscure for many years because ofgenetic redundancy In 2009 ABA receptors which aremembers of the START superfamily of ligand-bindingproteins were identified by chemical genetics and yeasttwo-hybrid screening (Ma et al 2009 Park et al 2009)The receptor family contains 14 members defined asPYRABACTIN RESISTANCE1 (PYR1) PYR1-LIKE(PYL) or REGULATORY COMPONENTS OF ABARECEPTORS (RCAR) The binding of ABA to PYRPYLRCAR (PYRPYL) induces physical interactionsbetween PYR1PYLs and PP2Cs which in turn inhibitPP2C activity and hence relieve SnRK2 to activate

1 This work was supported by the National Natural Science Foun-dation of China (grant nos 31171293 and 31371361 to YZ) and theChinese Academy of Sciences (to YZ and J-KZ)

2 These authors contributed equally to the article Address correspondence to zhu132purdueedu or yangzhaotoronto

sinacomThe author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (wwwplantphysiolorg) isYang Zhao (yangzhaotorontosinacom)

Y-JY L-JZ and YZ conceived the project Y-JY L-JZJ-KZ and YZ designed the research Y-JY L-JZ XL HLM-JC D-QL H-FC LX J-KZ and YZ performed the re-search andor analyzed data Y-JY L-JZ J-KZ and YZ wrotethe article

wwwplantphysiolorgcgidoi101104pp1601862

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Copyright copy 2017 American Society of Plant Biologists All rights reserved wwwplantphysiolorg on May 10 2017 - Published by wwwplantphysiolorgDownloaded from











Copyright copy 2017 American Society of Plant Biologists All rights reserved

downstream effectors to cause physiological responsesto ABA (Fujii et al 2009)Structural studies highlighted a conserved gate-

latch-lock mechanism underlying ABA perceptionand signal transduction (Melcher et al 2009 Miyazonoet al 2009 Nishimura et al 2009 Santiago et al 2009Yin et al 2009) In Arabidopsis (Arabidopsis thaliana)PYRPYLs are classified into two distinct categoriesdimeric receptors (PYR1 and PYL1ndashPYL3) and mono-meric receptors (PYL4ndashPYL12 Okamoto et al 2013)The dimeric receptors interact with and inhibit PP2Cactivity in an ABA-dependent manner The dimericreceptors possess an ABA-binding pocket flanked by agate and a latch loop and the apo-form receptors are inan open conformation permitting access of ABA to thebinding pocket (Melcher et al 2009) Upon ABAbinding the dimeric receptors close the gate which inturn creates the interaction surfaces that permit thedocking of PP2Cs onto the ABA-bound receptors TheABA-induced receptor-PP2C interaction induces a newconformation change of the protein complex whichlocks the gate of the receptors (Melcher et al 2009) Incontrast monomeric receptors are in equilibrium be-tween the gate-opened and gate-closed conformationsin the absence of ABA Thus monomeric receptors caninteract with and inhibit PP2C activity in an ABA-independent manner (Hao et al 2011 Sun et al 2012)The distinct roles of dimeric and monomeric receptorsin ABA signaling remain elusive However recent re-ports demonstrated that the selective chemical activa-tion of dimeric receptors could induce a full ABAresponse which indicated that dimeric receptors arekey factors in ABA signaling (Cao et al 2013 Okamotoet al 2013)Small molecules functioning as agonists or antago-

nists of ABA receptors promote the discovery of ABAreceptors and deepen our knowledge of the receptorsrsquomechanisms of action Pyrabactin the first identi-fied ABA receptor-related small molecule functionsas an agonist of PYR1 and PYL1 and as a weak antag-onist of PYL2 (Park et al 2009 Melcher et al 2010)Besides pyrabactin quinabactin (also known asAM1) working as a selective agonist toward dimericABA receptors is a promising agrochemical thatelicits stomatal closure and enhances crop droughtresistance (Cao et al 2013 Okamoto et al 2013) Inaddition mandipropamid a commercial agrochemicalfunctions as a selective agonist of engineered PYR1(Park et al 2015 Rodriguez and Lozano-Juste 2015)ABA antagonists have been identified and designedbefore (Kim et al 2011 Takeuchi et al 2014 Ito et al2015) DFPM a small molecule identified by chemicalgenetics screening down-regulates ABA-dependentgene expression and also inhibits ABA-induced sto-matal closure through the plant immune responsepathway and its target is still unclear (Kim et al 2011)Small molecules AS6 and RK460 are two antagonists ofABA receptors (Takeuchi et al 2014 Ito et al 2015)AS6 was designed according to the ABAPYR1 x-raystructure Because of the structural similarity of ABA

AS6 is not an entire PRYPYL antagonist which isrevealed by its intrinsic agonist activity toward ABAreceptors In additionAS6 functions as an antagonist notto all the PYRPYL-PP2C interactions The complexstructure of AS6 may result in a high production costand its chemical synthesis is complex limiting its avail-ability and utility as a chemical probe synthesized inlarge quantities (Takeuchi et al 2014) RK460 is an an-tagonist toward PYR1 but not other PYRPYLs whichcannot bypass the genetic redundancy of ABA receptorsand this may explain why RK460 has no antagonist ac-tivity to ABA-induced physiological processes (Ito et al2015)

The functional characterization of PYRPYL mem-bers using conventional molecular genetic approacheswould be daunting because of their genetic and func-tional redundancy In this situation a small moleculeacting as a broad-spectrum antagonist toward all ABAreceptors could be a powerful tool to sidestep this ge-netic redundancy Such an ABA antagonist is particu-larly needed for ABA signaling studies in other plantsystems with less available genetic resources More-over it has been reported that some pathogens such asPseudomonas syringae could hijack the ABA signalingpathway for their survival and cause plant disease(Fujita et al 2006) ABA is thought to be one of thephytohormones that promote leaf senescence ABAlevels can be observed in many plants during leaf se-nescence (Tan et al 2003) ABA induces expression ofseveral senescence-associated genes (SAGs) and yel-lowing of the leaves which are typical phenomena as-sociatedwith leaf senescence (Liang et al 2014)Whatrsquosmore a new study showed that overexpression of PYL9can promote Arabidopsis senescence via the core ABAsignaling pathway (Zhao et al 2016) Up-regulation ofgenes associated with ABA signaling and a dramaticincrease in endogenous ABA promote fruit ripeningand leaf senescence indicating a promising agriculturalapplication of ABA antagonist (Lim et al 2007)

Here we report a novel ABA antagonist AA1 whichwas uncovered using our plant chemical geneticsscreening using commercial compound libraries AA1releases ABA-induced seed germination inhibition andstomatal closure Biochemical assays demonstrated thatAA1 inhibits the interactions between all 13 PYRPYLsand PP2C by direct binding to PYRPYL which isa specific feature of all the PYRPYL-related smallmolecules Most importantly AA1 down-regulatessenescence-related gene expression and inhibits chlo-rophyll breakdown and thus delays plant leaf senes-cence which may provide some hints for how to applyABA antagonists to agriculture

RESULTS

Identification of an ABA Antagonist

To identify small molecules that inhibit ABA signal-ing we developed a chemical screen that relied on theseed-inhibitory effects of ABA (Zhao 2012 Ye et al

Plant Physiol Vol 173 2017 2357

Small Molecule AA1 Targets All ABA Receptors

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2016) Arabidopsis wild-type Columbia-0 (Col-0) seedswere grown in Murashige and Skoog (MS) mediumwith 07 mM ABA on 96-well microplates and thenscreened against 12000 structurally novel and diversesynthetic small molecules The resulting phenotypesafter 5 d of growth allowed the identification of onesmall molecule that could relieve the inhibitory effectsof ABA on seed germination and the chemical wasdesignated as AA1 (Fig 1 AndashC) We then assayed theeffects of AA1 against the effect of ABA on seed ger-mination by testing different ABA concentrationsThe results showed that 100 mM AA1 could reversethe effect of 50 mM ABA on seed germination (Fig 1Supplemental Fig S1) Next we determined the 50inhibitory concentration of AA1 by testing the activitiesof different concentrations of AA1 against 1 mM ABAand the result showed that the 50 inhibitory concen-tration of AA1 is 2546mM (Fig 1 A and B) Importantlythe suppression of the effect of ABA by AA1 was con-served in rice (Oryza sativa) and maize (Zea mays)which are monocotyledonous plants and this indicatesthat the effect of AA1 is not restricted to dicotyledonousplants (Supplemental Fig S1)

ABA controls stomatal aperture and transpirationrate which is one of its critical physiological roles thatcan be measured indirectly through the leaf tempera-ture (Kim et al 2010 Gonzalez-Guzman et al 2012)The leaf surface temperature increases when stomataclose because of reduced evaporative cooling TheABA-treated leaves displayed elevated leaf tempera-tures this did not occur when the leaves were treatedwith AA1 at the same time (Fig 1D) More importantlyAA1-treated leaves showed a decreased leaf tempera-ture in an AA1 dose-dependent manner indicating thatAA1 can function in guard cells and can antagonizeendogenous ABA (Fig 1D) Consistent with the factthat AA1 increased transpiration (Fig 1D) the AA1-treated leaves lost water more quickly (Fig 1E) Highsalt concentration induces ABA biosynthesis andhence inhibits seed germination (Gonzalez-Guzmanet al 2012) To examine the effect of AA1 on high-saltresponses Arabidopsis seeds were sown on MS me-dium containing 200 mM NaCl or AA1 plus NaCl Asshown in Supplemental Figure S1 the AA1-treatedseeds effectively resisted high salt Collectively AA1as an effective ABA antagonist functions in ABA-mediated seed germination and growth regulationWhatrsquosmore AA1 could be a useful tool for the study ofABA signaling in both monocots and dicots

AA1 Inhibits ABA-Responsive Gene Expression

ABA signal transduction results in the up-regulationof stress-response genes (Cao et al 2013) To charac-terize whether AA1 may affect ABA-induced gene ex-pression we detected the expression levels of RD29band RAB18 in seedlings under different chemicaltreatments These two genes were dramatically acti-vated by ABA treatment but their expression was

reduced when the seedlings were cotreated with AA1and ABA (Fig 1F) ABA-induced stress-response geneexpression also can be monitored in transgenic plantscontaining a firefly luciferase (LUC) reporter driven bythe RD29a promoter containing the DREC-repeat andABRE elements (Ishitani et al 1997 Cao et al 2014)The resulting transgenic plants emit bioluminescence inresponse to cold osmotic stress or exogenous appli-cation of ABA (Ishitani et al 1997) ABA treatmentenhanced the expression of RD29A-LUC whereas AA1reversed the effect of ABA in a dose-dependent manner(Fig 1G) Therefore our physiology and gene expres-sion results show that AA1 is a promising ABA antag-onist for the dissection of the ABA signaling pathway

AA1 Interferes with All the Interactions between PYRPYLand PP2C to Release PP2C Activity

To test whether the antagonist activity of AA1 resultsfrom inhibited ABA biosynthesis we measured theendogenous ABA concentration The result showedthat AA1 treatments had no effect on endogenous ABAcontent (Supplemental Fig S2) In addition weassessed the effects of AA1 on ABA transportation Weassessed ABA uptake inmesophyll protoplasts Indeeddifferent concentrations of AA1 had no effect on theuptake of ABA into protoplasts (Supplemental Fig S2)Taken together the antagonist activity of AA1 is notdue to the blocking of ABA biosynthesis or ABAtransportation

The selective agonist activities of pyrabactin andquinabactin result from their ability to act as ABAfunctional analogs to induce PYRPYL-PP2C interac-tions and AS6 a synthetic antagonist is designed to bean antagonist of ABA-dependent PYRPYL-PP2C in-teractions (Park et al 2009 2015 Cao et al 2013Okamoto et al 2013) We tested the possibility that theABA antagonist activity of AA1 results from a pertur-bation of PYRPYL-PP2C interactions Consistent withprevious reports ABA induced PYR1 and PYL1 toPYL4 interactions withHAB1 amember of the groupAsubfamily of plant PP2Cs (Fig 2A) and the monomericreceptors interacted with HAB1 in the absence of ABAAs expected AA1 blockedABA-dependent interactionsof PYR1 and PYL1 to PYL4 with HAB1 (Fig 2ASupplemental Fig S2) In addition AA1 attenuatedABA-independent PYRPYL-HAB1 interactions evenin the absence of ABA (Fig 2A Supplemental Fig S2)indicating that AA1 is possibly a potent broad-spectrum antagonist of PYRPYL-PP2C interactionsUp to 100 mM AA1 could completely inhibit the inter-actions between PYR1 and PYL1 to PYL4 with HAB1induced by 2 mM ABA However at this concentrationAA1 could not completely suppress the interactionsbetween PYL5 to PYL12 and HAB1 in yeast cells (Fig2A Supplemental Fig S2) However AS6 the designedantagonist of ABA could induce the interactions ofdimeric PYR1PYLs with HAB1 in yeast two-hybridassays which is similar to the effects of ABA on

2358 Plant Physiol Vol 173 2017

Ye et al


PYR1PYL interactions (Supplemental Fig S3) Andup to 100 mM AS6 could not block the interactions be-tween monomeric PYLs and HAB1 (Supplemental FigS3) These results may be explained by the fact that AS6also is a weak agonist of ABA receptors and the weak

agonist activity may be a side effect of AS6 Most im-portantly AA1 is a complete antagonist of PYRPYL-PP2C interactions without agonist activity To furthercharacterize the antagonist activity of AA1 for PYRPYL-PP2C interactions we carried out AlphaScreen

Figure 1 AA1 antagonizes the effects of ABA A AA1 relieves the ABA-induced inhibition of seed germinationWild-type (Col-0)seeds were sown on one-half-strength MS medium containing the indicated chemicals The concentrations of chemicals areshown as ABAAA1 (mMmM) Bar = 12 cm B Quantification of the seed germination rates in A (n = 3 error bars = SE) C Mo-lecular structures of AA1 D Leaf surface temperature as assessed by infrared thermal imaging About 1-month-old Arabidopsisleaves were sprayed with the indicated chemicals Leaf temperature was imaged by thermography after 12 h of chemicaltreatment Bar = 4 cm E AA1 promotes water loss Three-week-old Col-0 plants were sprayed with the indicated chemicals Theloss of fresh weight was measured after 12 h of chemical treatment (n = 3 error bars = SE) F Expression of ABA-responsive genesafter chemical treatment (DMSO as mock) Col-0 seedlings were sprayed with the indicated chemicals for 6 h after which geneexpression levels were determined by quantitative real-time reverse transcription-PCR (n = 3 error bars = SE) G AA1 attenuatesABA-induced RD29a expression Five-day-old pRD29-LUC transgenic seedlings were sprayedwith the indicated chemicals andthe photographs were taken after 6-h chemical treatments The concentrations of chemicals are shown as ABAAA1 (mMmM)




assays (Melcher et al 2009 Cao et al 2013) Theseassays showed that AA1 inhibits the interaction inten-sity of dimeric PYL1 and PYL2 with HAB1 and that thisinhibitory activity is dose dependent (Fig 2B) ABAenhances the interaction of the monomeric receptorPYL6withHAB1 this is revealed by a higher number ofphoton counts detected after ABA treatment (Fig 2B)and such an increase in the interaction intensity re-quired a higher dose of AA1 to reverse it (Fig 2B) Insummary AA1 is a broad-spectrum antagonist of PYRPYL-PP2C interactions and AA1 acts on more PYRPYL-PP2C interactions than AS6 which blocks ABA-dependent PYRPYL-PP2C interactions Like ABA theeffects of AA1 on dimeric and monomeric PYRPYL-PP2C interactions display different modes and themode in each type of PYRPYL is conserved

Next the antagonist activity of AA1 was character-ized by examining the way in which it reverses ABA-induced PP2C inhibition through PYRPYL All13 Arabidopsis ABA receptors are activated after ABAbinding and ABA-bound receptors are responsiblefor PP2C inhibition Hence receptor activation can bemonitored through PP2C activity inhibition In thephosphatase assays AA1 failed to activate any of the13 receptors This indicates that unlike pyrabactinwhich selectively activates (eg PYR1 and PYL1) orrepresses (eg PYL2) certain receptors AA1 has nonatural agonist activity (Fig 2C) AA1 could repressall 13 receptors activated by ABA revealed by itsability to reverse ABA-mediated PP2C inhibition tocertain levels (Fig 2C) In addition AA1 displayedunbiased antagonist activity to both dimeric receptors(PYR1 and PYL1ndashPYL3) and monomeric receptors(PYL4ndashPYL12 Fig 2 C and D) which is consistentwith the fact that AA1 interferes with all the interac-tions of 13 receptors with PP2C (Fig 2A SupplementalFig S2) More importantly unlike AS6 or pyrabactinAA1 is a broad-spectrum PYRPYL antagonist with-out intrinsic agonist activity andAA1 functions throughboth dimeric and monomeric receptor-PP2C interac-tions but not dimeric PYRPYL-PP2C interactionslike AS6

Figure 2 AA1 interfereswith PYRPYL-PP2C interactions and PYRPYL-dependent inhibition of PP2C activity A AA1 blocks ABA-dependentPYRPYL-HAB1 interactions in yeast two-hybrid assays Dimeric re-ceptors (PYR1 and PYL1ndashPYL3) and monomeric receptors (PYL4ndashPYL6)were constructed as binding domain fusion proteins HAB1 was fused

with the activation domain The yeast cells were grown on SD medium(-Leu-Trp-His plus 50 mgL X-a-Gal and 5 mM 3-amino-triazole) withthe indicated chemicals for 3 dWorking concentrations of the chemicalswere 2 mM for ABA and 100 mM for AA1 The yeast cells were diluted into110 (01) and 1100 (001) using water B Binding affinity of PYRPYLproteins to HAB1 measured by AlphaScreen assays Dimeric receptors(PYL1 and PYL2) and a monomeric receptor (PYL6) were fused with His-SUMO HAB1was fused with biotin The reactions were conducted withandwithout 100mM ABA and the indicated concentrations of AA1 (n = 3error bars = SE) IC50 Fifty percent inhibitory concentration C AA1 at-tenuates the ABA-dependent inhibition of HAB1 activity via various ABAreceptors Various PYRPYL-HAB1 combinations were incubated withthe indicated chemicals (2 mM ABA 100 mM AA1 or DMSO n = 3 errorbars = SE) D Dose-dependent effects of AA1 on PYRPYL-dependentinhibition of PP2C activity HAB1 activity was tested with dimeric re-ceptors (PYR1 and PYL2) ormonomeric receptors (PYL5 and PYL10)with2mMABA and the indicated concentrations of AA1 (n= 3 error bars = SE)


Ye et al


PYRPYLs Are the Molecular Targets of AA1

Given the results that AA1 antagonized the effects ofABA by interfering with PYRPYL-PP2C interactionsand relieved PP2C activity we speculated that AA1might act as an antagonist of PYRPYL by binding tothem directly To test this hypothesis we used a thermalshift assay (TSA) to quantify the changes in the thermaldenaturation temperature of PYRPYL under variousconditions including ligand-bound and ligand-free sta-tus (Soon et al 2012) ABA-receptor complexes are morethermally stable than apo receptors (PYR1 and PYL1)and the melting temperatures (Tms) are up-regulated inan ABA dose-dependent manner (Fig 3A) For AA1 theTSA dose-response plots showed an AA1 concentration-dependent decrease inDTmofAA1-bound receptors (Fig3A) indicating that AA1 might interact with PYRPYLMore importantly the receptors become more vulnerableupon AA1 binding compared with apo or ABA-boundreceptors this was revealed by the decreased DTm ofAA1-bound receptors Interestingly ABA andAA1 haveopposite effects on the thermostability of PYRPYLTo explore whether AA1 also may interact with

monomeric ABA receptors we measured the DTm ofPYR1H60P the monomeric form of PYR1 with Pro intro-duced at position His-60 (Dupeux et al 2011) The re-sults showed that AA1 interacted with PYR1H60P whichshared a similar DTm trend with PYR1 Monomeric re-ceptors have a higher binding affinity for ABA becausethey do not need a dimer dissociation step (Hao et al2011) Indeed ABA affects the DTm of PYR1H60P abouttwice as strongly as that of wild-type PYR1 and PYL1indicating that the introduction of this mutation leads toa significant increase in ABA binding affinity Howeverthis is not the case for AA1 Figure 3A shows that theDTm of PYR1H60P is higher than that of dimeric PYL1(Fig 3A) and this suggests that the binding of ABA andAA1 to PYRPLY is through different action modesTo further explore the binding of AA1 to ABA recep-

tors we developed a microscale thermophoresis (MST)assay and used it to determine the dissociation constant(Kd) for this binding The MST assays were conductedusing all 13 recombinant receptors including dimericand monomeric forms with a wide range of AA1 con-centrations The results revealed that AA1 bound to allthe receptors tested with different Kd values (Fig 3BSupplemental Fig S4) The acquired Kd values rangedfrom 179 mM (PYR1) to 17 mM (PYL5) and indicated thatPYL5 had the highest binding affinity of all 13 receptorstested (Fig 3B Supplemental Fig S4) AA1 displayedlow affinities to PYR1 PYL6 PYL7 PYL8 PYL9 andPYL10 To compare the binding affinity of AA1 andABA we used MST to measure the binding constant ofABA to two dimeric receptors (PYR1 and PYL2) and twomonomeric receptors (PYL5 and PYL9 SupplementalFig S4) The Kd values wemeasured are close to those inprevious reports (Miyazono et al 2009 Yin et al 2009Hao et al 2011 Sun et al 2012) In terms of Kd ABAprefers monomeric receptors over dimeric receptorsbut this preference was not observed for AA1 (Fig 3B

Supplemental Fig S4) The binding constants of AA1 toPYL2 and PYL5 are smaller than that of ABA Howeverwith regard to PYR1 and PYL10 ABA has strongerbinding affinity Taken together these results demon-strate that AA1 acts as an ABA antagonist by bindingdirectly to all ABA receptors and the binding affinities ofABA and AA1 are different toward given receptors

Molecular Docking of AA1 to PYL2

To understand the molecular basis for the action ofAA1 as an antagonist of PYRPYL we tried our assayon the crystal structure of AA1PYRPYLs but wefailed This may be explained by our TSA whichdemonstrated that AA1 will decrease the stability ofPYRPYLs (Fig 3B) and the addition of AA1 to thePYRPYL crystals could dissolve the crystal

Finally we predicated the structure of the AA1PYL2 complex bymolecular docking using the reportedcrystal structure of PYL2 (Melcher et al 2009) AA1was docked into the closed PYL2 (Protein Data Bank[PDB] no 3KDI) and the open PYL2 (PDB no 3KDH)The free binding energy values of AA1 to open andclosed PYL2were almost the same (Supplemental TableS1) Like ABA AA1 is centered in the ligand-bindingpocket of both closed and open PYL2 (Fig 4A) Inter-estingly AA1 undergoes an induced fit to accommo-date the shape of the ligand-binding pockets of openand closed PYL2 (Fig 4A Supplemental Fig S5)

The carboxylate group of ABA interacts with the innerend of the pocket through hydrogen bonding and thecyclohexene ring of ABA also interacts with the gatelatch loops via hydrogen bonding (Melcher et al 2009)In quinabactinPYL2 and pyrabactinPYL1 complexesthe sulfonamide of quinabactinpyrabactin functionslike this carboxylate on ABA to dock into the inner sideof the ligand-binding sites (Hao et al 2010 Okamotoet al 2013) ABA interacts with some of the same aminoacids of PYL2 asAA1 especially those at the inner end ofthe ligand-binding pocket (Supplemental Tables S2 andS3) In the open PYL2AA1 complex the diazepin-8-oneon AA1 docks into the inner end of the ligand-bindingpocket by interacting with Lys-64 Glu-98 Glu-147and Asn-173 through hydrogen bonding (Fig 4B)Like ABA AA1 also interacts with the latch and gateloops of PYL2 but via different amino acids (Fig 4 Band C) Overall AA1 enters into the ligand-bindingpocket of PYL2 (Supplemental Fig S5)

To confirm our molecular docking results wedetected the binding of AA1 to a PYL2 variant (K64RN173A) Lys-64 and Asn-173 are key amino acids of theligand-binding pocket of PYL2 and these two aminoacids locate in the inner face of PYL2

AA1 enters into the ligand-binding pocket of PYL2and forms hydrogen bonds with Lys-64 and Asn-173(Fig 4C Supplemental Table S2) The mutations ofLys-64 and Asn-173 abolish the binding of ABA toPYL2 (Melcher et al 2009 Nishimura et al 2009) Wetested the binding affinity ofAA1 toPYL2K64RN173A byMST




assays and the results showed that AA1 could not bindto this PYL2 variant (Fig 4D) Next we performedcompetitive binding assays to further confirm that AA1enters into the ABA-binding pocket of PYL2 AA1competed efficiently with [3H]ABA for binding to PYL2in a dose-dependent manner (Fig 4E) These resultsconfirmed our molecular docking results and illustratedthat AA1 enters into the binding pocket of PYL2

Structure-Activity Relationship Study of AA1

The molecular docking results display that thediazepin-8-one group of AA1 enters into the binding

pocket of PYL2 indicating that this is an importantgroup of AA1s We performed a structure-activity re-lationship study to find the core substructure of AA1We tested five AA1 analogs with various structures(Fig 5 A and B Supplemental Fig S6) Analogs 1 and2 have different side chains compared with AA1 andsuch differences did not change the activity of AA1(Supplemental Fig S6) Most importantly the coregroup without any side chains shows similar antag-onist activity to AA1 (Fig 5 A and B SupplementalFig S6) According to the docking modeling theoxygen atom the nitrogen atom of the heptatomicring and the sulfur atom form hydrogen bonds with

Figure 3 PYRPYLs are direct targets of AA1 A The TSA Plots of DTm (degC) are shown for the effects of AA1 and ABA on PYR1PYR1H60P and PYL1 at the indicated chemical concentrations The thermal stability of the chemical-PYRPYL complex shows thatthe ABA-PYRPYL complexes are more stable than the AA1-PYRPYL complexes B MST data for the binding affinity of AA1 toABA receptors Plots show the fraction of dimeric receptors (PYR1 PYL2 and PYL3) and monomeric receptors (PYL4 PYL5 andPYL10) bound to AA1 at each tested AA1 concentration (n = 3 error bars = SE)


Ye et al


PYL2 Consistent with the results of docking theantagonist activity disappeared when the core grouplost these atoms (Fig 5 A and B Supplemental FigS5) These results indicate that the diazepin-8-onegroup which docks into the ligand-binding pocketof PYL2 is necessary for the activity of AA1 and wefound a novel group that functions as the carboxylategroup of ABA and sulfonamide of quinabactinpyrabactin which can dock into the ligand-bindingpocket of ABA receptors (Supplemental Fig S5)

Genetic Relationship Study of AA1 with Core Componentsof ABA Signaling

Multilocus loss-of-function mutants are required forstrong ABA signaling phenotypes (Park et al 2009Gonzalez-Guzman et al 2012) which is due to theextensive redundancy between the receptors and suchgenetic redundancy impedes the genetic study of ABAreceptors with other components of ABA signaling The

ABA core signaling pathway includes receptors PP2CsSnRKs and downstream transcription factors (Fujiiet al 2009) However due to the difficulty of obtainingthe multilocus loss-of-function mutants of ABA recep-tors with other core components the genetic relation-ship study of ABA receptors with other componentshas not been realized yet

AA1 the broad-spectrum PYRPYLRCAR antago-nist could block all 13 members and this feature makesAA1 an effective genetic tool with which to study thegenetic relationships between the core components ofABA signaling Thus we tested the genetic relationship ofABA receptors with other components by treating therelated mutants with AA1 Our results showed that AA1could enhance the insensitivities of all theABA-insensitivemutants we tested including pyr1pyl1pyl2 abi2-1 abi3-8and abi4-1 (Fig 5C Supplemental Fig S6) Such results aresimilar to the fact that abi1 and abi2 could significantlyenhance the resistance of transcription factor mutants(abi3 and abi4) to ABA (Finkelstein and Somerville 1990Finkelstein 1994) Our results and previous studies

Figure 4 Molecular docking of AA1 to PYL2 A Overall structure of the AA1PYL2 complex The docking of AA1 to open PYL2(PDB no 3KDH) and closed PYL2 (PDB no 3KDI) is shown The AA1closed PYL2 complex is in green the AA1open PYL2complex is in blue ABA is in cyan the latch loop of PYL2 is in red and the gate loop is in magenta B Network of ABA (cyan-stickmodel with red oxygen atoms) inside the PYL2-binding pocket The amino acids that form hydrogen bonds with ABA are in redC Network of AA1 (green-stick model with red oxygen atoms blue nitrogen atoms and yellow sulfur atoms) inside the openPYL2-binding pocket The amino acids that form hydrogen bonds with AA1 are in red D Mutations in key ligand-binding pocketresidues of PYL2 compromise AA1 binding as determined by MST (n = 3 error bars = SE) E Competitive binding of [3H]ABA toPYL2 in the presence of AA1 The reactionswere performed in the presence of 01mM [3H]ABA and the indicated amounts of AA1Each assay was replicated three times and the results were normalized relative to no AA1 (n = 3 error bars = SE)




showed that the upstream genetic factors (receptors andPP2Cs) could enhance the phenotypes of downstreamgenetic factors (transcription factors) indicating that theABA signaling pathway is not a simple straightforwardgenetic pathway and the members at each level (13 re-ceptors nine PP2Cs three SnRKs and several transcrip-tion factors) may form complex genetic networks

AA1 Delays Leaf Senescence and Fruit Ripening

Besides its role in seed germination and stress regu-lation ABA is an important regulator in fruit ripening

(Zhang et al 2009 Leng et al 2014 Weng et al 2015)ABA production is gradually elevated to its maximallevel when ripening occurs (Zhang et al 2009) ABAcontent-decreased mutants display shortened fruitripening and the delayed ripening may prolong thestorage time for fresh fruits We tested whether AA1may delay fruit ripening by treating tomato (Solanumlycopersicum) fruits of the green mature stage The ap-plication of ABA accelerated the changes in skin colorcompared with the control (Fig 6A) In contrast tomock and ABA treatment AA1 delayed the ripeningtime of tomato in a dose-dependent manner indicatingits potential application in fruit storage

Figure 5 Structure-activity relationship and genetic study of AA1 A Structures of AA1 analogs B Germination rates of Col-0seeds under different combinations of ABAchemical treatments (n = 3 error bars = SE) The seeds were sown on the indicatedchemical-containing one-half-strength MS plates and the results were collected after 4 d of growth C Germination rates ofdistinct mutants under the indicated chemical treatments Seeds of different backgrounds were sown on the indicated chemical-containing one-half-strengthMS plates and the results were collected after 5 d of growth pyr1pyl1pyl2 abi3-8 and abi4-1 are inthe Col-0 background and abi2-1 is in the Landsberg erecta (Ler) background (n = 3 error bars = SE)


Ye et al


Due to the role of ABA in leaf senescence AA1 as ageneral antagonist of PYRPYLs may delay leaf se-nescence To test this hypothesis we used dark-inducedleaf senescence as a system in which to study the effectof AA1 ABA accelerated the yellowing of detached

Arabidopsis leaves While AA1 had the opposite effectscomparedwithABAAA1 can delay the yellowing of thedetached leaves (Fig 6B) The most striking feature ofleaf senescence is the breakdown of chlorophyll duringchloroplast degeneration The content of chlorophyll in

Figure 6 AA1 delays fruit ripening and leaf senescence AMorphological differences between tomato fruits treatedwith exogenousABAorAA1 andDMSO-treated fruits (Mock) In the greenmature stage tomato fruitswere harvested and treatedwith 05mLper fruitof 20mMABA or 50 or 100mMAA1 Tomato fruitswere treatedwith the indicated chemicals every 4 d for 2weeks B AA1 delays thedark-induced leaf senescence of Arabidopsis Detached leaves from Col-0 were incubated with the indicated chemicals (DMSO asmock 20 mM ABA or 50 or 100 mM AA1) for 5 d in darkness C Chlorophyll content in leaves of Col-0 treated with the indicatedchemicals (n = 3 error bars = SE) D Expression levels of SAG12 in Col-0 leaves treated with the indicated chemicals (n = 3 errorbars = SE) E One-month-old plants were treatedwith the indicated chemicals everyweek for 1 month Photographs were taken after1 month of chemical treatment F AA1 delays dark-induced leaf senescence in Zhonghua 11 rice Detached flag leaves from wild-type plants at the heading stagewere incubatedwith the indicated chemicals (DMSO asmock 20mMABA or 50 or 100mMAA1) for5 d in darkness G Chlorophyll content in leaves of rice treated with the indicated chemicals (n = 3 error bars = SE) H Expressionlevels of Osh36 and Osl85 in rice leaves treated with the indicated chemicals (n = 3 error bars = SE)




ABA-treated leaves was only one-fifth of that of non-chemical-treated leaves (Fig 6B) However AA1 inhibi-ted the breakdown of chlorophyll which was consistentwith the delayed leaf yellowing demonstrating thatchlorophyll degradation proceeded at a significantlyslower pace in the AA1-treated leaves (Fig 6 B and C)SAGs are molecular markers of ABA-induced senes-cence (Zhao et al 2016) Consistent with the delayedyellowing of AA1-treated leaves SAG12 the chloro-phyll degradation-related gene was down-regulatedby AA1 (Fig 6D) Whatrsquos more AA1 also delayedthe age-dependent leaf senescence in Arabidopsis(Fig 6E) AA1 also delayed the senescence of detachedflag leaves of rice in a dose-dependent manner (Fig6F) Similarly the content of chlorophyll in 100 mM

AA1-treated leaves was 3 times greater than that incontrol leaves (Fig 6G) Osh36 and Osl85 the senes-cence marker genes (Liang et al 2014) were down-regulated by AA1 treatment (Fig 6H) It is known thatcytokinin antagonizes ABA and cytokinin potentlysuppresses senescence (Ha et al 2012) To test whetherAA1 delays leaf senescence through cytokinin signalingwe detected the effects of AA1 on cytokinin-responsivegene expression (Bhargava et al 2013) Cytokinin sig-nificantly induced cytokinin-responsive gene expres-sion butAA1 had no such effects (Supplemental Fig S7)These results showed that AA1 delays leaf senescencenot through cytokinin signaling

DISCUSSION

Several small molecules that act as agonists or antag-onists of phytohormone receptors have been identifiedusing a chemical genetics approach and have advancedthe study of plant hormones the indispensable regula-tors in plant biology (Hayashi et al 2008 Park et al2009 Hicks and Raikhel 2012 Meesters et al 2014)Active molecules uncovered through chemical geneticsstudies have provided unique molecular genetic toolswith which to study specific life processes in tissues andtheir development and in general their use is not limitedto a particular species (Hicks and Raikhel 2012) In thisresearch using a high-throughput chemical geneticsscreen we identified a novel ABA antagonist AA1 thatacts on bothmonocots and dicots (Fig 1A SupplementalFig S1) with a potential application in agriculture tohelp solve problems related to stresses such as droughtand salinity (Supplemental Fig S1)

AA1 was demonstrated to act as an ABA antagonistthat targets all 13 PYRPYLs and affects their interac-tions with PP2Cs (Fig 2 A and B Supplemental FigS2) Compared with AS6 and other antagonists wethink AA1 has some advantages (1) AA1 is the firstsmall molecule acting on all PYRPYL-PP2C interac-tions while AS6 acts only on ABA-dependent PYRPYL-PP2C interactions (Figs 2 and 3 SupplementalFigs S2ndashS4) (2) AA1 is the first broad-spectrum ABAreceptor antagonist (3) AA1 has no intrinsic agonistactivity while acting as a weak antagonist (this may be

a side effect of AS6) it could induce the interactions ofPYRPYLs with PP2C The structure of AA1 is noveland we can design more agonists or antagonists ofPYRPYL according to AA1 In addition this noveltymakes AA1 a more powerful tool with which to studyABA receptors (4) To our knowledge for the first timewe have studied the genetic relationships betweenABAreceptors with other components of ABA signaling (5)Also to our knowledge for the first time we havedemonstrated that plant senescence can be chemicallycontrolled through ABA receptors indicating the po-tential application of the antagonist of ABA receptorsWe believe that this is a major advance in agricultureMost importantly for agricultural application thestructure of AA1 is simple and easy to synthesize atlower cost allowing its availability and utility as achemical probe synthesized in large quantitiesmaking ita very promising agrochemicalwhich is a big advantageover AS6 Previous reports showed the cocrystal struc-ture of AS6-PYL2 and the observations provided directevidence that like the agonist AS6 induced the gate-closed conformation of PYL2 leading to a more stablecomplex AS6 has intrinsic antagonist activity allowingthe conformation change of PYL2 tomake it more stablewhile AA1 is a complete antagonist and this differencemay be the reason why we failed to determine thecocrystal structure of AA1 with PYRPYLs Howeverwe believe that in the future the crystal structure willpromote our understanding of ABA receptors espe-cially for analog 3 which has the simplest structure

Considering the difficulty particularly in nonmodelplants of simultaneously obtaining multigene knock-out mutants of ABA receptors and the high functionalredundancy among these receptors it would be verydifficult to analyze their possible epistasis or hypostasisroles in certain genetic pathways (Hicks and Raikhel2012) As AA1 targets all ABA receptors the functionalredundancy among PYRPYL members can be over-come thus making such research more expedient andeffective The commercial AA1 can be used as a pow-erful tool with which to study nonmodel plants and theeffects of ABA signaling at specific developmentalstages In this study to our knowledge for the first timewe used AA1 to study the genetic relationship of ABAreceptors with other ABA signaling components

The MST and TSA demonstrated that PYRPYLs aredirect molecular targets of AA1 More importantlyAA1 binds to all 13 ABA receptors of Arabidopsiswhich is consistent with its role in suppressing all PYRPYL-PP2C interactions and its broad-spectrum activity(Fig 3 Supplemental Fig S4) Several structural studieshave demonstrated a gate-latch-lock mechanism forABA receptors to recognize ABA in which the dimericABA receptors have an inner ligand-binding pocketthat is guarded by two functionally important b-loopstermed the gate and latch loops The ligand-bindingpocket is blocked in the receptor homodimers there-fore binding of ABA to the receptor requires receptordimer dissociation which results in the ABA-bindingaffinity being approximately 2 orders of magnitude


Ye et al


lower than that of the monomeric ABA receptors(Melcher et al 2009 Miyazono et al 2009 Nishimuraet al 2009 Santiago et al 2009 Yin et al 2009)However we did not observe a similar result for AA1which indicates that AA1 has an unbiased binding af-finity toward dimeric and monomeric ABA receptors(Figs 2B and 3) The molecular docking assays dem-onstrated that AA1 enters into the ligand-bindingpocket of PYL2 through its diazepin-8-one group (Fig4) indicating that such a group may be promising forthe design of agonists toward PYRPYLABA responses are blocked in plants without stress

however the concentration of ABA in vivo is sufficientfor PYRPYL activation to initiate ABA signaling Thisobservation suggested that if not all at least some of theABA receptors need to be inhibited in plants withoutstress however how plants manage this is unknown atpresent We believe that AA1 could shed light on thisquestion and we imagine that there might be somenaturalmolecules other thanABA that target PYRPYLto inhibit ABA responses in unstressed plants Thesemolecules could function similarly to AA1 and evenshare some structural similarity with AA1 The study ofthe mechanism of the effects of AA1 provides a newvision for ABA signaling research and indicates thatexciting results may yet emerge in this field in the futureA previous report demonstrated that down-

regulated ABA content leads to delayed leaf senes-cence and an extended grain-filling period resulting inincreased grain yield in rice (Liang et al 2014) Our re-sults showed that AA1 can delay leaf senescence in bothArabidopsis and rice Thus the application of AA1should be a useful strategy for improving crop yield inthe future In summary our chemical genetics screenidentified AA1 which acts as an ABA antagonist on allABA receptors by binding to them directly AA1 mayprovide an essential key to address important unan-swered questions in ABA signaling research

Significance

We describe the discovery and mechanistic character-ization of a smallmolecule targeting to all ABA receptorsAA1 which acts in plants by repressing ABA-inducedresponses We have demonstrated that plant senescencecan be chemically controlled through ABA receptorsindicating the potential application of antagonists ofABA receptorsWe believe that this is a major advance inagriculture Most importantly for agricultural applica-tion the structure of AA1 is simple and easy to synthe-size at lower cost allowing its availability and utility as achemical probe synthesized in large quantities which is abig advantage over other antagonists of ABA receptors

MATERIALS AND METHODS

Plant Chemical Genetics Screening

The chemical genetics screens were conducted as described previously(Zhao 2012 Ye et al 2016) A small synthetic molecular chemical library of

12000 structurally novel and diverse chemicals was used (Life Chemical) Toidentify the small molecules reversal of the seed germination inhibitory activityof ABA was assessed in which Col-0 seeds were grown on 96-well microplatescontaining one-half-strength MS agar medium supplemented with 07 mM ABAand 100 mM of the chemical The seed plates were incubated at 4degC for 3 d forstratification and grown at 22degC for an additional 6 d The chemical hitsuncovering seed germination under ABA treatment were retested and thestrongest hit and its analogs were used for further study The chemical namesfrom Life Chemical are as follows AA1 (F0544-0152) Analog1 (F0544-0123)Analog2 (F0544-0096) Analog3 (F0544-0123) and Analog4 (F2163-0147)

Seed Germination Assay

The seedswere sterilizedwith aNaClOHClmixture for 2 h Then the seedswere planted on one-half-strength MS agar medium in the presence or absenceof the indicated amounts of chemicals After 3 d of synchronization in a 4degCfreezer the plates were grown in a phytotron (23degC 16 h of light 8 h of dark)Seed germination rates were determined by calculating the average germina-tion rates of three replicates and about 100 seeds in each replicate were tested

Thermal Imaging

Three-month-old Arabidopsis (Arabidopsis thaliana) seedlings were treatedwith the indicated chemicals for 12 h Thermal images were obtained usingTesto 881-2 thermography (Testo) Images were saved on a computer memorycard and analyzed using IRSoft software (Testo)

Gene Expression Analysis

TotalRNAwas isolatedusing theTRIzol reagent (Invitrogen)Up to 500ngofRNA was used for the synthesis of cDNA according to the manufacturerrsquos in-structions (Prime Script RT-PCR kit with gDNA Eraser Takara) Quantitativereal-time RT-PCR was carried out using the Bio-Rad CFX96 with Realtime PCRMaster Mix (SYBR Green Toyobo) To quantify the expression level of ABA-responsive genes total RNAs were extracted from 5-d-old seedlings whichwere treated with the indicated chemicals for 6 h qRT-PCR was carried outusing the primers described before (Cao et al 2013)

pRD29A-LUC Expression

One-week-old seedlings with pRD29a-LUC (C24 ecotype) grown on one-half-strength MS solid medium were transferred to filter paper soaked withDMSO control or the indicated chemicals for 6 h LUC imaging was carried outas described before (Ye et al 2016)

[3H]ABA Uptake into Arabidopsis Protoplasts

Arabidopsis protoplasts were incubated in 1 mL of a bathing solution con-taining 45 nM [3H]ABA (Perkin-Elmer) and the indicated amounts of AA1 (withDMSO as a control) After washing with 33 washing buffer the mixture wasresuspended in 100 mL of water and scintillation fluid The radioactivity of thebound [3H]ABAwasmeasuredusing a scintillation counter The chlorophyll contentof the protoplasts was used as an internal control to normalize the protoplast input

Yeast Two-Hybrid Assay

The yeast two-hybrid assaywas carriedout as describedpreviously (Cao et al2013 Ye et al 2016) The coding sequences of 13 PYRPYL genes andHAB1wereamplified from cDNA of Col-0 plants by PCR and cloned into pBD-GAL4 (Stra-tagene) and pGADT7 (Clontech) respectively The vectors were cotransformedinto AH109 (Clontech) with corresponding PYRPYL and HAB1 vector combi-nations After 2 d of incubation at 30degC on SD(-Leu-Trp) agar plates well-grownclones were diluted further to 110 and 1100 dilution gradients Up to 1 mL of thedilution was added onto SD(-Leu-Trp-His) agar plates containing 1 mM

3-amino-triazole 50 mg L21 X-a-Gal 2 mM ABA and 100 mM quinabactin Pho-tographs were taken after 3 d of growth at 30degC incubation

AlphaScreen Assay

The AlphaScreen assay was conducted according to previous reports(Melcher et al 2009 Cao et al 2013) Briefly 100 nM recombinant




H6-SUMO-PYRPYL bound to a nickel acceptor and 100 nM biotin-PP2Csbound to streptavidin acceptor beads were mixed with the indicated chemi-cals The interactions were assessed byAlphaScreen technology (Perkin-Elmer)

HAB1 Phosphatase Activity Assay

Phosphatase assays were performed according to a previous report (Parket al 2015) All the receptors and HAB1 were expressed in Escherichia coli BL21as H6-SUMO fusion proteins as described before (Cao et al 2013) except thatPYL11 and PYL12 were expressed as fusion proteins in vector pET51b Briefly60 pmol of receptors and 60 pmol of His-SUMO-HAB1 were mixed in 80 mL ofreaction buffer (100 mM Tris [pH 79] and 100 mM NaCl) and then the probemolecules were added for 30 min Reactions were started by adding 20 mL of5mM 4-methylumbelliferyl phosphate (Sigma) in 156mM Tris-OAc (pH 79) and330 mM KOAc substrate buffer The phosphatase activity was detected imme-diately using a microplate reader (Thermo Scientific Varioskan flash 355 nmexcitation and 460 nm emission)

TSAs

All the reactions were conducted as described previously (Soon et al 2012)The reactions were in final volumes of 10 mL on 96-well plates with SYPROOrange (Invitrogen) and incubated with compounds on ice for 30 min Thermalmelting experiments were carried out using the StepOnePlus Real-Time PCRSystem (Applied Biosystems) melt curve program with a ramp rate of 1degC anda temperature range of 25degC to 90degC for all other experiments All experimentswere conducted in triplicate

Microscale Thermophoresis

The MST assays were conducted according to the user manual (NanoTemperTechnologies) Approximately 10 mM recombinant receptors was labeled with redfluorescent dye (NT-647-NHS NanoTemper Technologies) A range of concentra-tions of the required ligand (ranging from 002 to 500 mM) was incubated with 1 mM

purified protein for 1 h in assay buffer (20 mM Bis-Tris [pH 79] and 150 mM NaCl)The samplewas loaded intoNanoTemperglass capillaries andmicrothermophoresiswas carried out using 20LEDpower and 80MST TheKdwas calculated usingthe mass action equation via NanoTemper software from duplicate reads oftriplicate experiments The instrument usedwas aNanoTempermonolithNT115

Molecular Docking

TheAutoDock40Vina packagewasused tomodel the docking ofAA1withopen PYL2 (PDB no 3KDH) and closed PYL2 (PDB no 3KDI) The principle ofAutoDock40Vina has been described previously (Morris et al 2009 Trott andOlson 2010) During the docking process the exhaustiveness of the globalsearchwas set to 20 and themaximumnumber of conformers was set to 15 Thedocking results were analyzed by the Pymol software (httppymolorg)

Competitive Binding Assays

His-PYL2 bound to Ni-NTA beads was incubated with 01 mM [3H]ABA(Perkin-Elmer) alone or with the indicated amounts of AA1 for 1 h Afterwashing three times the beads were resuspended in 100 mL of water andmixedwith scintillation fluid The radioactivity of the bound [3H]ABA was measuredusing a scintillation counter

Dark-Induced Leaf Senescence

About 3-week-old Col-0 leaves were detached and incubated with the in-dicated chemicals For rice (Oryza sativa) fully expanded flag leaves were ex-cised carefully Detached leaves were cut into 3-cm pieces and treated withthe indicated chemicals in petri dishes The samples were incubated at 28degC indarkness for 5 d

Supplemental Data

The following supplemental materials are available

Supplemental Figure S1 Col-0 seeds sown in the presence of the indicatedchemicals

Supplemental Figure S2 Ten-day-old seedlings incubated with the indi-cated chemicals

Supplemental Figure S3 Effects of AS6 on the interactions of HAB1-PYR1PYLs by yeast two-hybrid assay

Supplemental Figure S4 AA1 binding to ABA receptors in MST assays

Supplemental Figure S5 Overall structures of the PYL2AA1ABAcomplexes

Supplemental Figure S6 Structure-activity relationship study of AA1

Supplemental Figure S7 Effects of AA1 on cytokinin-responsive geneexpression

Supplemental Table S1 Binding free energy of AA1 to PYI2

Supplemental Table S2 The interacting amino acids of open PYL2 withAA1

Supplemental Table S3 The interacting amino acids of closed PYL2 withAA1

ACKNOWLEDGMENTS

We thankDr Yasushi Todoroki (ShizuokaUniversity) for the gift of AS6 andthe National Center for Protein Science Shanghai (Protein Expression andPurification System) for support with the instruments and technical assistance

ReceivedDecember 9 2016 accepted February 10 2017 published February 132017

LITERATURE CITED

Bhargava A Clabaugh I To JP Maxwell BB Chiang Y Schaller EGCarolina N Loriaine A Kiber J (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-seq in Ara-bidopsis Plant Physiol 162 272ndash294

Cao M Liu X Zhang Y Xue X Zhou XE Melcher K Gao P Wang F ZengL Zhao Y et al (2013) An ABA-mimicking ligand that reduces waterloss and promotes drought resistance in plants Cell Res 23 1043ndash1054

Cao MJ Wang Z Zhao Q Mao JL Speiser A Wirtz M Hell R Zhu JKXiang CB (2014) Sulfate availability affects ABA levels and germinationresponse to ABA and salt stress in Arabidopsis thaliana Plant J 77 604ndash615

Cutler SR Rodriguez PL Finkelstein RR Abrams SR (2010) Abscisic acidemergence of a core signaling network Annu Rev Plant Biol 61 651ndash679

Dupeux F Santiago J Betz K Twycross J Park SY Rodriguez LGonzalez-Guzman M Jensen MR Krasnogor N Blackledge M et al(2011) A thermodynamic switch modulates abscisic acid receptor sen-sitivity EMBO J 30 4171ndash4184

Finkelstein RR (1994) Mutations at two new Arabidopsis ABA responseloci are similar to the abi3 mutations Plant J 5 765ndash771

Finkelstein RR Somerville CR (1990) Three classes of abscisic acid (ABA)-insensitive mutations of Arabidopsis define genes that control over-lapping subsets of ABA responses Plant Physiol 94 1172ndash1179

Fujii H Chinnusamy V Rodrigues A Rubio S Antoni R Park SY CutlerSR Sheen J Rodriguez PL Zhu JK (2009) In vitro reconstitution of anabscisic acid signalling pathway Nature 462 660ndash664

Fujita M Fujita Y Noutoshi Y Takahashi F Narusaka Y Yamaguchi-Shinozaki K Shinozaki K (2006) Crosstalk between abiotic and bioticstress responses a current view from the points of convergence in thestress signaling networks Curr Opin Plant Biol 9 436ndash442

Gonzalez-Guzman M Pizzio GA Antoni R Vera-Sirera F Merilo EBassel GW Fernaacutendez MA Holdsworth MJ Perez-Amador MAKollist H et al (2012) Arabidopsis PYRPYLRCAR receptors play amajor role in quantitative regulation of stomatal aperture and tran-scriptional response to abscisic acid Plant Cell 24 2483ndash2496

Ha S Vankova R Yamaguchi-Shinozaki K Shinozaki K Tran LS (2012)Cytokinins metabolism and function in plant adaptation to environ-mental stresses Trends Plant Sci 17 172ndash179

Hao Q Yin P Li W Wang L Yan C Lin Z Wu JZ Wang J Yan SF Yan N(2011) The molecular basis of ABA-independent inhibition of PP2Cs by asubclass of PYL proteins Mol Cell 42 662ndash672


Ye et al


Hao Q Yin P Yan C Yuan X Li W Zhang Z Liu L Wang J Yan N (2010)Functional mechanism of the abscisic acid agonist pyrabactin J BiolChem 285 28946ndash28952

Hayashi K Tan X Zheng N Hatate T Kimura Y Kepinski S Nozaki H(2008) Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling Proc NatlAcad Sci USA 105 5632ndash5637

Hicks GR Raikhel NV (2012) Small molecules present large opportunitiesin plant biology Annu Rev Plant Biol 63 261ndash282

Ishitani M Xiong L Stevenson B Zhu JK (1997) Genetic analysis of os-motic and cold stress signal transduction in Arabidopsis interactions andconvergence of abscisic acid-dependent and abscisic acid-independentpathways Plant Cell 9 1935ndash1949

Ito T Kondoh Y Yoshida K Umezawa T Shimizu T Shinozaki K OsadaH (2015) Novel abscisic acid antagonists identified with chemical arrayscreening ChemBioChem 16 2471ndash2478

Kim TH Boumlhmer M Hu H Nishimura N Schroeder JI (2010) Guard cellsignal transduction network advances in understanding abscisic acidCO2 and Ca2+ signaling Annu Rev Plant Biol 61 561ndash591

Kim TH Hauser F Ha T Xue S Boumlhmer M Nishimura N Munemasa SHubbard K Peine N Lee BH et al (2011) Chemical genetics revealsnegative regulation of abscisic acid signaling by a plant immune re-sponse pathway Curr Biol 21 990ndash997

Leng P Yuan B Guo Y (2014) The role of abscisic acid in fruit ripening andresponses to abiotic stress J Exp Bot 65 4577ndash4588

Liang C Wang Y Zhu Y Tang J Hu B Liu L Ou S Wu H Sun X Chu Jet al (2014) OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice Proc Natl Acad Sci USA 111 10013ndash10018

Lim PO Kim HJ Nam HG (2007) Leaf senescence Annu Rev Plant Biol 58115ndash136

Ma Y Szostkiewicz I Korte A Moes D Yang Y Christmann A Grill E(2009) Regulators of PP2C phosphatase activity function as abscisic acidsensors Science 324 1064ndash1068

Meesters C Moumlnig T Oeljeklaus J Krahn D Westfall CS Hause B JezJM Kaiser M Kombrink E (2014) A chemical inhibitor of jasmonatesignaling targets JAR1 in Arabidopsis thaliana Nat Chem Biol 10 830ndash836

Melcher K Ng LM Zhou XE Soon FF Xu Y Suino-Powell KM Park SYWeiner JJ Fujii H Chinnusamy V et al (2009) A gate-latch-lockmechanism for hormone signalling by abscisic acid receptors Nature462 602ndash608

Melcher K Xu Y Ng LM Zhou XE Soon FF Chinnusamy V Suino-PowellKM Kovach A Tham FS Cutler SR et al (2010) Identification and mech-anism of ABA receptor antagonism Nat Struct Mol Biol 17 1102ndash1108

Miyazono K Miyakawa T Sawano Y Kubota K Kang HJ Asano AMiyauchi Y Takahashi M Zhi Y Fujita Y et al (2009) Structural basisof abscisic acid signalling Nature 462 609ndash614

Morris GM Huey R Lindstrom W Sanner MF Belew RK GoodsellDS Olson AJ (2009) AutoDock4 and AutoDockTools4 automateddocking with selective receptor flexibility J Comput Chem 30 2785ndash2791

Nishimura N Hitomi K Arvai AS Rambo RP Hitomi C Cutler SRSchroeder JI Getzoff ED (2009) Structural mechanism of abscisic

acid binding and signaling by dimeric PYR1 Science 326 1373ndash1379

Okamoto M Peterson FC Defries A Park SY Endo A Nambara EVolkman BF Cutler SR (2013) Activation of dimeric ABA receptorselicits guard cell closure ABA-regulated gene expression and droughttolerance Proc Natl Acad Sci USA 110 12132ndash12137

Park SY Fung P Nishimura N Jensen DR Fujii H Zhao Y Lumba SSantiago J Rodrigues A Chow TF et al (2009) Abscisic acid inhibitstype 2C protein phosphatases via the PYRPYL family of START pro-teins Science 324 1068ndash1071

Park SY Peterson FC Mosquna A Yao J Volkman BF Cutler SR (2015)Agrochemical control of plant water use using engineered abscisic acidreceptors Nature 520 545ndash548

Rodriguez PL Lozano-Juste J (2015) Unnatural agrochemical ligands forengineered abscisic acid receptors Trends Plant Sci 20 330ndash332

Santiago J Dupeux F Round A Antoni R Park SY Jamin M Cutler SRRodriguez PL Maacuterquez JA (2009) The abscisic acid receptor PYR1 incomplex with abscisic acid Nature 462 665ndash668

Soon FF Suino-Powell KM Li J Yong EL Xu HE Melcher K (2012)Abscisic acid signaling thermal stability shift assays as tool to ana-lyze hormone perception and signal transduction PLoS ONE 7e47857

Sun D Wang H Wu M Zang J Wu F Tian C (2012) Crystal structures ofthe Arabidopsis thaliana abscisic acid receptor PYL10 and its complexwith abscisic acid Biochem Biophys Res Commun 418 122ndash127

Takeuchi J Okamoto M Akiyama T Muto T Yajima S Sue M Seo MKanno Y Kamo T Endo A et al (2014) Designed abscisic acid analogsas antagonists of PYL-PP2C receptor interactions Nat Chem Biol 10477ndash482

Tan BC Joseph LM Deng WT Liu L Li QB Cline K McCarty DR (2003)Molecular characterization of the Arabidopsis 9-cis epoxycarotenoiddioxygenase gene family Plant J 35 44ndash56

Trott O Olson AJ (2010) AutoDock Vina improving the speed and ac-curacy of docking with a new scoring function efficient optimizationand multithreading J Comput Chem 31 455ndash461

Weng L Zhao F Li R Xu C Chen K Xiao H (2015) The zinc finger tran-scription factor SlZFP2 negatively regulates abscisic acid biosynthesisand fruit ripening in tomato Plant Physiol 167 931ndash949

Ye Y Gong Z Lu X Miao D Shi J Lu J Zhao Y (2016) Germostatin re-sistance locus 1 encodes a PHD finger protein involved in auxin-mediated seed dormancy and germination Plant J 85 3ndash15

Yin P Fan H Hao Q Yuan X Wu D Pang Y Yan C Li W Wang J Yan N(2009) Structural insights into the mechanism of abscisic acid signalingby PYL proteins Nat Struct Mol Biol 16 1230ndash1236

Zhang M Yuan B Leng P (2009) The role of ABA in triggering ethylenebiosynthesis and ripening of tomato fruit J Exp Bot 60 1579ndash1588

Zhao Y (2012) A chemical genetics method to uncover small molecules fordissecting the mechanism of ABA responses in Arabidopsis seed ger-mination Methods Mol Biol 876 107ndash116

Zhao Y Chan Z Gao J Xing L Cao M Yu C Hu Y You J Shi H Zhu Yet al (2016) ABA receptor PYL9 promotes drought resistance and leafsenescence Proc Natl Acad Sci USA 113 1949ndash1954

Zhu JK (2002) Salt and drought stress signal transduction in plants AnnuRev Plant Biol 53 247ndash273




A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50

Figure S1 A Col-0 seeds were sown in the presence of the indicated chemicals The images were acquired after seed stratification and growth for 5 days B Seed germination rates in (a) (n = 3 error bars = SD) C Rice (Zhonghua 11) seeds or (D) maize seeds were incubated with the indicated chemicals after 3 days of growth the chemicals were used at the following concentrations 10 μM ABA 100 μM AA1 or DMSO (mock) E AA1 antagonizes the high salt-induced inhibition of seed germination Col-0 seeds were sown on 12 MS plates with indicated chemicals Pictures were taken after 5 days of growth

60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO

ABA Concentration (μM）

Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

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M A

A1

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M A

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100

μM A

A1

0

2

4

6

8

AB

A c

on

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t

(n

gg

fre

sh

we

igh

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Figure S2 AEffects of AA1 on endogenous ABA concentration Ten-day old seedlings incubated with indicated chemicals (DMSO as control) for 6 hours (n = 3 error bars = SE) B Effects of AA1 on ABA transportation The isolated mesophyll cells were incubated in a bathingsolution containing 5 nM [3H]-ABA at pH 57 The chlorophyll content of the protoplasts was used tonormalize [3H]-ABA uptake (n = 3 error bars = SE)C AA1 inhibits the interactions of ABA monomeric receptors with HAB1 in yeast two-hybrid assays PYL8ndash12 were constructed in BD vectors and HAB1 in the AD vector Yeast cells were grown on SD medium (-Leu-Trp-His plus X-α-Gal (50 mgL) and 5 mM 3-AT) with indicated chemicals for 3 days

Mock 10 20 50 10

0

0

50

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AA1 concentration (microM)

[3H

]-A

BA

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A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C

Figure S3 The effects of AS6 on the interactions on HAB1-PYR1PYLs by yeast two hybrid assays PYR1PYLs were constructed in BD vectors and HAB1 in the AD vector Yeast cells were grown on SD medium (-Leu-Trp-His 5 mM 3-AT) with indicated chemicals (ABA 2 μM AS6 100 μM) for 3 days

PYR1

PYL1

PYL2

PYL3

PYL4

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Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

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188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

Kd 21plusmn 95 (μM)Kd 95 plusmn 38 (μM)

Kd 94 plusmn 38 (μM)

Kd 91 plusmn 54 (μM) Kd 101 plusmn 34 (μM) Kd 55 plusmn 2 (μM)

-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12


Figure S4 A AA1 binding to ABA receptors in microscale thermophoresis assays (MST) Kd stands for the dissociation constant (n = 3 error bars = SE) B ABA binding to ABA receptors in microscale thermophoresis assays (MST) Kd stands for the dissociation constant (n = 3 error bars = SE)

AA1 concentration (μM)

AA1 concentration (μM)AA1 concentration (μM)AA1 concentration (μM)

AA1 concentration (μM) AA1 concentration (μM) AA1 concentration (μM)

A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]

ABA Concentration [μM ]

-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]

ABA Concentration [μM]

-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]


Kd 103plusmn 95 (μM)

PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9

Figure S5 A B Overall structures of the PYL2AA1ABA complexesC Close-up overlay of ABA (red) AA1 (blue) in open-PYL2 and AA1 (green) in closed-PYL2D Molecular structures of ABA-related small molecules The groups with similar function are shown in blue frames

A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025

Figure S6 A Structure-activity-relationship (SAR) study of AA1The effects of the analogs of AA1 on ABA-induced seed germination Wild-type (Col-0) seeds were sown on 12 MS

medium containing the indicated chemicals and the pictures were taken after 4-day growthB AA1 decreases the sensitivities of the ABA-related mutants to ABA-induced seed germination inhibitionThe seeds of different backgrounds were sown in indicated chemical- containing 12 MS plates and the pictures were taken after 5-day growth pyr1pyl1pyl2 abi3-8 abi4-1are in Col-0 background abi2-1 is in Ler background

125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B

Figure S7The effects of AA1 on cytokinin-responsive gene expressions 10-day old seedlings were treated with 10 μM BA or 100 μMAA1 for 5 hours and detected by qRT-PCR

ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l

Table S1 Binding free energy of AA1 to PYL2

Complex 3KDH_AA1 3KDI_AA1

Energy (kcalmol) -4778 -5679

Table S2 The interacting amino acids of open-PYL2 with AA1

Amino Acid Interaction

Glu 147 Hydrogen bond


Lys 64 Hydrogen bond

Asn 173 Hydrogen bond

Ser 89 Polar

His 119 Polar

Ser 96 Polar

Phe 66 Hydrophobic interaction

Ile 88 Hydrophobic interaction


Val 169 Hydrophobic interaction


Leu 121 Hydrophobic interaction



Tyr 124 Hydrophobic interaction



Table S3 The interacting amino acids of closed-PYL2 with AA1


Arg 83 Hydrogen bond




Asn 157 Polar

His 119 Polar

Ser 96 Polar

Pro 154 Hydrophobic interaction




Ala 93 Hydrophobic interaction









downstream effectors to cause physiological responsesto ABA (Fujii et al 2009)Structural studies highlighted a conserved gate-

latch-lock mechanism underlying ABA perceptionand signal transduction (Melcher et al 2009 Miyazonoet al 2009 Nishimura et al 2009 Santiago et al 2009Yin et al 2009) In Arabidopsis (Arabidopsis thaliana)PYRPYLs are classified into two distinct categoriesdimeric receptors (PYR1 and PYL1ndashPYL3) and mono-meric receptors (PYL4ndashPYL12 Okamoto et al 2013)The dimeric receptors interact with and inhibit PP2Cactivity in an ABA-dependent manner The dimericreceptors possess an ABA-binding pocket flanked by agate and a latch loop and the apo-form receptors are inan open conformation permitting access of ABA to thebinding pocket (Melcher et al 2009) Upon ABAbinding the dimeric receptors close the gate which inturn creates the interaction surfaces that permit thedocking of PP2Cs onto the ABA-bound receptors TheABA-induced receptor-PP2C interaction induces a newconformation change of the protein complex whichlocks the gate of the receptors (Melcher et al 2009) Incontrast monomeric receptors are in equilibrium be-tween the gate-opened and gate-closed conformationsin the absence of ABA Thus monomeric receptors caninteract with and inhibit PP2C activity in an ABA-independent manner (Hao et al 2011 Sun et al 2012)The distinct roles of dimeric and monomeric receptorsin ABA signaling remain elusive However recent re-ports demonstrated that the selective chemical activa-tion of dimeric receptors could induce a full ABAresponse which indicated that dimeric receptors arekey factors in ABA signaling (Cao et al 2013 Okamotoet al 2013)Small molecules functioning as agonists or antago-

nists of ABA receptors promote the discovery of ABAreceptors and deepen our knowledge of the receptorsrsquomechanisms of action Pyrabactin the first identi-fied ABA receptor-related small molecule functionsas an agonist of PYR1 and PYL1 and as a weak antag-onist of PYL2 (Park et al 2009 Melcher et al 2010)Besides pyrabactin quinabactin (also known asAM1) working as a selective agonist toward dimericABA receptors is a promising agrochemical thatelicits stomatal closure and enhances crop droughtresistance (Cao et al 2013 Okamoto et al 2013) Inaddition mandipropamid a commercial agrochemicalfunctions as a selective agonist of engineered PYR1(Park et al 2015 Rodriguez and Lozano-Juste 2015)ABA antagonists have been identified and designedbefore (Kim et al 2011 Takeuchi et al 2014 Ito et al2015) DFPM a small molecule identified by chemicalgenetics screening down-regulates ABA-dependentgene expression and also inhibits ABA-induced sto-matal closure through the plant immune responsepathway and its target is still unclear (Kim et al 2011)Small molecules AS6 and RK460 are two antagonists ofABA receptors (Takeuchi et al 2014 Ito et al 2015)AS6 was designed according to the ABAPYR1 x-raystructure Because of the structural similarity of ABA

AS6 is not an entire PRYPYL antagonist which isrevealed by its intrinsic agonist activity toward ABAreceptors In additionAS6 functions as an antagonist notto all the PYRPYL-PP2C interactions The complexstructure of AS6 may result in a high production costand its chemical synthesis is complex limiting its avail-ability and utility as a chemical probe synthesized inlarge quantities (Takeuchi et al 2014) RK460 is an an-tagonist toward PYR1 but not other PYRPYLs whichcannot bypass the genetic redundancy of ABA receptorsand this may explain why RK460 has no antagonist ac-tivity to ABA-induced physiological processes (Ito et al2015)

The functional characterization of PYRPYL mem-bers using conventional molecular genetic approacheswould be daunting because of their genetic and func-tional redundancy In this situation a small moleculeacting as a broad-spectrum antagonist toward all ABAreceptors could be a powerful tool to sidestep this ge-netic redundancy Such an ABA antagonist is particu-larly needed for ABA signaling studies in other plantsystems with less available genetic resources More-over it has been reported that some pathogens such asPseudomonas syringae could hijack the ABA signalingpathway for their survival and cause plant disease(Fujita et al 2006) ABA is thought to be one of thephytohormones that promote leaf senescence ABAlevels can be observed in many plants during leaf se-nescence (Tan et al 2003) ABA induces expression ofseveral senescence-associated genes (SAGs) and yel-lowing of the leaves which are typical phenomena as-sociatedwith leaf senescence (Liang et al 2014)Whatrsquosmore a new study showed that overexpression of PYL9can promote Arabidopsis senescence via the core ABAsignaling pathway (Zhao et al 2016) Up-regulation ofgenes associated with ABA signaling and a dramaticincrease in endogenous ABA promote fruit ripeningand leaf senescence indicating a promising agriculturalapplication of ABA antagonist (Lim et al 2007)

Here we report a novel ABA antagonist AA1 whichwas uncovered using our plant chemical geneticsscreening using commercial compound libraries AA1releases ABA-induced seed germination inhibition andstomatal closure Biochemical assays demonstrated thatAA1 inhibits the interactions between all 13 PYRPYLsand PP2C by direct binding to PYRPYL which isa specific feature of all the PYRPYL-related smallmolecules Most importantly AA1 down-regulatessenescence-related gene expression and inhibits chlo-rophyll breakdown and thus delays plant leaf senes-cence which may provide some hints for how to applyABA antagonists to agriculture

RESULTS

Identification of an ABA Antagonist

To identify small molecules that inhibit ABA signal-ing we developed a chemical screen that relied on theseed-inhibitory effects of ABA (Zhao 2012 Ye et al













Ye et al













Ye et al






















Ye et al

















Ye et al










DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar























Ye et al













Ye et al






















Ye et al

















Ye et al










DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar

























Ye et al






















Ye et al

















Ye et al










DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar


































Ye et al

















Ye et al










DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar





























Ye et al










DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar






















DISCUSSION







Ye et al





Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar

















Significance








Thermal Imaging











AlphaScreen Assay








TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar

















TSAs





Molecular Docking






Supplemental Data












ACKNOWLEDGMENTS



LITERATURE CITED














Ye et al








































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar




















































A

B

AA1

100 μM

DMSO

0 1 5 10 20 30 40 50


60ABA

(μM)

0 1 5 10 20 30 40 50 60

0

20

40

60

80

100

AA1 DMSO


Ge

rmin

ati

on

Ra

te (

)

Mock ABA ABAAA1

Mock ABA ABAAA1

C

D

MOCK200 mM

Nacl

200 mM Nacl

100 μM AA1

E

Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar














Mock

10 μ

M A

A1

50 μ

M A

A1

100

μM A

A1

0

2

4

6

8

AB

A c

on

ten

t

(n

gg

fre

sh

we

igh

t)


Mock 10 20 50 10

0

0

50

100


[3H

]-A

BA

Co

nte

nt

(

)

A B

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL4

PYL8

PYL9

PYL10

PYL11

PYL12

ABA AA1AA1ABAMOCK

C


PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar















PYR1

PYL1

PYL2

PYL3

PYL4

PYL5

PYL6

PYL7

PYL9

PYL10

PYL11

PYL12

PYL8

ABA AS6AS6ABAMOCK

Diolution 1 01 001 1 01 001 1 01 001 1 01 001

PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar














PYL1

38

88

138

188

238

288

01 1 10 100 1000

Δ

Fn

orm

[permil

] PYL6

-31

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL7

-03

17

37

57

77

97

117

01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL8

-17

22

42

62

82

102

122

142

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

-02

PYL9

-19

30

80

130

180

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]

PYL10

-082

12

52

92

132

172

001 01 1 10 100 1000

Δ

Fn

orm

[permil

]




-20

-004

19

39

59

79

99

119

139

159

Δ

Fn

orm

[permil

]

001 01 1 10 100 1000

PYL12






A

B

-312

188

688

1188

1688

2188

001 1 100

Δ F

norm

[permil

]


-143

357

857

001 01 1 10 100 1000

Δ F

norm

[permil

]


-132

368

868

001 01 1 10 1001000

Δ F

norm

[permil

]


-312

688

1688

2688

3688

4688

01 10 1000

Δ F

norm

[permil

]



PYR1


PYL2

Kd 2plusmn 05 (μM)

PYL5


PYL9


A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar















A

AA1Closed PYL2

B

H119

R120

L91 P92 A93

E98

R83

K64

Pryabactin

Quinabactin

(AM1)

ABA

AA1

C D

AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar














AA1

Analog 1

Analog 2

Analog 5

Analog 3

Analog 4

11 12 13 15 110 115 120 ABA Chemical

(μM)025



125

A

Pyr1pyl1 pyl2

abi2-1

abi3-8

abi4-1 + 100 μM AA1

Col-0

abi3-8 + 100 μM AA1

Pyr1pyl1 pyl2

+ 100 μM AA1

abi2-1 + 100 μM AA1

abi4-1

100 μM AA1

Ler

100 μM AA1

ABA (μM) 1 15 20 50 75 100 1500 1252 5 10

B


ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar















ARR4

ARR5

ARR6

ARR15

0

5

10

15

20Mock BA AA1

Re

lati

ve

exp

ressio

n le

ve

l










Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar























Ser 89 Polar

His 119 Polar

Ser 96 Polar


















Asn 157 Polar

His 119 Polar

Ser 96 Polar




















Asn 157 Polar

His 119 Polar

Ser 96 Polar














a novel chemical inhibitor of aba signaling targets all

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