WO2023067192A1

WO2023067192A1 - Methods for improving abiotic stress resistance

Info

Publication number: WO2023067192A1
Application number: PCT/EP2022/079479
Authority: WO
Inventors: Armando Joaquín ALBERT DE LA CRUZ; Pedro Luis RODRIGUEZ; Lourdes INFANTES SAN MATEO; Jorge LOZANO-JUSTE
Original assignee: Consejo Superior De Investigaciones Cientificas (Csic); Universidad Politècnica de València
Priority date: 2021-10-21
Filing date: 2022-10-21
Publication date: 2023-04-27

Abstract

The invention relates to methods of improving/enhancing abiotic stress tolerance, and in particular methods for improving/enhancing drought tolerance. Also described are new ABA mimicking molecules, as well as genetically altered plants with increased susceptibility to the ABA mimicking molecule.

Description

Methods for improving abiotic stress resistance

FIELD OF THE INVENTION

The invention relates to methods of improving/enhancing abiotic stress tolerance, and in particular methods for improving/enhancing drought tolerance. Also described are new ABA mimicking molecules, as well as genetically altered plants with increased susceptibility to the ABA mimicking molecule. As such, the invention relates to a combined genetic-chemical approach to optimising water usage and improving a plant’s response to drought.

BACKGROUND OF THE INVENTION

Drought is a major limitation for crop productivity. The global warming and climate change exacerbate the effect of ordinary seasonal weather variations and atmospheric phenomena that limit fresh water availability, constituting a major threat in crop production. Plant transpiration through stomata is the major source of water loss during gas exchange for photosynthesis. Under drought stress, the phytohormone ABA controls stomatai aperture and modulates plant transpiration as well as water uptake by roots. Consequently it has been shown that ABA responses can be modulated to improve water use efficiency (WUE) of crop plants.

Enhanced ABA levels elicited in response to drought are perceived by the PYR/PYL/RCAR family of ABA receptors and the clade A subfamily of protein phosphatases type-2C (PP2Cs), which act as necessary ABA co-receptors in ternary complexes. This leads to PP2C inhibition and concomitant activation of three ABA- activated Snf1 -related protein kinases (SnRK2s). ABA-activated SnRK2s phosphorylate ABFs/AREBs transcription factors and the chromatin-remodeller ATPase BRAHMA for activation of ABA transcriptional response. In the plasma membrane, phosphorylation of different K+ transporters leads to inhibition of K+ influx and activation of K+ efflux, which together with activation of R- and S-type anion channels and aquaporins lead to loss of turgor in guard cells and stomatai closure.

The available structural information on ABA receptors suggests the mechanism behind ABA sensing is complicated. ABA receptors are distributed into three families - subfamily III includes dimeric receptors, whereas subfamily I and II include monomeric receptors. In the resting state, PYLR/PYL/RCAR receptors display an open ABA-binding cavity flanked by two highly conserved loops, named as gate/CL2/p3- p 4 loop and latch/CL3/ P 5- p 6 loop. ABA-induced conformational rearrangements are required for dissociation of dimeric receptors and activation of monomeric receptors. Thus both gate and latch loops define a surface that enables the receptor to dock into the PP2C active site. The formation of receptor-ABA-phosphatase complexes causes the dissociation of different PP2C-SnRK2 complexes and abolishes PP2C-mediated inhibition of the SnRK2s, triggering the ABA response. Additionally, RAF-like MAPKKKs are required to reactivate SnRK2s that have been previously dephosphorylated by PP2Cs.

The detailed knowledge of this pathway has been harnessed for the development of genetic and chemical strategies to cope with drought stress. Among them, the generation of agrochemical compounds mimicking ABA action (but that are more stable than ABA) as well as genetic approaches aimed at constitutively activating ABA-mediated plant responses by overexpressing ABA receptors or inactivating clade A PP2C repressors. In addition, there is an emerging field for the development of chemical compounds that act as ABA agonists or antagonists to modulate ABA signalling dynamically and exogenously. However, this chemical approach has generally been found to be less effective than the genetic one, unless these ABA mimicking molecules are specific enough as to target those receptors involved in regulation of plant transpiration, root water uptake and hydrotropism. Furthermore, the experimental identification of new ABA mimicking molecules requires the use of expensive compound libraries and equipment. A shortcut based on the vast amount of structural information available for ABA receptors takes advantage of inexpensive in silico techniques that enable the screening of large collections of commercially available chemical compounds. However, these techniques often produce large amounts of false positives that require validation or weak-interacting molecules that need extensive chemical optimization. An additional issue arises from the multigenic nature of PYR/PYL/RCAR ABA receptors, which show subtle sequence and structural differences. These variations usually led to a limited range of agonist activity over ABA receptors.

There therefore exists a need to identify new methods to modulate ABA responses and consequently increase resistance to abiotic stresses such as drought. The present invention addresses this need. SUMMARY OF THE INVENTION

Fresh water stores are compromised by the effect of global warming and the concomitant climate change. As agriculture represents about 70% of total fresh water consumption, optimization of crop production is required to increase water use efficiency. In particular, major water-loss occurs through transpiration at open stomata, which is regulated by the phytohormone ABA. The interaction of ABA with the family of pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of ABA receptors (RCAR) ABA receptors activates the signalling pathway that controls stomatai closure and is also required for root hydrotropism. Thus, a strategy to activate PYR/PYL/RCARs and enhance ABA signalling is a promising biotechnological tool to regulate transpiration and foster water foraging by roots. In the present invention we have combined chemical and genetic approaches to generate new sulfonamide-based ABA agonist molecules and engineer a CsPYLI ABA receptor, named CsPYL1^5m, that efficiently binds iSB09. Spraying of iSB09 over Arabidopsis plants overexpressing CsPYLI ^5m leads to activation of ABA signalling and marked drought tolerance, including a strong antitranspirant effect. Additionally, genome-wide transcriptional analysis reveals a powerful induction of ABA response in CsPYLI ^5m plants by iSB09. Given that CsPYLI ^5m is a dimeric receptor and displays lower ABA affinity than its wild type version, no constitutive activation of ABA signalling and hence growth penalty was observed in transformed plants. Therefore, we have achieved conditional and efficient activation of ABA signalling to optimise crop water usage through this genetic-chemical approach.

In a first aspect of the invention, there is provided a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof:

wherein Ri to R3 are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl;

R4 to R? are independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl;

at least one of R4 to R? has a structure according to Formulae 1-1 or 1-2; wherein in Formulae 1-1 and 1-2,

Rs to Rn are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl;

Li and L2 are independently selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene and optionally substituted heteroarylene; a1 and a2 are independently selected from 1 to 6; represents a single or double bond; and represents a connection point to the rest of the compound; wherein the compound is not Compound 1 :

In another aspect of the invention there is provided the use of a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, or a composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier, in enhancing abiotic stress resistance in a plant, wherein Ri to Rn, Li , L2, a1 and a2 are as defined herein.

In another aspect of the invention there is provided a method of enhancing abiotic stress resistance in a plant, comprising applying a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, or a composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier, wherein R1 to Rn, Li, L2, a1 and a2 are as defined herein.

In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYL1 polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more of V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1. In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYR1 polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more of V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5. In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYL2 polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more of V87L, F1121, T128I and V145A of SEQ ID 7. In another aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYL3 polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more ofV107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence. In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 2, 11 , 12 or 13 or a functional variant or homologue thereof.

In another aspect of the invention there is provided a nucleic acid construct comprising the isolated nucleic acid as described herein, wherein the nucleic acid is operably linked to a regulatory sequence.

In a further aspect of the invention, there is provided a host cell comprising the nucleic acid construct. In another aspect of the invention, there is provided a genetically altered plant or plant part thereof, expressing the isolated nucleic acid as described herein or the nucleic acid construct as described herein.

In another aspect of the invention, there is provided a genetically altered plant or part thereof, wherein the plant comprises at least one mutation in at least one PYL/PYR gene, wherein the mutation results in at least one of the following mutations: V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1 ; or at least one of the following mutations V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5; or at least one of the following mutations V87L, F112I, T128I and V145A of SEQ ID NO: 7; or at least one of the following mutations V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence.

In another aspect of the invention, there is provided a method of inhibiting seed germination/prolonging seed dormancy in a plant, the method comprising applying a compound or composition as described herein.

In another aspect of the invention, there is provided a method of activating ABA signalling and/or activating or increasing an ABA response, the method comprising applying a compound or composition as described herein.

In another aspect of the invention, there is provided a method of producing a plant with increased abiotic stress resistance, the method comprising introducing and expressing a nucleic acid as described herein or a nucleic acid construct as described herein.

In another aspect of the invention, there is provided a method of producing a plant with increased abiotic stress resistance, the method comprising introducing at least one mutation into a plant genome, wherein the mutation is the addition of one or more additional copy of a mutated PYR/PYL polypeptide, wherein preferably, the mutated polypeptide comprises SEQ ID NO: 2, 11 , 12 or 13 or a functional variant or fragment thereof, and wherein the one or more additional copy of the mutated PYL/PYR polypeptide is operably linked to a regulatory sequence.

In another aspect of the invention, there is provided a plant obtained or obtainable by the methods described herein. The plant may be selected from a crop plant or biofuel plant, preferably maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures.

Figure 1 shows identification of SB as selective ABA receptor agonist and structural analysis of the CsPYL1-SB-AtHAB1 complex. (A) PP2C inhibition assay in presence of 100 μM SB and the indicated ABA receptors. (B) Chemical structure of SB and IC50 values for ABA or SB-dependent inhibition of HAB1 by AtPYLI , AtPYL5 and AtPYLIO. (C) Inhibition of seedling establishment by SB in wild-type Col-0 and either PYL5 or PYL10 overexpressing lines. (D) Quantification of ABA or SB-mediated inhibition of root growth in the indicated genotypes. Seedlings were grown in plates supplemented with 1 % DMSO (mock), 50 μM SB or 10 μM ABA. Values are mean ± SD of three independent experiments (n=20 each). * indicates p-value < 0.05 in Student’s t-test. (E) Ribbon representation of the overall CsPYLI -SB-AtHAB1 AN ternary complex. Ligand (SB, QB or ABA) binding in the ternary complex occurs in the closed conformation of the receptor that fits into the active site of the HAB1 phosphatase. (F) Superimposition of SB and QB in the ligand binding site of CsPYLI . The steric hinderance between the oxygen of SB’s SO2 group and Val110 of CsPYLI is represented as red arcs. (G) A detailed section of the SB (left) and QB (right) binding sites showing hydrogen bonding pattern of interactions between the ligand and CsPYLI :HAB1 complex.

Figure 2 shows structure-guided design of a synthetic CsPYLI ^5m receptor that shows enhanced sensitivity to SB. (A) Superimposition of the ligand binding pocket of CsPYLI - SB-AtHAB1AN (wheat) and either PYL10-ABA (green) (left panel) or CsPYLI ^5m-SB- AtHABIAN (grey) (center and right panels). The concatenated interactions of the four PYL10 residues (labeled from 2 to 5) are indicated as black arcs. The right panel highlights the higher water network (grey spheres) and methyl contacts of Leu112 with SB (lacking in Val112 of CsPYLI) in the ligand binding site of CsPYL1^5m. Residues changed in CsPYLI ^5m are labeled in grey (center and right panels). (B) The chain of interacting residues along the beta sheet in PYR1 to PYL10 receptors is labelled from 2 to 5, according to the structural detail showed in A. Number 1 corresponds to Leu79 of PYL10 and the equivalent position in other receptors. (C) Determination of the IC50 (nM) for inhibition of HAB1 by ABA and SB in presence of CsPYLI (circles) or CsPYL1^5m (triangles). Dose response curves are shown in presence of the indicated concentrations of ABA or SB. (D) Immunoblot analysis of protein extracts obtained from CsPYLI and CsPYLI ^5m lines. The epitope-tagged receptor was detected using anti-HA antibodies. Ponceau staining serves as a protein loading control. (E) Inhibition of seedling establishment by SB in wild-type Col-0 and either CsPYLI or CsPYLI ^5m overexpressing lines. Representative images are shown in the left panel and quantification of the experiment in the right panel. Values are mean ± SD of three independent experiments (n=20 each). * indicates p-value < 0.05 in Student’s t-test.

Figure 3 shows that iSB07 and iSB09 are SB derivatives that show improved agonist activity. (A) Chemical structure of iSB07 and iSB09 showing the swap of the SO2 group and CH2 of the benzyl group with respect to SB structure. The table shows the IC50 (nM) for inhibition of HAB1 by iSB07 and iSB09 in presence of CsPYLI or CsPYLI ^5m using pNPP as substrate. (B) PP2C inhibition assay in presence of 1 μM iSB07 or iSB09 and the indicated Arabidopsis ABA receptors. For HAB1 , pNPP was used as a substrate; for ABI1 , phosphopeptide was used as a substrate. (C) (D) The binding of ABA to CsPYLI in the presence of ANHAB1 shows similar affinity to the binding of iSB09 to CsPYLI ^5m. ITC data were obtained by repeated injections of ABA or iSB09 into a 1 :1 mixture of receptor: ANHAB1. (E, F) Native Red Electrophoresis (NRE) analysis of ligand-induced ternary complexes. Dose-response NRE analysis of ABA-induced (E) or iSB09-induced (F) AtPYL5-ligand-ANHAB1 complex. The fraction of ligand bound in the ternary complex was represented against free ABA or free iSB09 concentration in order to calculate apparent Kd.

Figure 4 shows structural insights into iSB-receptor-phosphatase complexes. (A) (B) Superimposition of the ligand binding pocket in CsPYL1-iSB07-AtHAB1AN, CsPYL1^5m- iSB07-AtHAB1AN, CsPYLI -iSB09-AtHAB1 AN and CsPYLI ^5m-iSB09-AtHAB1 AN complexes. The A panel shows interactions at the Trp lock (top) and the hydrogen bond network in the opposite part of the ligand (bottom). The B panel shows the hydrophobic tunnel of the receptors and interactions of the alkyl group close to the carbonyl oxygen. (C) Two-dimensional schematic representation of the iSB09 interactions in the ligand binding pocket of the CsPYLI ^5m-ligand-AtHAB1 AN ternary complex. The ligplot shows interactions of iSB09 at the Trp lock as well as H-bonds with Lys88 and Arg 108. (D) The conformations of the sulfonamide linker of SB, iSB07 and iSB09 represented as the a and p torsion angles of the pure compounds (Xtal) and in complex with CsPYLI and CsPYL1^5m is compared with the mean value of the observed torsion angles for identical fragments recorded at CSD. The ligands in the CsPYLI ^5m complexes relax to a more stable conformation than that observed in the CsPYLI complexes, showing torsion values closer to the mean value observed for chemical fragments in the CSD and in the crystals of pure compounds (Xtal).

Figure 5 shows that iSB07 and iSB09 compounds show enhanced agonist potency in vivo combined with the synthetic CsPYL1^5m receptor. (A) Determination of the IC50 for inhibition of seed germination by iSB07 and iSBiO9 in wild-type Col-0 or in lines expressing CsPYLI or CsPYL1^5m receptors. (B) Inhibition of seedling establishment by iSB07 and iSB09 in wild-type Col-0 or in lines expressing CsPYLI or CsPYLI ^5m receptors. (C) Quantification of ABA or iSB-mediated inhibition of root growth in the indicated genotypes. Seedlings were grown in plates supplemented with 0.1 % DMSO (mock), 10 μM SB or 10 μM ABA. Values are mean ± SD of three independent experiments (n=20 each). * indicates p-value < 0.05 (Student’s t-test) when compared to the same genotype mock-treated. (D) Representative photographs of the experiment described in (C) are shown in the right panel.

Figure 6 shows whole plant gas exchange analysis of stomatai conductance in CsPYLI ^5m plants treated with iSB07 or iSB09. (A) Stomatai conductance values of wildtype Col-0 and two transgenic lines expressing CsPYLI ^5m before and 56 min after spraying with different compounds (5 μM iSB07, 5 μM iSB09 or control). Asterisk denotes significant difference with respect to the pretreatment value of stomatai conductance (repeated measures ANOVA, GLM). Values show averages ± SE, n=5-7. (B) Timecourses of stomatai conductance after spraying with 5 μM iSB07, 5 μM iSB09 or control solutions at time 0. Values show averages ± SE, n=5-7. (C) Time-courses of stomatai conductance in relative units of wild-type Col-0 after spraying with 5 μM iSB07, 5 μM iSB09, 20 μM iSB07 or 20 μM iSB09. Only treatment with 20 μM iSB09 led to a significant reduction of stomatai conductance (repeated measures ANOVA, GLM). Values show averages ± SE, n=5-7. (D) Stomatai conductance values of wild-type Col- 0 and two transgenic lines before or 24 and 48 hours after spraying with different compounds (5 μM iSB07, 5 μM iSB09 or control). Asterisk denotes significant difference with respect to the pretreatment value of stomatai conductance (repeated measures ANOVA, GLM). Values show averages ± SE, n=4-7. (E) IR-images of representative Arabidopsis CsPYL15m plants 24 hours after being treated with 0.1 % DMSO (mock- treated control), or 50 μM ABA, iSB07 or iSB09. (F) Quantification of the temperature difference of the experiment described in E. (G) IR-images of representative N. benthamiana wild-type plants treated with DMSO (mock), 50 μM ABA or 100 μM iSB07/iSB09. (H) Quantification of the experiment described in G. (I) IR-images of representative wheat plants 72 hours after being treated with 0.1 % DMSO (mock-treated control) or 10 μM iSB09. Right, quantification of the increase in leaf temperature induced by iSB09 treatment.

Figure 7 shows drought resistance of 5m plants under long (LD) or short day (SD) conditions in greenhouse or plant growth chamber, respectively. (A) Col-0 and CsPYL1^5m plants were grown under LD conditions in greenhouse as described in methods and DMSO- (mock), 50 /zM ABA- or 50 /zM iSB09-treated. Three independent experiments were performed (n=10 each). Photographs represent plants observed in at least 50% of the total cases. Survival of CsPYL1^5m plants ABA-treated was over 50% in one experiment. (B) Gravimetric analysis of water loss in pots containing CsPYL1^5m plants reveals reduced water consumption in plants treated with ABA and iSB09 compared to mock-treated plants. Weight is shown at the indicated days after stopping irrigation and reflects water remaining in soil at each day compared to day 0. (C) Survival rate of Col- 0 and CsPYL1^5m plants 6 days after rewatering. (D) Col-0 and CsPYL1^5m plants were grown under SD conditions in growth chamber as described in methods and DMSO- (mock), 50 /zM ABA or 50 /zM iSB09-treated. Three independent experiments were performed (n=10 each). Photographs represent plants observed in at least 50% of the total cases. Drought indicates that watering was removed for 15 days. Five days after rewatering, photographs were taken and representative plants are shown. (E) Gravimetric analysis of water loss in pots containing CsPYL1^5m plants reveals reduced water consumption in plants treated with ABA and iSB09 compared to mock-treated plants. Weight is shown at the indicated days after stopping irrigation. (F) Survival rate of Col-0 and CsPYL1^5m plants 12 days after rewatering. (G) Enhanced growth of leaves in CsPYL1^5m plants treated with iSB09 compared to mock- or ABA-treated plants.

Figure 8 shows that the iSB09-CsPYL1^5m combination strongly induces an ABA-like transcriptional response in transgenic plants. (A) (B) iSB compounds induce the μMAP3K18-LUC reporter gene in wild type. (C) Volcano plots of RNA seq data obtained in wild type or CsPYL1^5m transgenic plants that were iSB09 or mock-treated. Genes upregulated (log2FC>1) or downregulated (log2FC<-1) with a false discovery rate<0.5 and pvalue were ploted. (D) Selected ABA responsive markers were plotted.

Figure 9 shows in vitro and in vivo activity of SB derivatives. (A) PP2C inhibition assays show enhanced inhibition of HAB1 by SB-01 with both CsPYL1^5m and CsPYLI . * indicates p<0.05 (Student’s t test) compared to SB at the same dosage. (B) Quantification of seedling establishment inhibition by 10 μM SB derivatives in CsPYLI ^5m compared to Col-0 wild type at 72 h (left) or 7 d (right).

Figure 10 shows that iSB09 induces ABA-responsive promoters and requires PYR1 and PYL1 for inhibition of seedling establishment. (A) RT-qPCR analysis of RAB18 and RD29B upregulation induced by ABA and iSB09 in the indicated genotypes. (B) Inhibition of seedling establishment by iSB09 in different Arabidopsis mutants lacking the indicated ABA receptors. 112458 is the abbreviation for pyr1 pyl1 pyl2 pyl4 pyl5 pyl8 sextuple mutant.

Figure 11 shows that CsPYL1^5m has lower affinity for ABA binding than CsPYLI . (A) A comparison of the ABA binding pocket in the CsPYLI ^5m-ABA-AtHAB1 AN (left) and CsPYLI -ABA-AtHAB1 AN (right) complexes. The corresponding sections of the unbiased omit Fo-Fc maps contoured at 3s are also shown. Note a reduction of the water mediated hydrogen bonds to the receptor in the vicinity of the carboxylate group for CsPYLI ^5m that leads to a looser binding of this moiety as shown by the weaken electron density. (B) PP2C inhibition assays (phosphopeptide as a substrate) show that ABA was less effective in CsPYLI ^5m than CsPYLI to inhibit HAB1 and ABI1 phosphatase activity. (C) Lower affinity of ABA for CsPYL1^5m than CsPYLI . ITC data were obtained by repeated injections of ABA into a 1 :1 mixture of receptor: ADNHAB1.

Figure 12 shows the numbering of the amino acid residues of each receptor described along the main text, including the five mutations introduced in CsPYLI ^5m, which do not alter the dimeric nature of the modified receptor. (A) Amino acid sequence alignment of Arabidopsis, CsPYLI and SIPYL1 ABA receptors identifies unique changes in PYL10 that were engineered into the synthetic CsPYLI ^5m receptor. The position of the five amino acid substitutions introduced in CsPYLI ^5m are indicated. Alignment was generated using GeneDoc and ClustalW software. The predicted secondary structure of the receptors is indicated, taking as a model the crystallographic structure of CsPYLI (Protein DataBank Code XXX) and using the ESPRIPT program(http://espript. ibcp.fr/ESPript/ESPript. (B) Engineering of the above mutations into CsPYLI ^5m does not affect the dimeric nature of the receptor. Native Red Electrophoresis (NRE) analysis was performed using AtPYLIO (monomeric receptor) and dimeric GST as protein markers.

Figure 13 shows a superimposition the bioinformatic models of PYR1_5m (wheat), PYL1_5m (pale green), PYL2_5m (light blue) and PYL3_5m (pale yellow) in complex with iSB09 and the experimentally determined crystal structure of CsPYL1_^5m (pale pink).

Figure 14 shows the effect of 10 mM iSB09 treatment on Gs in WT and PYL15m wheat plants (IRGA measurements). The LICOR-6400 system (LICOR Biosciences, NE, USA) was used to measure stomatai conductance and transpiration rate in 28-day-old plants grown in the greenhouse. At 2 h after the beginning of the light period, instantaneous measurements (n = 5) were taken under steady-state conditions at saturating light (1000 mmol m-2 s-1), 400 ppm CO2, 1-2.5 kPa vapor pressure difference, and ambient temperature (25 °C). Wild-type wheat plants were treated with 0.1 % DMSO (mock- treated control) or 10 μM iSB09. PYL1^5m overexpressing wheat plants were treated with 10 μM iSB09.

Figure 15 shows the control of stomatai aperture by iSB09 in tomato measured by IR thermography. (A) IR-images of representative tomato plants 90 min after being treated with 0.1 % DMSO (mock-treated control) or 10 mM iSB09. (B, C) Quantification of the temperature difference at 1 .5, 4 and 24 h or (B) 3 and 6 days after treatment.

Figure 16 shows tomato plants that were mock- or iSB09-treated and submitted to WD for 2d. (A) Well-watered tomato plants. Photographs of representative tomato plants under well-watered conditions. (B) Tomato plants submitted to water deficit. Plants were mock- or 10 mM iSB09-treated and watering was withheld for 2 d.

DETAILED DESCRIPTION

We have demonstrated that crop receptors can be tailored to enhance the binding of potential agrochemicals. This approach has not been addressed previously in crops. Given that ABA receptors can be functionally exchanged between different plant species, we suggest that the use of the orthogonal module formed by CsPYL1^5m and the iSB09 molecule could be effective to enhance drought resistance in many crops. The combined use of the CsPYL1^5m receptor-iSB09 chemical module presents a number of advantages. First, being a dimeric receptor, we did not observe constitutive activation of ABA signalling in vivo, which otherwise could be detrimental in the absence of stress. Even we could measure in vitro a reduction in ABA sensitivity of CsPYL1^5m compared to wild type (Fig 11). Second, iSB09 showed a powerful antitranspirant effect in drought experiments, either under short-day growth chamber conditions or under long-day greenhouse conditions. Third, it is a dynamic approach, which enables flexibility in the timing and intensity of the application. Fourth, persistence of the iSB09 molecule is expected since it is not an endogenous molecule that might follow pre-established catabolic pathways as ABA. Indeed, application of a relatively low dosage of iSB09 (5 μM) lasted at least for 48 h to induce reduction of stomatai conductance in CsPYL1^5m plants (Figure 5). Thus, in genetically altered plants, low dosage of chemical was required whereas in non-genetically altered plants spraying with 20 μM iSB09 was necessary to observe a significant reduction in stomatai conductance. Therefore, the CsPYL1^5m/iSB09 module would be even safer from an ecological perspective focused to limited use of agrochemicals. Finally, chemical synthesis of iSB09 is very simple, cheap and easily scalable.

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature. As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. In one embodiment, the isolated nucleic acid and the isolated nucleic acid used in the various methods and plants according to the invention is PYL/PYR cDNA. Examples of such sequences are given herein.

The terms “peptide”, "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

For the purposes of the invention, "genetically altered ", “transgene” or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or

(c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50bp, preferably at least 500bp, especially preferably at least 1000bp, most preferably at least 5000bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a genetically altered expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815 both incorporated by reference.

The aspects of the invention involve recombination DNA technology, mutagenesis or genome editing and exclude embodiments that are solely based on generating plants by traditional breeding methods.

General Chemical Definitions

The term “hydroxyl” or “hydroxy” as used herein refers to the group -OH.

The term “halo” or “halogen” as used herein refers to any radical of fluorine, chlorine, bromine or iodine.

The term “cyano” as used herein refers to the group -ON.

The term “alkyl” or “alkylene” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent/divalent radicals of up to twelve carbons. For example, an alkyl or alkylene group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Non-limiting examples of C1-C12 alkyl or alkylene groups include methyl(ene), ethyl(ene), propyl(ene), isopropyl(ene), butyl(ene), sec-butyl(ene), tertbutylene), 3-pentyl(ene), hexyl(ene) and octyl(ene) groups. Preferably, the term "alkyl" or “alkylene” as used herein, by itself or as part of another group, may refer to a straight or branched chain univalent/divalent radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms. An “optionally substituted alkyl” or “optionally substituted alkylene” group may include the substituents as described below for the term “optionally substituted”.

For example, an “optionally substituted alkyl” or “optionally substituted alkylene” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted alkyl” or “optionally substituted alkylene” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. The term “haloalkyl” as used herein, by itself or as part of another group, refers to both straight and branched chain radicals of up to twelve carbon atoms, comprising at least one halogen atom. For example, a haloalkyl group may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "haloalkyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, and comprising at least one halogen atom. For example, a “haloalkyl” group may be a fluoroalkyl or perfluoroalkyl group. Preferably, a “haloalkyl” group may be a C1-C6 fluoroalkyl group, or a C1-C6 perfluoroalkyl group. Even more preferably, a “haloalkyl” group may be a C₁-C₄ fluoroalkyl group, or a C₁-C₄ perfluoroalkyl group. For example, a “haloalkyl” group may include difluoromethyl, trifluoromethyl or pentafluoroethyl. The term “alkenyl” or “alkenylene” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent/divalent radicals of up to twelve carbons, and which comprise at least one carbon-carbon double bond. For example, an alkenyl or alkenylene group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "alkenyl" or “alkenylene” as used herein, by itself or as part of another group, may refer to a straight or branched chain univalent/divalent radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, and which comprise at least one carbon-carbon double bond. An “optionally substituted alkenyl” or “optionally substituted alkenylene” group may include the substituents as described below for the term “optionally substituted”. 12568980-1 For example, an “optionally substituted alkenyl” or “optionally substituted alkenylene” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted alkenyl” or “optionally substituted alkenylene” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. More preferably, an “optionally substituted alkenyl” or “optionally substituted alkenylene” group may include a C1-C6 fluoroalkenyl(ene) group, or a C2-C6 perfluoroalkenyl(ene) group. Even more preferably, an “optionally substituted alkenyl” or “optionally substituted alkenylene” group may include a C₂-C₄ fluoroalkenyl(ene) group, or a C₂-C₄ perfluoroalkenyl(ene) group. The term “alkynyl” or “alkynylene” as used herein, by itself or as part of another group, refers to both straight and branched chain univalent/divalent radicals of up to twelve carbons, and which comprise at least one carbon-carbon triple bond. For example, an alkynyl or alkynylene group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. For example, the term "alkynyl" or “alkynylene” as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, and which comprise at least one carbon-carbon triple bond. An “optionally substituted alkynyl” or “optionally substituted alkynylene” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted alkynyl” or “optionally substituted alkynylene” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. 12568980-1 Preferably, an “optionally substituted alkynyl” or “optionally substituted alkynylene” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. The term “cycloalkyl” as used herein refers to an alkyl group comprising a closed ring comprising from 3 to 8 carbon atoms, for example, 3 to 6 carbon atoms. For example, a cycloalkyl group may contain 3, 4, 5, 6, 7 or 8 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, (cyclohexyl)methyl, and (cyclohexyl)ethyl. An “optionally substituted cycloalkyl” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted cycloalkyl” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted cycloalkyl” group may include at least one substituent selected from halogen, hydroxy, a C₁-C₆ alkyl group, a C₁-C₆ haloalkyl group, a C₁-C₆ alkoxy group and a C₁-C₆ haloalkoxy group. The term “cycloalkenyl” as used herein refers to a closed non-aromatic ring comprising from 3 to 8 carbon atoms, for example, 3 to 6 carbon atoms, and which contains at least one carbon-carbon double bond. For example, a cycloalkenyl group may contain 3, 4, 5, 6, 7 or 8 carbon atoms. Non-limiting examples of cycloalkenyl groups include 1- cyclohexenyl, 4-cyclohexenyl, 1-cyclopentenyl, 2-cyclopentenyl. An “optionally substituted cycloalkenyl” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted cycloalkenyl” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. 12568980-1 Preferably, an “optionally substituted cycloalkenyl” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. The term “heterocycloalkyl” as used herein refers to a saturated or partially saturated 3 to 7 membered monocyclic, or 7 to 10 membered bicyclic ring system, which consists of carbon atoms and from one to four heteroatoms independently selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur heteroatoms may be optionally oxidised, the nitrogen may be optionally quaternised, and includes any bicyclic group in which any of the above-defined rings is fused to a benzene ring, and wherein the ring may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. Non-limiting examples of common saturated or partially saturated heterocycloalkyl groups include azetinyl, oxetanyl, tetrahydrofuranyl, pyranyl, piperidinyl, piperazinyl, pyrrolidinyl, imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, isochromanyl, chromanyl, pyrazolidinyl, pyrazolinyl, tetronoyl and tetramoyl groups. An “optionally substituted heterocycloalkyl” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted heterocycloalkyl” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted heterocycloalkyl” group may include at least one substituent selected from halogen, hydroxy, a C₁-C₆ alkyl group, a C₁-C₆ haloalkyl group, a C₁-C₆ alkoxy group and a C₁-C₆ haloalkoxy group. The term “alkoxy” as used herein, by itself or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. For example, an alkoxy group may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the "alkoxy" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, appended to the parent molecular moiety through an oxygen atom. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, 2-propoxy, 12568980-1 butoxy, tert-butoxy, pentyloxy, and hexyloxy. An “optionally substituted alkoxy” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted alkoxy” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted alkoxy” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. The term “haloalkoxy” as used herein, by itself or as part of another group, refers to both straight and branched chain radicals of up to twelve carbon atoms, comprising at least one halogen atom and being appended to the parent molecular moiety through an oxygen atom. For example, a haloalkoxy group may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. Preferably, the term "haloalkoxy" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from one to eight carbon atoms, more preferably one to six carbon atoms and even more preferably one to four carbon atoms, comprising at least one halogen atom and being appended to the parent molecular moiety through an oxygen atom. For example, a “haloalkoxy” group may be a fluoroalkoxy or perfluoroalkoxy group. Preferably, a “haloalkoxy” group may be a C₁-C₆ fluoroalkoxy group, or a C₁-C₆ perfluoroalkoxy group. Even more preferably, a “haloalkoxy” group may be a C₁-C₄ fluoroalkoxy group, or a C₁- C4 perfluoroalkoxy group. For example, a “haloalkyl” group may include difluoromethoxy, trifluoromethoxy or pentafluoromethoxy. The term “alkanoyl” as used herein by itself or as part of another group, refers to an alkyl group, as defined herein, and appended to the parent molecular moiety through an R^x- C(=O)O- group via the oxygen atom, where R^x represents the alkyl group. For example, an alkanoyl group may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 carbon atoms. 12568980-1 Preferably, the term "alkanoyl" as used herein, by itself or as part of another group, may refer to a straight or branched chain radical comprising from two to eight carbon atoms, more preferably two to six carbon atoms and even more preferably two to four carbon atoms, and being appended to the parent molecular moiety through an R^x-C(=O)O- group via the oxygen atom, where R^x represents the alkyl group. Non-limiting examples of alkanoyl groups include acetoxy, propionyloxy, butyryloxy and pentanoyloxy. An “optionally substituted alkanoyl” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted alkanoyl” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted alkanoyl” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. The term “amino” or “amine” as used herein refers to the group -NH₂. The term “aryl” or “arylene” as used herein by itself or as part of another group refers to monocyclic, bicyclic or tricyclic aromatic univalent/divalent radicals containing from 6 to 14 carbon atoms in the ring. Common aryl(ene) groups include C₆-C₁₄ aryl(ene), for example, C₆-C₁₀ aryl(ene). Non-limiting examples of C₆-C₁₄ aryl groups include phenyl(ene), naphthyl(ene), phenanthrenyl(ene), anthracenyl(ene), indenyl(ene), azulenyl(ene), biphenyl(ene), biphenylenyl(ene) and fluorenyl(ene) groups. An “optionally substituted aryl” or “optionally substituted arylene” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted aryl” or “optionally substituted arylene” group may include at least one substituent selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. 12568980-1 Preferably, an “optionally substituted aryl” or “optionally substituted arylene” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. More preferably, an “optionally substituted aryl” or “optionally substituted arylene” group may include at least one substituent selected from halogen, hydroxy and a C1-C6 alkyl group. The term “heteroaryl” or “heteroarylene” as used herein refers to aromatic univalent/divalent radicals having 5 to 14 ring atoms (for example, 5 to 10 ring atoms) and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms. Examples of heteroaryl(ene) groups include thienyl(ene) (thiophenyl(ene)), benzo[b]thienyl(ene), naphtho[2,3-b]thienyl(ene), thianthrenyl(ene), furyl(ene) (furanyl(ene)), pyranyl(ene), isobenzofuranyl(ene), chromenyl(ene), xanthenyl(ene), phenoxanthiinyl(ene), pyrrolyl(ene), including without limitation 2H-pyrrolyl(ene), imidazolyl(ene), pyrazolyl(ene), pyridyl(ene) (pyridinyl(ene)), including without limitation 2-pyridyl(ene), 3-pyridyl(ene), and 4-pyridyl(ene), pyrazinyl(ene), pyrimidinyl(ene), pyridazinyl(ene), indolizinyl(ene), isoindolyl(ene), 3H-indolyl(ene), indolyl(ene), indazolyl(ene), purinyl(ene), 4H-quinolizinyl(ene), isoquinolyl(ene), quinolyl(ene), phthalazinyl(ene), naphthyridinyl(ene), quinozalinyl(ene), cinnolinyl(ene), pteridinyl(ene), carbazolyl(ene), β-carbolinyl(ene), phenanthridinyl(ene), acrindinyl(ene), perimidinyl(ene), phenanthrolinyl(ene), phenazinyl(ene), isothiazolyl(ene), phenothiazinyl(ene), isoxazolyl(ene), furazanyl(ene), phenoxazinyl(ene), 1,4- dihydroquinoxaline-2,3-dione(ene), 7-aminoisocoumarin(ene), pyrido[1,2-α]pyrimidin-4- one(ene), pyrazolo[1,5-α]pyrimidinyl(ene), including without limitation pyrazolo[1,5- α]pyrimidin-3-yl(ene), 1,2-benzoisoxazol-3-yl(ene), benzimidazolyl(ene), 2- oxindolyl(ene) and 2-oxobenzimidazolyl(ene). Where the heteroaryl(ene) group contains a nitrogen atom in a ring, such nitrogen atom may be in the form of an N-oxide, e.g., a pyridyl(ene) N-oxide, pyrazinyl(ene) N-oxide and pyrimidinyl(ene) N-oxide. An “optionally substituted heteroaryl” or “optionally substituted heteroarylene” group may include the substituents as described below for the term “optionally substituted”. For example, an “optionally substituted heteroaryl” or “optionally substituted heteroarylene” group may include at least one substituent selected from hydroxy, 12568980-1 halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, an “optionally substituted heteroaryl” or “optionally substituted heteroarylene” group may include at least one substituent selected from halogen, hydroxy, a C1-C6 alkyl group, a C1-C6 haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. As described herein, compounds may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogen atoms of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisaged by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. For example, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from hydroxy, halogen, cyano, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkoxy, haloalkoxy, alkanoyl, amino, aryl and heteroaryl. Preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from halogen, hydroxy, a C₁-C₆ alkyl group, a C₁-C₆ haloalkyl group, a C1-C6 alkoxy group and a C1-C6 haloalkoxy group. More preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from halogen, hydroxy, a C1-C4 alkyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group and a C1-C4 haloalkoxy group. 12568980-1 Even more preferably, the term “optionally substituted” as used herein may refer to when at least one substituent is selected from fluoro, chloro, hydroxy, a methyl group, a trifluoromethyl group, a methoxy group and a trifluoromethoxy group. As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined. The term “salt” as used herein refers to salts of the compounds as described herein that are derived from suitable inorganic and organic acids and bases. Examples of salts of a basic group include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p- toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C₁-C₄ alkyl)₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate. Certain compounds of the present disclosure may exist in unsolvated forms as well as solvated forms, including hydrated forms. “Hydrate” refers to a complex formed by combination of water molecules with molecules or ions of the solute. “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the 12568980-1 solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Solvate is meant to include hydrate. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetra hydrofuran, dimethylsulfoxide, and water. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

“Tautomer” means compounds produced by the phenomenon wherein a proton of one atom of a molecule shifts to another atom (See, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms and Structures, Fourth Edition, John Wiley & Sons, pages 69-74 (1992)). The tautomers also refer to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. Examples include keto-enol tautomers, such as acetone/propen-2-ol, imine-enamine tautomers and the like, ring-chain tautomers, such as glucose/2, 3,4,5, 6- penta hydroxyhexanal and the like, the tautomeric forms of heteroaryl groups containing a -N=C(H)- NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Where the compound contains, for example, a keto or oxime group or an aromatic moiety, tautomeric isomerism (‘tautomerism’) may occur. The compounds described herein may have one or more tautomers and therefore include various isomers. A skilled person would recognise that other tautomeric ring atom arrangements are possible. All such isomeric forms of these compounds are expressly included in the present disclosure.

“Isomers” mean compounds having identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. “Stereoisomer” and “stereoisomers” refer to compounds that exist in different stereoisomeric forms if they possess one or more asymmetric centres or a double bond with asymmetric substitution and, therefore, may be produced as individual stereoisomers or as mixtures. Stereoisomers include enantiomers and diastereomers. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric centre, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer may be characterised by the absolute configuration of its asymmetric centre and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarised light and designated as dextrorotatory or laevorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound may exist as either individual enantiomers or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”. Unless otherwise indicated, the description is intended to include individual stereoisomers as well as mixtures. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of A^DVANCED O^RGANIC C^HEMISTRY, 6th edition J. March, John Wiley and Sons, New York, 2007) differ in the chirality of one or more stereocentres. The term “deuterated” as used herein alone or as part of a group, means substituted by deuterium atoms. The term “deuterated analogue” as used herein alone or as part of a group, means deuterium atoms substituted in place of hydrogen atoms. The deuterated analogue of the disclosure may be a fully or partially deuterium substituted derivative. In some embodiments, the deuterium substituted derivative of the disclosure holds a fully or partially deuterium substituted alkyl, aryl or heteroaryl group. The disclosure also embraces isotopically-labelled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that may be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶Cl, and ¹²⁵I. Unless otherwise stated, when a position is designated specifically as “H” or “hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition or its isotopes, such as deuterium (D) or tritium (³H). Certain isotopically-labelled compounds of the present disclosure (e.g., those labelled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) and fluorine-18 (i.e., ¹⁸F) isotopes are useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., 12568980-1 increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labelled compounds of the present disclosure may generally be prepared by following procedures analogous to those described in the Schemes and in the Examples herein below, by substituting an isotopically labelled reagent for a non-isotopically labelled reagent. Compounds In an embodiment, a compound may have a structure according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof:

In Formula I, R¹ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; especially optionally substituted alkyl. For example, R¹ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C6-C14 aryl and optionally substituted 5-14 membered heteroaryl; especially optionally substituted C1-C6 alkyl. Preferably, R¹ may be independently selected from hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, C6-C14 aryl and 5-14 membered heteroaryl; especially C1-C6 alkyl. 12568980-1 More preferably, R¹ may be hydrogen, C1-C6 alkyl or C1-C6 haloalkyl; especially C1-C6 alkyl. Even more preferably, R¹ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R¹ is methyl. In Formula I, R² may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. For example, R² may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C₁-C₆ alkoxy, optionally substituted C₂-C₆ alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R² may be independently selected from hydrogen, hydroxy, halogen, cyano, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 3-10 membered heterocycloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkanoyl, amino, C₆-C₁₄ aryl and 5-14 membered heteroaryl. More preferably, R² may be hydrogen, C₁-C₆ alkyl or C₁-C₆ haloalkyl. Even more preferably, R² may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R² is hydrogen. In Formula I, R³ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted 12568980-1 heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; especially optionally substituted alkyl and optionally substituted cycloalkyl. For example, R³ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C6-C14 aryl and optionally substituted 5-14 membered heteroaryl; especially optionally substituted C1-C6 alkyl and optionally substituted C3-C8 cycloalkyl. Preferably, R³ may be independently selected from hydrogen, hydroxy, halogen, cyano, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 3-10 membered heterocycloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, C6-C14 aryl and 5-14 membered heteroaryl; especially C1-C6 alkyl and C₃-C₈ cycloalkyl. More preferably, R³ may be hydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl or C₃-C₈ cycloalkyl; especially C₁-C₆ alkyl or C₃-C₈ cycloalkyl. In a preferred embodiment, R₃ may be C₂-C₆ alkyl, more preferably C₂-C₄ alkyl. In a preferred embodiment, the C₁-₆ alkyl, C₂-C₆ alkyl or C₂-C₄ alkyl group may be a straight-chain. Even more preferably, R³ may be methyl, ethyl or cyclopropyl. It is particularly preferred that R³ is ethyl. In Formula I, R⁴ may be selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. 12568980-1 For example, R⁴ may be independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C6-C14 aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R⁴ may be independently selected from a structure according to Formulae 1- 1 or 1-2, hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1- C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkanoyl, amino, C₆-C₁₄ aryl and 5-14 membered heteroaryl. More preferably, R⁴ may be hydrogen, C₁-C₆ alkyl or C₁-C₆ haloalkyl. Even more preferably, R⁴ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R⁴ is hydrogen. In Formula I, R⁵ may be selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. For example, R⁵ may be independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl. 12568980-1 Preferably, R⁵ may be independently selected from a structure according to Formulae 1- 1 or 1-2, hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1- C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, C6-C14 aryl and 5-14 membered heteroaryl. More preferably, R⁵ may be hydrogen, C1-C6 alkyl or C1-C6 haloalkyl. Even more preferably, R⁵ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R⁵ is hydrogen. For example, R⁶ may be independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C₂-C₆ alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R⁶ may be independently selected from a structure according to Formulae 1- 1 or 1-2, hydrogen, hydroxy, halogen, cyano, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 3-10 membered heterocycloalkyl, C₁- C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkanoyl, amino, C₆-C₁₄ aryl and 5-14 membered heteroaryl. More preferably, R⁶ may be a structure according to Formulae 1-1 or 1-2, hydrogen, C₁- C₆ alkyl or C₁-C₆ haloalkyl. Even more preferably, R⁶ may be a structure according to Formulae 1-1 or 1-2. It is particularly preferred that R⁶ is a structure according to Formula 1-1. In Formula I, R⁷ may be selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted 12568980-1 alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. For example, R⁷ may be independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R⁷ may be independently selected from a structure according to Formulae 1- 1 or 1-2, hydrogen, hydroxy, halogen, cyano, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1- C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, C6-C14 aryl and 5-14 membered heteroaryl. More preferably, R⁷ may be hydrogen, C₁-C₆ alkyl or C₁-C₆ haloalkyl. Even more preferably, R⁷ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R⁷ is hydrogen. In Formula 1-1, R⁸ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. For example, R⁸ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substituted C₃-C₈ cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally 12568980-1 substituted C1-C6 alkoxy, optionally substituted C2-C6 alkanoyl, optionally substituted amino, optionally substituted C6-C14 aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R⁸ may be independently selected from hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, C6-C14 aryl and 5-14 membered heteroaryl. More preferably, R⁸ may be hydrogen, C1-C6 alkyl or C1-C6 haloalkyl. Even more preferably, R⁸ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R⁸ is hydrogen. In Formula 1-1, R⁹ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; especially optionally substituted aryl. For example, R⁹ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈ cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C₁-C₆ alkoxy, optionally substituted C₂-C₆ alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl; especially optionally substituted C6-C14 aryl. Preferably, R⁹ may be independently selected from hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkanoyl, amino, optionally substituted C₆-C₁₄ aryl and 5-14 membered heteroaryl; especially optionally substituted C6-C14 aryl. 12568980-1 More preferably, R⁹ may be hydrogen or optionally substituted C6-C14 aryl; especially optionally substituted C6-C14 aryl. Even more preferably, R⁹ may be optionally substituted phenyl. It is particularly preferred that R⁹ is selected from phenyl, halogen-substituted phenyl and hydroxy-substituted phenyl. In Formula 1-2, R¹⁰ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl. For example, R¹⁰ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C₂-C₆ alkynyl, optionally substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₈ cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C₁-C₆ alkoxy, optionally substituted C₂-C₆ alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl. Preferably, R¹⁰ may be independently selected from hydrogen, hydroxy, halogen, cyano, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 3-10 membered heterocycloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkanoyl, amino, C₆-C₁₄ aryl and 5-14 membered heteroaryl. More preferably, R¹⁰ may be hydrogen, C1-C6 alkyl or C1-C6 haloalkyl. Even more preferably, R¹⁰ may be hydrogen, methyl, ethyl, propyl or butyl. It is particularly preferred that R¹⁰ is hydrogen. 12568980-1 In Formula 1-2, R¹¹ may be selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; especially optionally substituted aryl. For example, R¹¹ may be independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C3-C8 cycloalkenyl, optionally substituted 3-10 membered heterocycloalkyl, optionally substituted C₁-C₆ alkoxy, optionally substituted C₂-C₆ alkanoyl, optionally substituted amino, optionally substituted C₆-C₁₄ aryl and optionally substituted 5-14 membered heteroaryl; especially optionally substituted C₆-C₁₄ aryl. Preferably, R¹¹ may be independently selected from hydrogen, hydroxy, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 3-10 membered heterocycloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₂-C₆ alkanoyl, amino, optionally substituted C₆-C₁₄ aryl and 5-14 membered heteroaryl; especially optionally substituted C₆-C₁₄ aryl. More preferably, R¹¹ may be hydrogen or optionally substituted C₆-C₁₄ aryl; especially optionally substituted C₆-C₁₄ aryl. Even more preferably, R¹¹ may be optionally substituted phenyl. It is particularly preferred that R¹¹ is selected from phenyl, halogen-substituted phenyl and hydroxy-substituted phenyl. In Formula 1-1, L1 may be selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene and optionally substituted heteroarylene; especially optionally substituted alkylene. For example, L₁ may be selected from optionally substituted C₁-C₆ alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 alkynylene, optionally 12568980-1 substituted C6-C14 arylene and optionally substituted 5-14 membered heteroarylene; especially optionally substituted C1-C6 alkylene. Preferably, L1 may be selected from C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene, C6-C14 arylene and 5-14 membered heteroarylene; especially C1-C6 alkylene. More preferably, L1 may be selected from C1-C4 alkylene, C2-C4 alkenylene and C2-C4 alkynylene; especially C1-C4 alkylene. Even more preferably, L1 may selected from methylene, ethylene, propylene and butylene. It is particularly preferred that L₁ is methylene. In Formula 1-2, L₂ may be selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene and optionally substituted heteroarylene; especially optionally substituted alkylene. For example, L₂ may be selected from optionally substituted C₁-C₆ alkylene, optionally substituted C₂-C₆ alkenylene, optionally substituted C₂-C₆ alkynylene, optionally substituted C₆-C₁₄ arylene and optionally substituted 5-14 membered heteroarylene; especially optionally substituted C₁-C₆ alkylene. Preferably, L₂ may be selected from C₁-C₆ alkylene, C₂-C₆ alkenylene, C₂-C₆ alkynylene, C₆-C₁₄ arylene and 5-14 membered heteroarylene; especially C₁-C₆ alkylene. More preferably, L₂ may be selected from C₁-C₄ alkylene, C₂-C₄ alkenylene and C₂-C₄ alkynylene; especially C₁-C₄ alkylene. Even more preferably, L2 may selected from methylene, ethylene, propylene and butylene. It is particularly preferred that L2 is methylene. In Formula 1-1, a1 may be 1 to 6. 12568980-1 Preferably, a1 may be 1 to 4. More preferably, a1 may be 1 or 2. Even more preferably, a1 may be 1. In Formula 1-2, a2 may be 1 to 6. Preferably, a2 may be 1 to 4. More preferably, a2 may be 1 or 2. Even more preferably, a2 may be 1. In Formula I, at least one of R4 to R7 may be a structure according to Formulae 1-1 or 1- 2. Preferably, in Formula I, one of R₄ to R₇ may be a structure according to Formulae 1-1 or 1-2. Preferably, the compound may have a structure according to Formula II:

wherein R1 to R11, L1, L2, a1 and a2 are as defined above. Preferably, the compound may have a structure according to Formula III:

wherein R₁, R₃, R₆, R₈ to R₁₁, L₁, L₂, a1 and a2 are as defined above. 12568980-1 Preferably, the compound may be selected from any one of Compounds 1 to 9:

In embodiments of the present invention relating to compounds as described herein, the compound may not be Compound 1 :

In embodiments of the present invention relating to compositions as described herein, uses in enhancing abiotic stress resistance in a plant as described herein, and/or methods of enhancing abiotic stress resistance in a plant as described herein, the compound may not be Compound 1. However, the compositions as described herein, uses in enhancing abiotic stress resistance in a plant as described herein, and/or methods of enhancing abiotic stress resistance in a plant as described herein are not necessarily limited thereto.

As used herein, may refer to a single or double bond.

As used herein, may refer to a connection point to the rest of the compound. Compositions

In an embodiment, a composition as described herein may contain a compound as described herein and a carrier.

A carrier in a composition as described herein is any material with which the active ingredient is formulated to facilitate application to a surface, or to facilitate storage, transport or handling. A carrier may be a solid or a liquid, including a material which is normally gaseous but which has been compressed to form a liquid.

In an embodiment, the composition may be formulated for agricultural use.

An agrochemically acceptable carrier may be used.

Any of the carriers normally used in formulating agrochemical (e.g. herbicidal, fungicidal or pesticidal) compositions may be used.

Suitable solid carriers include natural and synthetic clays and silicates, for example natural silicas such as diatomaceous earths; magnesium silicates, for example talcs; magnesium aluminium silicates, for example attapulgites and vermiculites; aluminium silicates, for example kaolinites, montmorillonites and micas; calcium carbonate; calcium sulfate; ammonium sulfate; synthetic hydrated silicon oxides and synthetic calcium or aluminium silicates; elements, for example carbon and sulfur; natural and synthetic resins, for example coumarone resins, polyvinyl chloride, and styrene polymers and copolymers; solid polychlorophenols; bitumen; waxes; and solid fertilisers, for example superphosphates.

Suitable liquid carriers include water; alcohols, for example isopropanol and glycols; ketones, for example acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone; ethers; aromatic or araliphatic hydrocarbons, for example benzene, toluene and xylene; petroleum fractions, for example kerosene and light mineral oils; chlorinated hydrocarbons, for example carbon tetrachloride, perchloroethylene and trichloroethane. Mixtures of different liquids are often suitable. Agricultural compositions are often formulated and transported in a concentrated form which is subsequently diluted by the user before application. The presence of small amounts of a carrier which is a surface-active agent facilitates this process of dilution. Thus, at least one carrier in a composition as described herein may be a surface-active agent. For example, the composition may contain at least two carriers, at least one of which is a surface-active agent.

A surface-active agent may be an emulsifying agent, a dispersing agent or a wetting agent; it may be nonionic or ionic. Examples of suitable surface-active agents include the sodium or calcium salts of polyacrylic acids and lignin sulfonic acids; the condensation of fatty acids or aliphatic amines or amides containing at least 12 carbon atoms in the molecule with ethylene oxide and/or propylene oxide; fatty acid esters of glycerol, sorbitol, sucrose or pentaerythritol; condensates of these with ethylene oxide and/or propylene oxide; condensation products of fatty alcohol or alkyl phenols, for example p-octylphenol or p-octylcresol, with ethylene oxide and/or propylene oxide; sulfates or sulfonates of these condensation products; alkali or alkaline earth metal salts, preferably sodium salts, of sulfuric or sulfonic acid esters containing at least 10 carbon atoms in the molecule, for example sodium lauryl sulfate, sodium secondary alkyl sulfates, sodium salts of sulfonated castor oil, and sodium alkylaryl sulfonates such as dodecylbenzene sulfonate; and polymers of ethylene oxide and copolymers of ethylene oxide and propylene oxide.

The compositions as described herein may for example be formulated as wettable powders, dusts, granules, solutions, emulsifiable concentrates, emulsions, suspension concentrates and aerosols. Wettable powders usually contain 25, 50 or 75% w/w of active ingredient and usually contain in addition to solid inert carrier, 3-10% w/w of a dispersing agent and, where necessary, 0-10% w/w of stabiliser(s) and/or other additives such as penetrants or stickers. Dusts are usually formulated as a dust concentrate having a similar composition to that of a wettable powder but without a dispersant, and are diluted in the field with further solid carrier to give a composition usually containing 0.5-10% w/w of active ingredient. Granules are usually prepared to have a size between 10 and 100 BS mesh (1.676 - 0.152 mm), and may be manufactured by agglomeration or impregnation techniques. Generally, granules will contain 0.5-75% w/w active ingredient and 0-10% w/w of additives such as stabilisers, surfactants, slow release modifiers and binding agents. The so-called "dry flowable powders" consist of relatively small granules having a relatively high concentration of active ingredient. Of particular interest in current practice are the water-dispersible granular formulations. These are in the form of dry, hard granules that are essentially dust-free, and are resistant to attrition on handling, thus minimising the formation of dust. On contact with water, the granules readily disintegrate to form stable suspensions of the particles of active material. Such formulations contain 90% or more by weight of finely divided active material, 3-7% by weight of a blend of surfactants, which act as wetting, dispersing, suspending and binding agents, and 1-3% by weight of a finely divided carrier, which acts as a resuspending agent. Emulsifiable concentrates usually contain, in addition to a solvent and, when necessary, co-solvent, 10-50% w/v active ingredient, 2-20% w/v emulsifiers and 0-20% w/v of other additives such as stabilisers, penetrants and corrosion inhibitors. Suspension concentrates are usually compounded so as to obtain a stable, nonsedimenting flowable product and usually contain 10-75% w/w active ingredient, 0.5- 15% w/w of dispersing agents, 0.1-10% w/w of suspending agents such as protective colloids and thixotropic agents, 0-10% w/w of other additives such as defoamers, corrosion inhibitors, stabilisers, penetrants and stickers, and water or an organic liquid in which the active ingredient is substantially insoluble; certain organic solids or inorganic salts may be present dissolved in the formulation to assist in preventing sedimentation or as anti-freeze agents for water. Aerosol recipes are usually composed of the active ingredient, solvents, furthermore auxiliaries such as emulsifiers, perfume oils, if appropriate stabilisers, and, if required, propellants.

The specific choice of a carrier, if any, to be utilised in achieving the desired intimate admixture with the final product may be any carrier conventionally used in insect repellent formulations. The carrier, moreover, may also be one that will not be harmful to the environment. Accordingly, the carrier may be any one of a variety of commercially available organic and inorganic liquid, solid, or semi-solid carriers or carrier formulations usable in formulating insect repellent products. For example, the carrier may include silicone, petrolatum, lanolin or many of several other well-known carrier components.

Examples of organic liquid carriers include liquid aliphatic hydrocarbons (e.g., pentane, hexane, heptane, nonane, decane and their analogs) and liquid aromatic hydrocarbons. Examples of other liquid hydrocarbons include oils produced by the distillation of coal and the distillation of various types and grades of petrochemical stocks, including kerosene oils which are obtained by fractional distillation of petroleum. Other petroleum oils include those generally referred to as agricultural spray oils (e.g., the so-called light and medium spray oils, consisting of middle fractions in the distillation of petroleum and which are only slightly volatile). Such oils are usually highly refined and may contain only minute amounts of unsaturated compounds. Such oils, moreover, are generally paraffin oils and accordingly may be emulsified with water and an emulsifier, diluted to lower concentrations, and used as sprays. Tall oils, obtained from sulfate digestion of wood pulp, like the paraffin oils, may similarly be used. Other organic liquid carriers may include liquid terpene hydrocarbons and terpene alcohols such as alphapinene, dipentene, terpineol, and the like.

Other carriers include silicone, petrolatum, lanolin, liquid hydrocarbons, agricultural spray oils, paraffin oil, tall oils, liquid terpene hydrocarbons and terpene alcohols, aliphatic and aromatic alcohols, esters, aldehydes, ketones, mineral oil, higher alcohols, finely divided organic and inorganic solid materials.

In addition to the above-mentioned liquid hydrocarbons, the carrier may contain conventional emulsifying agents which may be used for causing the compounds to be dispersed in, and diluted with, water for end-use application.

Still other liquid carriers may include organic solvents such as aliphatic and aromatic alcohols, esters, aldehydes, and ketones. Aliphatic monohydric alcohols include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, and tert-butyl alcohols. Suitable alcohols include glycols (such as ethylene and propylene glycol) and pinacols. Suitable polyhydroxy alcohols include glycerol, arabitol, erythritol, sorbitol, and the like. Suitable cyclic alcohols include cyclopentyl and cyclohexyl alcohols.

Conventional aromatic and aliphatic esters, aldehydes and ketones may be used as carriers, and occasionally are used in combination with the above-mentioned alcohols. Still other liquid carriers include relatively high-boiling petroleum products such as mineral oil and higher alcohols (such as cetyl alcohol). Additionally, conventional or so- called “stabilisers” (e.g., tert-butyl sulfinyl dimethyl dithiocarbonate) may be used in conjunction with, or as a component of, the carrier or carriers comprising the compositions as described herein. Solid carriers which may be used in the compositions as described herein include finely divided organic and inorganic solid materials.

Suitable finely divided solid inorganic carriers include siliceous minerals such as synthetic and natural clay, bentonite, attapulgite, fuller's earth, diatomaceous earth, kaolin, mica, talc, finely divided quartz, and the like, as well as synthetically prepared siliceous materials, such as silica aerogels and precipitated and fume silicas. Examples of finely divided solid organic materials include cellulose, sawdust, synthetic organic polymers, and the like. Examples of semi-solid or colloidal carriers include waxy solids, gels (such as petroleum jelly), lanolin, and the like, and mixtures of well-known liquid and solid substances which may provide semi-solid carrier products, for providing effective repellency.

The compositions as described herein may be formulated and packaged so as to deliver the product in a variety of forms including as a solution, suspension, gel, film or spray, depending on the preferred method of use. The carrier may be an aerosol composition adapted to disperse the compounds into the atmosphere by means of a compressed gas.

In an embodiment, the compositions as described herein may comprise at least one additional active ingredient.

For example, the additional active ingredient may be a herbicide or a pesticide.

PYL mutant nucleic acid sequences and genetically altered plants

Thus, the term “PYL” or “PYL receptor polypeptide" refers to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL), and refers to a protein characterized in part by the presence of one or more or all of a polyketide cyclase domain 2 (PF 10604), a polyketide cyclase domain 1 (PF03364), and a Bet V I domain (PF03364), which in wildtype form mediates abscisic acid (ABA) and ABA analog signalling.

In another aspect of the invention there is provided an isolated plant PYL mutant nucleic acid and isolated mutant plant PYL polypeptides encoded by said mutant nucleic acid wherein said mutant polypeptide comprises one or more amino acid mutations or modifications, preferably one or more substitutions, compared to the wild type sequence. In one embodiment, the one or more mutations are in the ABA-binding pocket and/or indirectly affect the configuration or orientation of the ABA-binding pocket, as for example, explained in Example 3. In one embodiment, when the PYL is a dimer, the one or more mutations do not affect dimerization of the receptor.

In one embodiment, the “ABA-binding pocket” may comprise one or more of the following residues: R145, P117, E123, R108, K88, N196, H89, S121 , 191 Y149, 1139, L146, F188, V192, V112, A118, H144 V110, F90 and F137 in SEQ ID NO: 1 or a corresponding position in a homologous sequence, or a corresponding position in PYR1 , PYL2 and PYL3.

In one embodiment the one or more mutations may be in one or more p-loop of the receptor.

Thus, the mutant polypeptide/proteins according to the invention are non-naturally occurring peptides which can be generated by site-directed mutagenesis and introduced stably into plants and expressed in said plants to produce stable genetically altered plants with improved traits. Said plants are preferably homozygous for the transgene.

In one aspect of the invention, there is provided an isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYL or PYR polypeptide. In a preferred embodiment, the PYL or PYR is a dimeric receptor. More preferably, the PYL is selected from PYL1 , PYL2 and PYL3 and the PYR is PYR1. In one embodiment, the PYL is PYL1.

In one embodiment, the nucleic acid comprises at least one mutation in the nucleic acid sequence encoding the PYL/PYR polypeptide, compared to the wild-type sequence. In one embodiment, the mutation is at least one mutation selected from positions 334-336, 403-405, 409-411 , 457-459 and 502-504 of SEQ ID NO: 3 or a corresponding position in a homologue or orthologue sequence.

In one embodiment, the mutation leads to a substitution in a PYL1 polypeptide sequence. More preferably, the substitution is selected from one or more of the following substitutions: a V to L substitution at position 112 of SEQ ID NO: 1 or a corresponding position in a homologue or orthologue sequence; a F to I substitution at position 137 of SEQ ID NO: 1 or a corresponding position in a homologue or orthologue sequence; a T to L substitution at position 135 of SEQ ID NO: 1 or a corresponding position in a homologue or orthologue sequence; a T to I substitution at position 153 of SEQ ID NO: 1 or a corresponding position in a homologue or orthologue sequence; and a V to A substitution at position 168 of SEQ ID NO: 1 or a corresponding position in a homologue or orthologue sequence.

In one embodiment, the mutant PYL polypeptide comprises at least one, at least two, at least three or at least four of the above substitutions. In another embodiment, the mutant PYL polypeptide comprises all five substitutions. An isolated nucleic acid comprises all five mutations in PYL1 is referred to herein as PYL1^5m. In another embodiment, the PYL polypeptide does not contain any other mutations, compared to the wild-type (i.e. nonmutated sequence). In one embodiment, the PYL1^5m nucleic acid preferably comprises or consists of SEQ ID NO: 4 or a functional variant or homologue thereof. In another embodiment, the PYL1^5m- amino acid preferably comprises or consists of SEQ ID NO: 2 or a functional variant or homologue thereof.

As shown in the Examples, overexpression of PYL1^5m showed increased sensitivity to SB (at concentrations as low as 10μM) (as shown by a significant decrease in seeding establishment compared to plants expressing wild-type PYL1 ; Figure 2E). However, these plants showed an even greater level of sensitivity to the SB derivatives, iSB07 and iSB09. As shown in Figures 5 and 6, plants expressing PYL1^5m were markedly more sensitive to agonist-mediated inhibition of seed germination, seed establishment and stomatai conductance.

In another embodiment, the mutation leads to a substitution in a PYR1 polypeptide sequence. More preferably, the substitution is selected from one or more of the following substitutions: a V to L substitution at position 83 of SEQ ID NO: 5 or a corresponding position in a homologue or orthologue sequence; a F to I substitution at position 108 of SEQ ID NO: 5 or a corresponding position in a homologue or orthologue sequence; a T to L substitution at position 106 of SEQ ID NO: 5 or a corresponding position in a homologue or orthologue sequence; a T to I substitution at position 124 of SEQ ID NO: 5 or a corresponding position in a homologue or orthologue sequence; and a V to A substitution at position 139 of SEQ ID NO: 5 or a corresponding position in a homologue or orthologue sequence.

In one embodiment, the mutant PYR polypeptide comprises at least one, at least two, at least three or at least four of the above substitutions. In another embodiment, the mutant PYR polypeptide comprises all five substitutions. An isolated nucleic acid comprises all five mutations in PYR1 is referred to herein as PYR1^5m. In another embodiment, the PYR polypeptide does not contain any other mutations, compared to the wild-type (i.e. nonmutated sequence). In one embodiment, the PYR1^5m amino acid preferably comprises or consists of SEQ ID NO: 11 or a functional variant or homologue thereof. The wild-type (non-mutated) nucleic acid sequence is shown in SEQ ID NO: 6.

In another embodiment, the mutation leads to a substitution in a PYL2 polypeptide sequence. More preferably, the substitution is selected from one or more of the following substitutions: a V to L substitution at position 87 of SEQ ID NO: 7 or a corresponding position in a homologue or orthologue sequence; a F to I substitution at position 112 of SEQ ID NO: 7 or a corresponding position in a homologue or orthologue sequence; a T to I substitution at position 128 of SEQ ID NO: 7 or a corresponding position in a homologue or orthologue sequence; and a V to A substitution at position 145 of SEQ ID NO: 7 or a corresponding position in a homologue or orthologue sequence.

In one embodiment, the mutant PYL2 polypeptide comprises at least one, at least two or at least three of the above substitutions. In another embodiment, the mutant PYL2 polypeptide comprises all four substitutions. An isolated nucleic acid comprises all four mutations in PYL2 is referred to herein as PYL2^4m. In another embodiment, the PYL2 polypeptide does not contain any other mutations, compared to the wild-type (i.e. nonmutated sequence). In one embodiment, the PYL2^4m amino acid preferably comprises or consists of SEQ ID NO: 12 or a functional variant or homologue thereof. The wild-type (non-mutated) nucleic acid sequence is shown in SEQ ID NO: 8..

In another embodiment, the mutation leads to a substitution in a PYL3 polypeptide sequence. More preferably, the substitution is selected from one or more of the following substitutions: a V to L substitution at position 107 of SEQ ID NO: 9 or a corresponding position in a homologue or orthologue sequence; a F to I substitution at position 132 of SEQ ID NO: 9 or a corresponding position in a homologue or orthologue sequence; a T to I substitution at position 148 of SEQ ID NO: 9 or a corresponding position in a homologue or orthologue sequence; and a V to A substitution at position 168 of SEQ ID NO: 9 or a corresponding position in a homologue or orthologue sequence.

In one embodiment, the mutant PYL3 polypeptide comprises at least one, at least two, or at least three of the above substitutions. In another embodiment, the mutant PYL3 polypeptide comprises all four substitutions. An isolated nucleic acid comprises all four mutations in PYL3 is referred to herein as PYL3^4m. In another embodiment, the PYL3 polypeptide does not contain any other mutations, compared to the wild-type (i.e. nonmutated sequence). In one embodiment, the PYL3^4m amino acid preferably comprises or consists of SEQ ID NO: 13 or a functional variant or homologue thereof. The wild-type (non-mutated) nucleic acid sequence is shown in SEQ ID NO: 10.

Suitable homologues or orthologues and the corresponding positions therein can be identified by sequence comparisons and identification of conserved domains using databases such as NCBI and Plant ensemble and alignment programmes known to the skilled person. The function of the homologue or orthologue can be identified as described herein and a skilled person will thus be able to confirm the function when expressed in a plant. Thus, one of skill in the art will recognize that analogous amino acid substitutions listed above with reference to SEQ ID NO: 2, 5, 7 or 9 can be made in PYL1/2/3 or PYR1 receptors from other plants by aligning the receptor polypeptide sequences to be mutated with the CsPYL1/2/3 or PYR1 receptor polypeptide sequence set forth in SEQ ID NO: 1.

Thus, the nucleotide sequences of the invention and described herein can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly cereals. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Thus, for example, probes for hybridization can be made by labelling synthetic oligonucleotides based on the sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

The term “functional variant of a nucleic acid or peptide sequence” as used herein with reference to any of the SEQ ID NOs referred to herein or homologs/orthologues thereof as described herein refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full mutant sequence, for example confers increased abiotic stress resistance when expressed in a genetically altered plant. For example, a functional variant of SEQ ID NO: 2 nonetheless has at least one of the following substitutions: V112L, F137I, T135L, T153I and V168A, and preferably all substitutions, compared to the wild-type sequence given in SEQ ID NO: 1. A functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non- conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non- conserved residues, to the wild type sequences but which includes the target mutations as shown herein and is biologically active. Variants have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the wild type sequence. A variant may for example have restriction sites introduced in the coding sequence to facilitate cloning (see examples).

Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses, encompass not only a nucleic acid sequence comprising, consisting essentially or consisting of SEQ ID NO: 4, 11 , 12 or 13 ora nucleic acid sequence encoding a mutant polypeptide comprising, consisting essentially or consisting or SEQ ID NO: 2, but also functional variants of these sequences that do not affect the biological activity and function of the resulting mutant protein. In other words, the additional variations present in the variants do not affect, for example PP2C interaction or other biological functions and the phenotype of the genetically altered plant expressing the variant is that of the genetically altered plant expressing the mutant peptide as described above. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site but that do however not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

Also, the various aspects of the invention the aspects of the invention, including the methods and uses, encompass not only a PYL, but also a fragment thereof. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence encode protein fragments that retain the biological activity of the native protein and also have improved sensitivity to SB and SB derivatives, particularly, iSB07 and iSB09.

In another aspect, the invention relates to a nucleic acid construct or vector comprising an isolated nucleic acid as described herein. The terms “nucleic acid” and “vector” can be used interchangeably. Thus, in one embodiment, the nucleic acid construct comprises a nucleic acid sequence that encodes a mutant PYL/PYR polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more of V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1 ; or at least one mutation that corresponds to one or more of V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5; or at least one mutation that corresponds to one or more of V87L, F112I, T128I and V145A of SEQ ID NO: 7; or at least one mutation that corresponds to one or more of V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence. In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding a PYL1 polypeptide as defined in SEQ ID NO: 2 or a functional variant or fragment thereof. In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding a PYR1 polypeptide as defined in SEQ ID NO: 11 or a functional variant or fragment thereof. In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding a PYL2 polypeptide as defined in SEQ ID NO: 12 or a functional variant or fragment thereof. In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding a PYL3 polypeptide as defined in SEQ ID NO: 13 or a functional variant or fragment thereof. Preferably, the vector further comprises a regulatory sequence which directs expression of the nucleic acid.

The terms "regulatory element", "regulatory sequence", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissuespecific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences. The term "regulatory element" also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.

A "plant promoter" comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The "plant promoter" can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other "plant" regulatory signals, such as "plant" terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'- regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include but are not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize H3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS, SAD1 , SAD2, nos, V-ATPase, super promoter, G-box proteins and synthetic promoters.

A "strong promoter" refers to a promoter that leads to increased or overexpression of the gene. Examples of strong promoters include, but are not limited to, CaMV-35S, CaMV- 35Somega, Arabidopsis ubiquitin LIBQ1 , rice ubiquitin, actin, or Maize alcohol dehydrogenase 1 promoter (Adh-1). The term "increased expression" or "overexpression" as used herein means any form of expression that is additional to the control, for example wild-type, expression level. In one embodiment, the promoter is 35S.

In a one embodiment, the promoter is a constitutive or strong promoter. In a preferred embodiment, the regulatory sequence is an inducible promoter, a stress inducible promoter or a tissue specific promoter. The stress inducible promoter is selected from the following non limiting list: the HaHB1 promoter, RD29A (which drives drought inducible expression of DREB1A), the maize rabl7 drought-inducible promoter, P5CS1 (which drives drought inducible expression of the proline biosynthetic enzyme P5CS1), ABA- and drought-inducible promoters of Arabidopsis clade A PP2Cs (ABI1 , ABI2, HAB1 , PP2CA, HAI1 , HAI2 and HAI3) or their corresponding crop orthologs.

Other regulatory sequences, such as terminator sequences may also be included. The invention also relates to an isolated host cell transformed with an isolated nucleic acid or vector as described above. The host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described above.

The mutant nucleic acid or vector described above is used to generate genetically altered plants using transformation methods known in the art. Thus, according to the various aspects of the invention, a nucleic acid comprising a sequence encoding for a mutant PYL/PYR polypeptide as described herein, for example a mutant PYL1 as defined in SEQ ID NO: 2 is introduced into a plant and expressed as a transgene.

The nucleic acid sequence is introduced into said plant through a process called transformation. The terms "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Genetically altered plants, including genetically altered crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

In one embodiment, the progeny plant is stably transformed with the nucleic acid construct described herein and comprises the exogenous polypeptide or polypeptides that are heritably maintained in the plant cell (and progeny). The method may also comprise the additional step of collecting seeds from the selected progeny plant. Thus, the invention also relates to a genetically altered plant or part thereof comprising and expressing an isolated nucleic acid or vector of the invention. In one embodiment, the plant expresses a PYL polypeptide as defined in SEQ ID NO: 2 or a functional variant or fragment thereof. In embodiment, the plant expresses a PYL/PYR polypeptide as defined in any one of SEQ ID NOs: 2, 11 , 12 and 13 or a functional variant or fragment thereof.

Accordingly, in another aspect of the invention, there is provided a method of making a genetically altered plant that has improved abiotic stress tolerance, the method comprising introducing and expressing the isolated nucleic acid or nucleic acid construct of the invention. Also provided are plants obtained or obtainable by the methods described herein. These plants will have improved drought tolerance.

In one embodiment, there is therefore provided, a method of obtaining a genetically modified plant as described herein, the method comprising a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one nucleic acid construct as described herein; c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that show expression of the mutant PYL/PYR polypeptide.

In another aspect of the invention, there is provided a genetically altered plant, wherein the plant comprises one or more mutations, wherein the mutation is the introduction of one or more additional copy(ies) of a nucleic acid encoding a mutant PYL/PYR polypeptide of the invention, such that the said one or more additional copy(ies) is operably linked to a regulatory sequence. In one example, the mutation is the introduction of one or more additional copies of a nucleic acid sequence encoding a mutant PYL/PYR sequence as defined in one of SEQ ID NOs: 2, 11 , 12 or 13 or a functional variant or fragment thereof. In one embodiment, the additional copy of a mutant PYL/PYR polypeptide of the invention is introduced using genome editing, such as using CRISPR. Preferably, said mutation results in an increased level of expression of the mutant PYL/PYR polypeptide compared to a wild-type or control plant. Again, such plants will have improved abiotic stress tolerance, and in particular, improved drought tolerance. In this embodiment, the regulatory sequence may be the endogenous PYL/PYR promoter, a promoter for a strongly expressed gene, such as the chlorophyll A/B binding protein 1 promoter or the promoter of a stress-inducible gene.

Accordingly, in another aspect of the invention, there is provided, a method of obtaining a genetically modified plant as described herein, the method comprising a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one CRISPR construct designed to introduce at least one of the following mutations: V112L, F137I, T135L, T153I and V168A in SEQ ID NO: 1 ; and/or V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5; and/or V87L, F112I, T128I and V145A of SEQ ID NO: 7; and/or V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence; c. regenerating at least one plant derived from the transfected cell or cells; d. selectng one or more plants obtained according to paragraph (c) that show at least one of the above mutations in SEQ ID NO: 1 , 5, 7 or 9 or a homologous/orthologous sequence.

In one example of step (b), a sgRNA can be used with a modified Cas9 protein, such as nickase Cas9 (nCas9) or a “dead” Cas9 (dCas9) fused to a “Base Editor” - such as an enzyme, for example a deaminase such as cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR or APOBEC. These enzymes are able to substitute one base for another. As a result, no DNA is deleted, but a single substitution is made.

In a further embodiment, the method also comprises the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in at least one PYL/PYR gene. In one embodiment, the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect a mutation in at least one PYL/PYR gene. In a further embodiment, the methods comprise generating stable T2 plants preferably homozygous for the mutation.

A genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that does not contain a mutation in at least one of the PYL/PYR gene. The methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could be carried out as mentioned, but without having to regenerate a plant afterward.

Uses and Methods for Enhancing Abiotic Stress Resistance in a Plant

In an embodiment, compounds or compositions as described herein may be used to enhance abiotic stress resistance (or tolerance) in a plant. The plant may be a wild-type plant. Alternatively, the plant may be a genetically altered plant of the invention.

As used herein, the terms “improving” or “enhancing” (e.g. “improving abiotic stress resistance” or “enhancing abiotic stress resistance”) are used interchangeably.

The stress is preferably abiotic stress and may be selected from drought, salinity, freezing (caused by temperatures below 0°C), chilling (caused by low temperatures over 0°C) and heat stress (caused by high temperatures). Preferably, the stress is drought.

The stress may be severe or preferably moderate stress. In Arabidopsis research, stress is often assessed under severe conditions that are lethal to wild type plants. For example, drought tolerance is assessed predominantly under quite severe conditions in which plant survival is scored after a prolonged period of soil drying. However, in temperate climates, limited water availability rarely causes plant death, but restricts biomass and seed yield. Moderate water stress, that is suboptimal availability of water for growth can occur during intermittent intervals of days or weeks between irrigation events and may limit leaf growth, light interception, photosynthesis and hence yield potential. Leaf growth inhibition by water stress is particularly undesirable during early establishment. There is a need for methods for making plants with increased yield under moderate stress conditions. In other words, whilst plant research in making stress tolerant plants is often directed at identifying plants that show increased stress tolerance under severe conditions that will lead to death of a wild type plant, these plants do not perform well under moderate stress conditions and often show growth reduction which leads to unnecessary yield loss. Thus, in one embodiment of the methods of the invention, yield is improved under moderate stress conditions. The genetically altered plants according to the various aspects of the invention show enhanced tolerance to these types of stresses compared to a control plant and are able to mitigate any loss in yield/growth. The tolerance can therefore be measured as an increase in yield. The terms moderate or mild stress/stress conditions are used interchangeably and refer to non-severe stress. In other words, moderate stress, unlike severe stress, does not lead to plant death. Under moderate, that is non-lethal, stress conditions, wild type plants are able to survive, but show a decrease in growth and seed production and prolonged moderate stress can also result in developmental arrest. The decrease can be at least 5%-50% or more. Tolerance to severe stress is measured as a percentage of survival, whereas moderate stress does not affect survival, but growth rates. The precise conditions that define moderate stress vary from plant to plant and also between climate zones, but ultimately, these moderate conditions do not cause the plant to die.

Drought stress can be measured through leaf water potentials. Generally speaking, moderate drought stress is defined by a water potential of between -1 and -2 Mpa. Drought tolerance can be measured using methods known in the art, for example assessing survival of the genetically altered plant compared to a control plant, or by determining turgor pressure, rosette radius, water loss in leaves, growth or yield. Regulation of stomatai aperture by ABA is a key adaptive response to cope with drought stress. Thus, drought resistance can also be measured by assessing stomatai conductance (Gs) and leaf temperature in whole plants under basal conditions (see Fig. 6).

In an embodiment, a method of improving abiotic stress resistance (e.g. drought resistance) in a plant may comprise applying a compound or composition as described herein.

According to the invention, a plant has enhanced drought tolerance if stomatai conductance (Gs) is reduced by at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more, more preferably within the range 30 to 70%, and more preferably within the range 40 to 60% compared to the level of stomatai conductance if the plant is not exposed to a compound or composition of the invention. As shown in Figures 6C and 6D, a reduction in stomatai conductance is still present at least 24 and even at least 48 hours after application of the compound or composition of the invention. According to the invention, a plant also has enhanced drought conductance if the leaf temperature increases by at least 1 %, 2%, 3%, 4%, 5%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or more, more preferably between 1 and 5%, and even more preferably between 1 and 3% or 1 and 2% compared to the leaf temperature if the plant is not exposed to a compound or composition of the invention.

According to the invention, a plant also has enhanced drought resistance, if following drought conditions (for example lack of watering for at least 20 to 22 days under long day conditions or 13 to 15 days under short day conditions), water consumption of the soil is reduced (increasing water retention in the soil - “water banking”) and/or survival of the plant following re-watering is increased. The plant may be deprived of water for 10-30, for example 22 days and the re-watered. The increase of water banking may be at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more, preferably between 20 and 30% and more preferably around 25% compared to the level of water banking and/or the survival rate of a plant is not exposed to a compound or composition of the invention. As shown in Figure 7, application of iSB09 increased water retention of the soil and increased percent survival rate following re-watering.

Preferably, the compound or composition is applied to the plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown. In one embodiment, the method relates to improving drought tolerance of plant vegetative tissue. Of key importance is that the compounds or compositions of the invention can be administered upon demand (i.e. under environmental stress conditions), which will minimise the effect of the compound of any other stage of plant development or growth.

The compound or composition as described herein may be applied at a concentration of about 0.1 μM to about 1 mM. In some cases, the compound or composition as described herein may be applied at a concentration of about 0.1 μM to about 200 μM, preferably about 1 μM to about 100 μM, more preferably about 2 μM to about 50 μM, even more preferably about 5 μM to about 20 μM. In some cases, the compound or composition as described herein may be applied at a concentration of about 1 μM to about 1 mM, preferably about 5 μM to about 500 μM, more preferably about 10 μM to about 200 μM, more preferably about 20 μM to about 100 μM. For wild type plants, the compound or composition as described herein may be applied at a concentration of about 1 μM to about 1 mM, preferably about 5 μM to about 500 μM, more preferably about 10 μM to about 200 μM, more preferably about 20 μM to about 100 μM. In one embodiment, the compound or composition described herein may be applied at a concentration between 30 and 70g/Ha, more preferably between 40 and 60g/Ha and even more preferably around 50g/Ha.

For genetically altered plants, the compound or composition as described herein may be applied at a concentration of about 0.1 μM to about 200 μM, preferably about 1 μM to about 100 μM, more preferably about 2 μM to about 50 μM, even more preferably about 5 μM to about 20 μM. In one embodiment, the compound or composition described herein may be applied at a concentration between 1 and 10g/Ha, more preferably between 1 and 5g/Ha and even more preferably around 3g/Ha.

The compound or composition as described herein may be applied at least once, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. In cases where the compound or composition as described herein is applied at least twice, the length of time between applications may be between a day to a month (e.g. between 1 to 21 days, preferably between 2 to 14 days, more preferably between 5 to 14 days, and even more preferably around 7 days).

Compounds and compositions described herein can be administered to seeds or plants wherein the improvement in abiotic stress resistance (e.g. drought resistance) is desired.

As used herein, the term “seed” broadly encompasses plant propagating material such as, tubers cuttings, seedlings, seeds, and germinated or soaked seeds.

The compounds and compositions described herein can be administered to the environment of plants (e.g., soil) wherein the improvement in abiotic stress resistance (e.g. drought resistance) is desired.

A compound or composition as described herein may be supplied to a plant exogenously. The compound or composition may be applied to the plant and/or the surrounding soil through sprays, drips, and/or other forms of liquid application. The compounds described herein may penetrate the plant through the roots via the soil (systemic action); by drenching the locus of the plant with a liquid composition; or by applying the compounds in solid form to the soil, e.g. in granular form (soil application).

A compound or composition as described herein may be applied to a plant, including plant leaves, shoots, roots, or seeds. For example, compound or composition as described herein can be applied to a foliar surface of a plant. Foliar applications may require 50 to 500 g per hectare (Ha) of a compound as described herein, and in particular around 250g/Ha.

As used herein, the term "foliar surface" broadly refers to any green portion of a plant having surface that may permit absorption, including petioles, stipules, stems, bracts, flowerbuds, and leaves. Absorption commonly occurs at the site of application on a foliar surface, but in some cases, the applied compound or composition may run down to other areas and be absorbed there.

Compounds or compositions described herein can be applied to the foliar surfaces of the plant using any conventional system for applying liquids to a foliar surface. For example, application by spraying will be found most convenient. Any conventional atomisation method can be used to generate spray droplets, including hydraulic nozzles and rotating disk atomisers. In other instances, alternative application techniques, including application by brush or by rope-wick, may be utilised.

A compound or composition as described herein can be directly applied to the soil surrounding the root zone of a plant. Soil applications may require at most or at least 0.1 to 5 kg per hectare of a compound as described herein on a broadcast basis (rate per treated area if broadcast or banded).

For example, a compound or composition as described herein may be applied directly to the base of the plants or to the soil immediately adjacent to the plants.

In some embodiments, a sufficient quantity of the compound or composition is applied such that it drains through the soil to the root area of the plants. Generally, application of a compound or composition as described herein may be performed using any method or apparatus known in the art, including but not limited to hand sprayer, mechanical sprinkler, or irrigation, including drip irrigation.

A compound or composition as provided herein can be applied to plants and/or soil using a drip irrigation technique. For example, the compound or composition may be applied through existing drip irrigation systems. For example, this procedure can be used in connection with cotton, strawberries, tomatoes, potatoes, vegetables, and ornamental plants.

In other embodiments, a compound or composition as described herein can be applied to plants and/or soil using a drench application. For example, the drench application technique may be used in connection with crop plants and turf grasses.

A compound or composition as described herein may be applied to soil after planting. Alternatively, a compound or composition as described herein may be applied to soil during planting, or may be applied to soil before planting.

For example, a compound or composition as described herein may be tilled into the soil or applied in furrow.

In crops grown in water, such as rice, solid granulates comprising the compounds or compositions described herein may be applied to the flooded field or locus of the crop plants to be treated.

In another aspect of the invention, there is provided a method of inhibiting seed germination/prolonging seed dormancy in a plant, the method comprising applying a compound or composition of the invention. This can be an advantage if seeds germinate too early - i.e. pre-harvest sprouting (PHS). In particular, seeds sometimes fail to enter a period of dormancy and may germinate while still attached to the parent plants (for example, if there is abundant rainfall during seed maturation). This is associated with reduced ABA signalling in the maturing seeds. Obviously, this situation impairs seed harvesting and storage, and can be a serious problem for farmers under very humid conditions prior to harvesting. Accordingly, in another aspect of the invention, there is provided a method of inhibiting PHS. An inhibition of PHS can be determined using routine techniques in the art. Aerial spraying mainly focused to wheat/barley/rice spikes/panicles during harvest time (in humid periods or abundant rainfall) should prevent germination of the grains while attached to the parent plants. Therefore, inhibition of PHS will be measured by scoring frequency of germination in spikes/panicles of control/wild-type-versus compound- treated plants.

As used herein “inhibiting” may mean a reduction in % germination or PHS by between 50 and 100%, more preferably between 75% and 100%, and particularly 75%, 80%, 85%, 90%, 95% or 100% compared to the level in a wild-type or control plant. This % inhibition will depend on the compound dosage and its use combined with the mutant receptor. For example, 5-25 uM iSB09 is effective to inhibit germination in Col-0 wt plants, whereas 0.5-5 uM iSB09 can inhibit germination in mutant plants (Figure 5).

In another aspect of the invention, there is provided a method of activating ABA signalling and/or activating or increasing an ABA response, the method comprising applying a compound or composition of the invention.

The various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention. In one embodiment, the progeny comprise the nucleic acid or construct of the invention.

The invention also extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof. In one embodiment, the plant part is a seed. In one embodiment, the plant part comprises the nucleic acid or nucleic acid construct of the invention.

The plant according to the various aspects of the invention described herein may be a monocot or a dicot plant. Non-limiting examples of monocot or dicot plants are given below.

A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (eg Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine or citrus species. In one embodiment, the plant is oilseed rape.

Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as wheat, rice, barley, maize, oat, sorghum, rye, millet, buckwheat, turf grass, Italian rye grass, sugarcane or Festuca species, or a crop such as onion, leek, yam or banana or a citrus, such as an orange.

Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. Most preferred plants are maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, citrus (such as orange, lemon, grapefruit, pomelos and limes), sugar beet, broccoli or other vegetable brassicas or poplar. In another embodiment, the plant is selected from Nicotiana sp, including Nicotiana benthamiana.

Control plants as defined herein are plants that do not express the isolated nucleic acid or nucleic acid construct described above. For example, the control plant is a wild-type plant, such as Col-0. Alternatively, the control plant (over)expresses a wild-type PYL/PYR, for example CsPYLI .

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is now described in the following non-limiting examples.

EXAMPLES

Materials and Methods

Synthesis of SB-derivatives, iSB07 and iSB09 compounds

Sulfobactin (SB, Compound 1) was purchased from UAB Crea-Chim (Lithuania).

All reactions were carried out under air unless stated otherwise. Reactions were monitored by thin-layer chromatography (TLC) analysis on Merck® silica gel 60 F254 TLC plates. Spots were visualized by exposure to ultraviolet (UV) light (254 nm), or by staining with a 5% solution of phosphomolybdenic acid (PMA) in ethanol or basic aqueous potassium permanganate (KMnC ) and then heating. Flash chromatography was carried out using Merck® silica gel 60 (230-400 mesh). All solvents were of HPLC grade quality and used as received. All reagents were purchased at the highest commercial quality and used without further purification. 1 H-NMR spectra were recorded on a Varian Mercury (300 MHz) spectrometer. Chemical shifts are reported in parts per million (ppm) down field from TMS as an internal standard. Data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, dd=doublets of doublets, m=multiplet, br=broad), coupling constant(s) and integration. HPLC analyses were carried out on a Waters system model Alliance HT (Mass detector: Micromass ZQ 2000). A: water, B: CHsCIXkCHsOH (1 :1), C: 100 mM ammonium acetate solution (approx. pH 6.8). Method A (9 min): Analysis conditions: Luna C18(2) 5 pm, 2.0 x 50 mm. Gradient: A:B:C 30 s at 85:10:5; then from 85:10:5 to 0:95:5 in 4 min, finally 4.5 min at 0:95:5. Method B (15 min): Analysis conditions: Luna C18(2) 5 pm, 2.0 x 50 mm. Gradient: A:B:C 3 min at 85:10:5; then from 85:10:5 to 0:95:5 in 6 min, finally 7 min at 0:95:5. Method C (30 min): Analysis conditions: SunFire C18 3.5 pm, 2.1 x 100 mm. Gradient: A:B:C 5 min at 85:10:5; then from 85:10:5 to 0:95:5 in 15 min, finally 10 min at 0:95:5.

Reference Synthesis Example 1: 1-ethyl-4-methylquinolin-2(1H)-one (Reference Compound A-1) 4-methylquinolin-2(1H)-one (4.39 mmol) was suspended in DMF (10 mL) under N2 atmosphere. To this mixture was added NaH (60%, 3 equiv.) at rt, after which bubbling was observed in the grey-ish solution. The reaction mixture was left at rt for 20 min. Next, the corresponding alkylation reagent (EtBr) was added (2.5 equiv). The reaction was completed in 4-6 h, after which it was quenched with a few drops of water. Next, NaCl (sat aq, 30 mL) was added and extracted with AcOEt (2 x 30 mL). The combined organic phases were dried over Na2SO4, filtered off, and the solvent removed under vacuum. The obtained crude product was finally purified by chromatography (SiO2, 30% to 60% AcOEt/Hexane). Compound A-1 was obtained as a yellow oil, 70% yield. LC-MS (Method A): Purity=93.46%, M-Me=173.6 (ESI+). Reference Synthesis Example 2: 1-cyclopropyl-4-methylquinolin-2(1H)-one (Reference Compound A-2) 12568980-1 4-methylquinolin-2(1H)-one (3.76 mmol) was suspended in toluene (20 mL), after which cyclopropylboronic acid (2 equiv), (AcO)2Cu (1 equiv), and pyridine (5 equiv) were sequentially added. The mixture was deoxygenated with N2 and NaHMDS (1 equiv) was added. The reaction mixture was stirred at 100 °C under constant air flow for 15 h. After cooling down to rt, the mixture was filtered through celite with AcOEt washes (20 mL). The organic phase was further washed with water (20 mL) and subsequently dried over Na2SO4, filtered off, and the solvent removed under vacuum to give Compound A-2 as a brown oil, 89% yield. LC-MS (Method B): Purity=88.21%, M+1= 173.6 (ESI+). Reference Synthesis Example 3: 1,4-dimethylquinolin-2(1H)-one (Reference Compound A-3) The same procedure as Reference Synthesis Example 1 was used, except that MeI was used instead of EtBr. Compound A-3 was obtained as a white solid, 89% yield. LC-MS (Method A): Purity=98.84%, M+1=199.7 (ESI+). Reference Synthesis Example 4: 1-ethyl-4-methyl-2-oxo-1,2-dihydroquinoline-6-sulfonyl chloride (Reference Compound B-1) ClSO₃H (1.5 mL) was added dropwise to Compound A-1 (1.84 mmol) at low temperature (ice bath). The mixture was then heated to 50 °C and stirred for 4-6 h. Next, the mixture was poured over crushed ice and NaCl (sat aq, 30 mL) was added. This aqueous mixture was extracted with DCM (2 x 30 mL), and the organic phase dried over Na₂SO₄, filtered off, and the solvent removed under vacuum to give Compound B-1 as a light-brown solid, 77% isolated yield after chromatography (SiO₂, 30% AcOEt/Hex).1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 8.16 (d, J = 9.1 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 6.72 (s, 1H), 4.38 (q, J = 7.2 Hz, 2H), 2.54 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). Reference Synthesis Example 5: 1-cyclopropyl-4-methyl-2-oxo-1,2-dihydroquinoline-6- sulfonyl chloride (Reference Compound B-2) The same procedure as Reference Synthesis Example 4 was used, except that Compound A-2 was used as starting material. Compound B-2 was obtained as a brown solid, 27% isolated yield after chromatography (SiO2, 50% AcOEt/Hex). 12568980-1 Reference Synthesis Example 6: 1,4-dimethyl-2-oxo-1,2-dihydroquinoline-6-sulfonyl chloride (Reference Compound B-3) The same procedure as Reference Synthesis Example 4 was used, except that Compound A-3 was used as starting material. Compound B-3 was obtained as a brown solid, 60% isolated yield after chromatography (SiO2, 30% AcOEt/Hex). LCMS (Method A): Purity=89.32%, M+1 (acid)=252.2 (ESI+). Synthesis Example 7: N-benzyl-1-ethyl-4-methyl-2-oxo-1,2-dihydroquinoline-6- sulfonamide (Compound 2, SB-01) To a solution of Compound B-1 (0.73 mmol) in DMF (5 mL) was added pyridine (1.1 equiv) and benzylamine (1.05 equiv). The resulting mixture was stirred at rt for 1-2 h. The mixture was diluted with water (15 mL) and extracted with AcOEt (20 mL). The organic phase was separated and washed successively with HCl (10%, 10 mL), NaHCO3 (aq. sat, 10 ml), and NaCl (sat. aq., 10 mL), to be finally dried over Na2SO4, filtered off, and the solvent removed under vacuum. The residue was then washed with Et₂O or AcOEt to obtain Compound 2 as a white solid, 16% isolated yield; LC-MS (Method C): Purity=99.16%, M+1=357.1 (ESI+)).1H NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.27 (dd, J = 13.3, 7.6 Hz, 5H), 6.73 – 6.66 (m, 1H), 4.92 (t, J = 6.3 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 4.23 (d, J = 6.2 Hz, 2H), 2.50 (s, 3H), 1.45 – 1.34 (m, 3H). Synthesis Example 8: N-benzyl-1-cyclopropyl-4-methyl-2-oxo-1,2-dihydroquinoline-6- sulfonamide (Compound 3, SB-02) A solution of Compound B-2 (1.04 mmol) in DCM (10 mL) was cooled down in an ice bath, after which Et3N (1.1 equiv) and benzylamine (1.05 equiv) were added. The reaction mixture was stirred at low temperature for 1-3 h (until completion). The mixture was diluted with water (15 mL) and extracted with DCM (10 mL). The organic phase was separated and washed successively with HCl (10%, 10 mL), NaHCO3 (sat. aq., 10 mL), and NaCl (sat, 10 mL), to be finally dried over Na2SO4, filtered off, and the solvent removed under vacuum. The obtained residue was washed several times with Et₂O and AcOEt. The crude solid was further purified by chromatography to obtain Compound 3 12568980-1 as a white solid, 4% isolated yield after purification by reverse phase chromatography (C18, MeCN/buffer pH7 65% to 85%); LC-MS (Method C): Purity=81.25% +18.55%, M+1=369.1 (ESI+). 1 H NMR (300 MHz, CDCI3) 5 8.11 (s, 1 H), 7.95 (s, 2H), 7.30 - 7.16 (m, 6H), 6.57 (s, 1 H), 4.87 (s, 1 H), 4.19 (d, J = 5.9 Hz, 2H) 2.95 (s, 1 H), 2.43 (d, J = 3.2 Hz, 3H), 1.60 (s, 3H)1.41 (s, 2H), 0.89 (s, 2H).

Synthesis Example 9: 1 ,4-dimethyl-N-(4-methylbenzyl)-2-oxo-1 ,2-dihydroquinoline-6- sulfonamide (Compound 4, SB-03)

The same procedure as Synthesis Example 7 was used, except that Compound B-3 and 4-methylbenzylamine were used as starting materials. Compound 4 was obtained as a white solid, 18% isolated yield; LC-MS (Method C): Purity=96.32%, M+1=357.2 (ESI+)).1 H NMR (300 MHz, CDCI3) 5 8.14 (s, 1 H), 8.01 (dd, J = 8.7, 2.0 Hz, 1 H), 7.46 (d, J = 9.0 Hz, 1 H), 7.28 (s, 1 H), 7.06 (s, 4H), 6.69 (s, 1 H), 4.84 (s, 1 H), 4.16 (d, J = 5.6 Hz, 2H), 3.75 (s, 3H), 2.48 (s, 3H), 2.29 (s, 3H).

Synthesis Example 10: N-(2-fluorobenzyl)-1 ,4-dimethyl-2-oxo-1 ,2-dihydroquinoline-6- sulfonamide (Compound 5, SB-04)

The same procedure as Synthesis Example 7 was used, except that Compound B-3 and 2-fluorobenzylamine were used as starting materials. Compound 5 was obtained as a beige solid, 9% isolated yield; LC-MS (Method C): Purity=95.12%, M+1=361.0 (ESI+)). 1 H NMR (300 MHz, CDCI3) 5 8.09 (d, J = 2.3 Hz, 1 H), 7.94 (dd, J = 9.0, 2.3 Hz, 1 H), 7.37 (d, J = 8.6 Hz, 1 H), 7.30 - 7.09 (m, 2H), 7.04 - 6.93 (m, 1 H), 6.86 (dd, J = 10.5, 8.0 Hz, 1 H), 6.65 (s, 1 H), 5.03 (s, 1 H), 4.27 (d, J = 6.3 Hz, 2H), 3.70 (d, J = 1.3 Hz, 3H), 2.45 (q, J = 1.3 Hz, 2H).

Synthesis Example 11 : N-(2-chlorobenzyl)-1 ,4-dimethyl-2-oxo-1 ,2-dihydroquinoline-6- sulfonamide (Compound 6, SB-05)

The same procedure as Synthesis Example 7 was used, except that Compound B-3 and 2-chlorobenzylamine were used as starting materials. Compound 6 was obtained as an off-white solid, 33% isolated yield; LC-MS (Method C): Purity=97.28%, M+1=377.0 (ESI+)).1 H NMR (300 MHz, CDCI3) 5 8.09 (d, J = 2.1 Hz, 1 H), 7.95 (dd, J = 8.9, 2.1 Hz, 1 H), 7.38 (d, J = 8.9 Hz, 1 H), 7.31 - 7.24 (m, 2H), 7.21 (d, J = 4.1 Hz, 1 H), 7.18 - 7.08 (m, 2H), 6.67 (s, 1 H), 5.15 (t, J = 6.4 Hz, 1 H), 4.33 (d, J = 6.4 Hz, 2H), 3.72 (s, 3H), 2.47 (s, 3H).

Synthesis Example 12: N-(2-hydroxybenzyl)-1 ,4-dimethyl-2-oxo-1 ,2-dihydroquinoline-6- sulfonamide (Compound 7, SB-06)

The same procedure as Synthesis Example 8 was used, except that Compound B-3 and 2-hydroxybenzylamine were used as starting materials. Compound 7 was obtained as a white solid, 8% isolated yield after purification by chromatography (SiC>2, 5%MeOH/DCM); LC-MS (Method C): Purity=96.51%, M+1 =359.1 (ESI+). 1 H NMR (300

MHz, DMSO-d6) 5 8.10 - 8.03 (m, 1 H), 8.00 - 7.90 (m, 1 H), 7.65 (d, J = 8.9 Hz, 1 H), 7.12 (d, J = 7.4 Hz, 1 H), 6.96 (s, 1 H), 6.71 - 6.60 (m, 3H), 3.91 (s, 2H), 3.61 (d, J = 4.1 Hz, 3H), 2.42 (s, 2H).

Reference Synthesis Example 13: N-(4-bromophenyl)-3-oxobutanamide (Compound C) para-Bromoaniline (52.31 mmol) was dissolved in xylene (30 mL) and ethyl acetoacetate was added (1.2 equiv). The solution was heated at 135 °C for 24 h. After allowing the flask to naturally cool down to room temperature (rt), the reaction flask was placed in the freezer for 5 h, obtaining a white precipitate. The solid was filtered off, washed with hexane (3 x 20 mL) and dried under vacuum to afford Compound C as a light brown solid (22% isolated yield). LC-MS (Method B): Purity=96.37%, M+1=254.1 (ESI-). Reference Synthesis Example 14: 6-bromo-4-methylquinolin-2(1H)-one (Compound D) 12568980-1 Compound C (10.5 mmol) was dissolved in H2SO4 (10 mL, 97%) and the solution was heated at 100 °C for 2 h. After cooling down to rt, iced water was added dropwise (total volume of 20 mL). The resulting suspension was left stirring overnight at rt. The white solid was filtered off, washed with water (3 x 3 mL), Et2O (5 x 3 mL), and finally with hexane (3 x 3 mL), affording Compound D as a grey-ish solid (92% isolated yield). LC- MS (Method A): Purity=91.0%, M+1=239.1 (ESI+). Reference Synthesis Example 15: 6-bromo-1,4-dimethylquinolin-2(1H)-one (Compound E-1) Compound D (1.68 mmol) was suspended in DMF (25 mL), affording a grey-ish suspension. To this mixture was added NaH (60%, 3 equiv.) at rt, after which bubbling was observed. The reaction mixture was left at rt for 20 min. Next, the corresponding alkylation reagent (MeI) was added (1 equiv). The reaction was completed in 4-6 h, after which the solution was concentrated under vacuum, the residue was dissolved in DCM (15 mL), and water (20 mL) was added. The layers were separated, and the aqueous phase was further washed with DCM (2 x 15 mL). The combined organic layers were dried over Na₂SO₄, filtered off, and the solvent removed under vacuum, affording Compound E-1 as a light brown solid (63% isolated yield; LC-MS (Method B): Purity=98.68%, M+1=252.1 (ESI+)). Reference Synthesis Example 16: 6-bromo-1-ethyl-4-methylquinolin-2(1H)-one (Compound E-2) The same procedure as Reference Synthesis Example 15 was used, except that EtI was used instead of MeI. Compound E-2 was obtained as a yellow-ish solid (82% isolated yield; LC-MS (Method B): Purity=96.33%, M+1=265.8 (ESI+)). Reference Synthesis Example 17: 1,4-dimethyl-2-oxo-1,2-dihydroquinoline-6- carbonitrile (Compound F-1) Compound E-1 (1.31 mmol) was dissolved in DMF and then Zn(CN)2 (2 equiv) was added. The mixture was deoxygenated for 5 min with nitrogen, before adding Pd(PPh3)4 (0.1 equiv). The reaction was heated at 100 °C until completion (2-6 h). The mixture was allowed to cool down to rt and poured over an aqueous solution of sat. NaCl. This mixture 12568980-1 was extracted with AcOEt (3 x 10 mL) and the combined organic layers were dried over Na2SC>4, filtered off, and the solvent removed under vacuum. The resulting residue was washed with hexane (2 x 1 mL) and Et2<D (2 x 1 mL) to afford Compound F-1 as a white solid (64% isolated yield; LC-MS (Method B): Purity=98.87%, no ionization).

Reference Synthesis Example 18: 1-ethyl-4-methyl-2-oxo-1 ,2-dihydroquinoline-6- carbonitrile (Compound F-2)

The same procedure as Reference Synthesis Example 17 was used, except that Compound E-2 was used as starting material. Compound F-2 was obtained as a yellowish solid (91 % isolated yield; LC-MS (Method B): Purity=98.94%, M+1=213.2 (ESI+)).

Reference Synthesis Example 19: 6-(aminomethyl)-1 ,4-dimethylquinolin-2(1 H)-one hydrochloride salt (Compound G-1)

Compound F-1 (1.18 mmol) was suspended in MeOH (10 mL) and HCI (37%, 3 mL) was added. The mixture was bubbled with N2 for 5 min to remove oxygen, after which the catalyst Pd-C (10%, 0.1 % wt) was added. The reaction mixture was placed under atmospheric pressure of H2 (rubber balloon) and left stirring at rt until completion (1-2 days). Next, the mixture was filtered through Celite® with MeOH washes (4 x 5 mL). The solvent was removed under vacuum affording Compound G-1 as a white solid (98% isolated yield; LC-MS (Method B): Purity= 96.99%, M+1= 203.0 (ESI+).

Reference Synthesis Example 20: 6-(aminomethyl)-1-ethyl-4-methylquinolin-2(1 H)-one hydrochloride salt (Compound G-2)

The same procedure as Reference Synthesis Example 19 was used, except that Compound F-2 was used as starting material. Compound G-2 was obtained as a yellowish solid (94% isolated yield; LC-MS (Method B): Purity=93.41%, M+1=218.1 (ESI+).

Synthesis _ Example _ 21 : _ N-((1 ,4-dimethyl-2-oxo-1 ,2-dihydroquinolin-6- yl)methyl)benzenesulfonamide (Compound 8, iSB-07)

Compound G-1 (1 mmol) was suspended in DMF (7 mL) and DI PEA (3 equiv) was added, affording a yellow solution. Benzenesulfonyl chloride (1.2 equiv) was then added, and the reaction was stirred for 2 h at rt. The solvent was then removed under vacuum and the resulting residue dissolved in DCM (10 mL). Aqueous NH4Cl was added (20 mL) and the organic layer separated. The aqueous phase was further extracted with DCM (2 x 10 mL). The combined organic layers were dried over Na2SO4, filtered off, and the solvent removed under vacuum. The resulting oily solid was finally purified by silica column (MeOH 3% to 5%/DCM) affording Compound 8 as a white solid, 52% isolated yield; LC-MS (Method C): Purity=99.15%, M+1=343.1 (ESI+). 1H NMR (300 MHz, DMSO-d6): δ 8.25 (s, 1H), 7.84 – 7.70 (m, 2H), 7.65 – 7.37 (m, 6H), 6.51 (d, J = 1.4 Hz, 1H), 4.12 (s, 2H), 3.56 (s, 3H), 2.34 (s, 3H). Synthesis Example 22: N-((1-ethyl-4-methyl-2-oxo-1,2-dihydroquinolin-6- yl)methyl)benzenesulfonamide (Compound 9, iSB-09) The same procedure as Synthesis Example 21 was used, except that Compound G-2 was used as starting material. Compound 9 was obtained as a white solid, 45% isolated yield. LC-MS (Method C): Purity=99.82%, M+1=357.1 (ESI+). 1H NMR (300 MHz, DMSO-d6): δ 8.23 (s, 1H), 7.76 (dd, J = 8.0, 1.7 Hz, 2H), 7.62 – 7.43 (m, 6H), 6.49 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 4.12 (s, 2H), 2.34 (s, 3H), 1.16 (t, J = 7.0 Hz, 3H). Protein expression and purification Expression and purification of the sweet orange receptor CsPYL1 (amino acids 1-235), the quintuple mutant CsPYL1^5m and the phosphatase AtHAB1ΔN (amino acids 179-511) were conducted as described previously (Moreno-Alvero et al., 2017, Mol Plant 10, 1250- 1253). Crystallization, Diffraction Data Collection, and Structure Solution SB, iSB07, iSB09 and QB were dissolved in 40% DMSO, 30 mM Tris-HCl pH 8.5 and 6% PEG 400. ABA was dissolved in 2% DMSO, 50 mM Tris-HCl pH 8.5 and 10% PEG 400. Equal volumes of the solution containing ligand and a solution containing 5 mg/mL AtHAB1ΔN and 3 mg/ml of CsPYL1 or CsPYL1^5m were mixed to obtain a 1:1.4:10 ratio CsPYL1:AtHAB1ΔN:ABA or 1:1.4:10 ratio CsPYL1:AtHAB1ΔN:ligand. These mixtures were incubated during 1 hour at 4°C before the crystallization experiments. 12568980-1 Crystallization of the complexes were carried out using microbath under parafine-oil technique at 18°C on a 60 well Terasaki plate (Jena Bioscience).

The best X-ray data collected for each complex correspond to crystals growth after 4-8 days at the following conditions CsPYL1-SB-AtHAB1AN (30% PEG 3350, pH 7.5, ratio 1 :2), CsPYL1^5m-SB-AtHAB1AN (30% PEG 3350, pH 6.5, ratio 1 :1), CsPYLI- QBAtHABIAN (25% PEG 3350, pH 6.5, ratio 1 :1), CsPYL1^5m-ABA-AtHAB1AN (30% PEG 3350, pH 6.0, ratio 1 :1), CsPYL1-iSB07-AtHAB1AN (30% PEG 3350, pH 6.5, ratio 1 :1), CsPYL1^5m-iSB07- AtHABIAN (30% PEG 3350, pH 6.0, ratio 1 :1), CsPYL1-iSB09- AtHABIAN (35% PEG 3350, pH 6.5, ratio 1 :1) and CsPYL1^5m-iSB09-AtHAB1AN (35% PEG 3350, pH 6.5, ratio 1 :2). All crystals were cryo-protected in their corresponding crystallization solution which also contained 30% glycerol and flash-frozen in liquid nitrogen.

Diffraction data from were collected at 100 K at the ALBA synchrotron radiation source (BL13 beamline) and processed with XDS (Kabsch, 2010, Acta crystallographica. Section D, Biological crystallography 66, 125-132).

The structures of the complexes were solved by molecular replacement with Phaser (DiMaio et al., 2011 , Nature 473, 540-543) and using as search model the ligand-free protein structure (PDB ID 5mn0). Refinement was performed running several cycles of automated refinement with Phenix (Adams et al., 2010, Acta crystallographica. Section D, Biological crystallography 66, 213-221) followed by manual model building with COOT (Emsley and Cowtan, 2004, Acta crystallographica. Section D, Biological crystallography 60, 2126-2132). The ABA and QB dictionary with geometrical restraints was first generated with the eLBOW program from the Phenix package (Adams et al., 2010, Acta crystallographica. Section D, Biological crystallography 66, 213-221) and later on, the dictionary was improved using the information included in the Cambridge Structural Database, CSD. The iSB07 and iSB09 dictionary was generated using the Grade Web Server (http://grade.globalphasing.org) run on mol2 files from the X-ray structures of the protein-free small molecules. SB, iSB07 and iSB09 crystal structures were solved by direct methods using SIR2011 software (Burla et al., 2012, Appl Crystallogr 45, 357- 361), and refinement was performed with SHELXL (Sheldrick, 2015, Acta Crystallogr C Struct Chem 71 , 3-8). Analysis of the structures was done with CCP4 (Cowtan et al., 2011 , Acta crystallographica. Section D, Biological crystallography 67, 233-234) programs and the CSD (Groom et al., 2016, Acta Crystallogr B Struct Sci Cryst Eng Mater 72, 171-179). Images were drawn with Pymol (DeLano, 2002, The PyMOL Molecular Graphics System. L. Schrodinger, ed.) and Mercury programs (C. F. Macrae, 2020, J. Appl. Cryst. 53, 226- 235).

Generation of Arabidopsis transgenic plants

Arabidopsis thaliana plants were grown as described by Pizzio et al., (2013, Plant physiology 163, 441-455). The pAlligator2-35S:HA-CsPYL1 and pAlligator2-35S:HA- CsPYL1^5m constructs were transferred to Agrobacterium tumefaciens C58C1 (pGV2260) (Deblaere et al., 1985) by electroporation and used to transform Col-0 wild-type plants by the floral dip method (Clough and Bent, 1998). T1 transgenic seeds were selected based on seed GFP fluorescence (pAlligator2) and sowed in soil to obtain the T2 generation. Homozygous T3 progeny was used for further studies and expression of HA- tagged protein was verified by immunoblot analysis using anti-HA-HRP antibodies.

Whole plant gas exchange experiments

Arabidopsis seeds were planted in specific gas exchange pots where above- and below ground parts can be separated by glass into 2:1 (v:v) peat:vermiculite mixture. The plants were grown in growth chambers (Snijders Scientific, Drogenbos, Belgia) at 12/12 photoperiod, 23/18°C temperature, 160 pmol nr² s'¹ light and 70% relative humidity and were 23-28 days old during gas exchange experiments. Whole-rosette stomatai conductances were recorded with an 8-chamber custom-built temperature-controlled gas-exchange device as described before (Koilist et al. 2007). Plants were inserted into the measurement cuvettes and allowed to stabilize at standard conditions: ambient CO2 (-420 ppm), air temperature 24 ± 0.5 °C, light 160 pmol m^-2 s’¹, relative air humidity (RH) 66 ± 3%. For sprayings, plants were removed from gas exchange cuvettes, sprayed with respective solutions and put back into the cuvettes for stomatai conductance recordings for 56 minutes. To check the long-term effect of compounds on plants, stomatai conductance measurements were performed about 24 and 48 h after sprayings. In the meantime, plants were kept in growth chambers as described above. Photographs of plants were taken after the experiment and leaf rosette area was calculated using Imaged 1.37v (National Institutes of Health, USA). Stomatal conductance for water vapor was calculated with a custom written program as described in Kollist et al. (2007). PP2C inhibition assays The expression in bacteria and purification of 6His-ΔNHAB1, and the different Arabidopsis and sweet orange ABA receptors was performed as described in Santiago et al., (2009). His-tagged proteins were purified using Ni-NTA affinity chromatography, eluted and analyzed by SDS-PAGE, followed by Instant Blue staining. Phosphatase activity of ΔNHAB1 was measured using pNPP (15 mM) as substrate, 1 μM of the PP2C and 2 μM of the indicated receptors. Dephosphorylation of pNPP was monitored with a ViktorX5 reader at 405 nm (Antoni et al., 2012, Plant physiology 158, 970-980). Phosphatase activity of ABI1 was measured using RRA(phosphoT)VA peptide as a substrate, as described in Dupeux et al., 2011. Infrared thermography Plants were grown in a controlled environment growth chamber at 22ºC under a 12 h light, 12 h dark photoperiod at 100 E m^-2 s^-1 and 40-50% room humidity. Philips bulbs were used (TL-D Super 8036W, white light 840, 4000K light code). Infrared thermography images of rosette leaves were acquired from 6-week-old plants with a thermal camera FLIR E95 equipped with a 42° lens. Images were processed and quantified with the FLIR tools software. For quantification, the average temperature of 15 different sections corresponding to 4 leafs per plant were calculated. 5 plants per genotype were analyzed in each experiment. The mean temperature ± standard deviation of all the plants for each genotype was reported. Statistical comparisons among genotypes were performed by pairwise t-tests. Root growth assay Seedlings were grown on vertically oriented Murashige and Skoog (MS) plates for 4-5 days. Afterwards, 20 plants were transferred to new MS plates lacking or supplemented with 10 µM ABA. The plates were scanned on a flatbed scanner after 10 days to produce image files suitable for quantitative analysis of root growth using the NIH Image software ImageJ. 12568980-1 Drought resistance experiments Seeds from Col-0, pAlligator2-35S:CsPYL1^5m plants were grown in MS medium for 7 days. Then seedlings were transferred to individual 0.18 L pots (n = 20, per genotype, 3 independent experiments) containing equal amount of moist soil composed of peat, vermiculite and perlite at 1: 0.5: 0.5 ratio (v/v). Plants were grown under long-day conditions (LD) in a controlled environment growth chamber at 22ºC under 16 h light, 8 h dark photoperiod, 40-50% room humidity and standard watering for 3 weeks. Then, watering was withheld for 22 days. Spraying with a solution containing 10 mM MES pH 5.7, 0.02% Silwet L-77 and either DMSO (0.1%), 50 µM ABA or 50 µM iSB09 was performed once per week. A total of 2 sprays were applied. After 22 days of water deprivation, watering was restored and images were acquired to document water stress. Survival rate and foliar leaf area were measured 5 days after rewatering. Gravimetric analysis of water loss in pots was performed along the experiment. Seed germination and seedling establishment assays After surface sterilization of the seeds, stratification was conducted in the dark at 4ºC for 3 d. Approximately 100 seeds of each genotype were sowed on MS plates supplemented with different ABA concentrations per experiment. To score seed germination, radical emergence was analyzed at 72 h after sowing. Seedling establishment was scored as the percentage of seeds that developed green expanded cotyledons and the first pair of true leaves at 5 or 7 d. Seedling establishment assays in the presence of ABA agonists were performed in 24-well plates, where approximately 25 seeds of the indicated genotype (three independent experiments) were sown on wells lacking or supplemented with the indicated concentration of ABA agonists. Seedling establishment was scored for the presence of both green cotyledons and the first pair of true leaves after 7 d. Isothermal titration calorimetry (ITC) ITC measurements were performed at 35 °C with an Auto-iTC200 isothermal titration calorimeter (Micro-Cal-Malvern) in a buffer consisting of 50 mM Hepes pH 7.5, 200 mM NaCl, 5mM MgCl₂, 10 % glycerol, 1mM β-mercaptoethanol and 0.2 % DMSO. For binding measurements, a 1:1 mixture of receptor: ΔNHAB1 (20 μM) was titrated with a 12568980-1 solution of 200 μM of ABA or iSB09 as corresponds. To obtain the Kd values, the data were analyzed using nonlinear least-squares regression employing a model considering a single set of binding sites implemented in Origin 7.0 (OriginLab). Native red electrophoresis (NRE) A 1:1 mixture of receptor: ΔNHAB1 (18 μM) was mixed with increasing concentrations of ABA or iSB09, as indicated in Figure 3E and 3F, in a buffer consisting in 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5 mM DTT, 10 % glycerol and 0.02 % Ponceau Red S. Samples were incubated during 30 min at room temperature and were loaded onto a 13.5 % polyacrylamide gel prepared in 375 mM Tris–HCl (pH 8.8), 10% glycerol, 0.012% of Ponceau Red S. A solution of Tris–glycine (pH 8.8) with or without 3 mM of MgCl₂ and 0.012% Ponceau Red S was used as the cathode and anode buffer, respectively. Electrophoresis was performed at a constant current of 25 mA for 150 min at 4 °C. Proteins were detected in gel by Instant Blue staining and band quantification was performed using ImageJ software. To obtain apparent Kd values, the proportion of ternary complex formed (fraction bound) and the ABA or iSB09 free concentration in each lane were calculated and plotted as represented in Figures 3E and 3F. Then, the dose-response curves were fitted to the classical Hill equation using Graph Pad Prism software. Example 1: Identification of SB (Compound 1) as a selective ABA-receptor agonist The in vitro activity of SB with different ABA receptors, including Solanum lycopersicum (tomato) SlPYL1 and representative Arabidopsis thaliana ABA receptors (Figure 1A and 1B). ABA receptors are able to inhibit PP2Cs from different plant species and HAB1 was chosen for these studies. Regarding Arabidopsis receptors, representative members of the three receptor subfamilies were included, such as PYR1, PYL1, PYL2, PYL4, PYL5, PYL6, PYL8, PYL9 and PYL10. Using 100 µM SB, both PYL5 and PYL10 were able to inhibit the HAB1 phosphatase activity by more than 75%, whereas AtPYL1, CsPYL1 and SlPYL1 (PYL1 family) induced around 50% reduction of phosphatase activity. To quantitatively characterize SB activity with respect to ABA, the half maximal inhibitory concentrations (IC50) of HAB1 for either SB or ABA with AtPYL1, AtPYL5 and AtPYL10 12568980-1 were determined. Interestingly, SB activates efficiently AtPYL5 and AtPYLIO and moderately AtPYLI .

Next, the in vivo activity of SB on wild-type Col-0 and either PYL5 or PYL10 overexpressing (OE) lines was tested. SB reduced seedling establishment of both PYL5 and PYL10 OE lines (Figure 1C). These results suggest that SB is active in vivo, In particular, overexpression of these receptors in transgenic lines enhances SB activity. This effect was also observed in root growth assays since SB reduced root growth by 40 % in PYL5 and PYL10 OE lines and 20 % in wild-type seedlings (Figure 1 D). In contrast the sextuple 112458 mutant was resistant to SB, which indicates that SB effect in root growth is dependent on ABA receptors (Figure 1 D).

In summary, SB is an ABA-receptor agonist that shows activity against AtPYLI , AtPYL5 and AtPYLIO.

Example 2: Structural analysis of the SB binding pocket in CsPYLI

As a first step to improve binding of SB to CsPYLI , the crystal structure of CsPYLI -SB- HAB1 and CsPYL1-QB-HAB1 complexes were determined and compared (Figures 1 E, 1 F and 1G) (QB = quinabactin).

The gate and latch loops of CsPYLI display a closed conformation that fits into the active site of the HAB1 phosphatase (Figure 1 E). Both ternary complexes yield isomorphous crystals whose molecular structure was almost identical to a previously reported CsPYLI -ABA-HAB1 complex. SB and QB display a “U” shaped conformation into the ligand binding pocket of the receptor (Figure 1 F). However, superimposition of the structures of the CsPYL1-SB-HAB1 and CsPYL1-QB-HAB1 ternary complexes reveals a shallower insertion of SB in the ABA binding pocket with respect to that observed for QB. This may be explained by the different orientation in SB and QB of the SO2 group, which contains one oxygen atom that shows similar position in both compounds and interacts through a water bridge with both Asn196 and the backbone carbonyl of His89, whereas the other oxygen is located in different position in SB and QB. Thus, in the QB complex this latter oxygen forms a hydrogen bond with Arg108, whereas in the SB complex interacts with the Val110 side chain of CsPYLI (Figure 1 F and G). This difference in SO2 coordination may lead to different activity of SB compared to QB (Figure 1 B). Other than this major difference, QB and SB maintain otherwise a similar network of hydrogen bond interactions in the ABA binding pocket. These include the hydrogen bonds linking the sulfonamide NH to Glu123 side chain, the SO2 group to a conserved water molecule described above, as well as the hydrogen bond network forming the “Trp lock”. This is depending on a water molecule that is hydrogen bonded to the carbonyl oxygen of SB or QB, the backbone amine of Arg 145 at the latch and the carbonyl of Pro117 at the gate, and the side chain of Trp385 from HAB1 .

Example 3: Engineering a synthetic CsPYL1^5m receptor with enhanced sensitivity to SB

To translate the features of AtPYLIO into the ABA binding pocket of CsPYLI , we engineered a synthetic CsPYLI receptor including five substitutions: Val112Leu, Phe137lle, Thr135Leu, Thr153lle and Val168Ala (abbreviated as CsPYLI ^5m) (Figure 2A, centre, Figure 12A) and performed biochemical assays to analyse the sensitivity of CsPYLI ^5m to SB and ABA (Figure 2C). Previously we confirmed that introduction of these five mutations into CsPYLI ^5m does not alter the dimeric state of the receptor (Figure 12B). Interestingly, we observed increased sensitivity to SB of CsPYL1^5m with respect to the wild type as the IC50 of SB was 12-fold lower for the synthetic receptor (Figure 2C). In contrast, a 4-fold increase in the IC50 of ABA was determined in the synthetic receptor compared to wild type (Figure 2C). To further explain these results, we solved the crystal structures of CsPYLI ^5m-SB-HAB1 and CsPYLI ^5m-ABA-HAB1 complexes, and compared them with those obtained previously for wild-type CsPYLI (Figure 2A, center; Figure 11). The comparison of CsPYLI and CsPYL^5m binding pockets reveals that CsPYLI ^5m displays a closer interaction with SB at mutated Leu112, whose methyl groups show favorable contacts with the SB’s aromatic bicyclic ring (Figure 2A, center and right panels). In addition, an enlarged binding pocket is observed at Ile137 (Figure 2A, right). This free space is filled with water molecules that interact with the polar side chain of Lys88, which is otherwise buried in a more hydrophobic environment in the wild-type receptor bound to SB (Figure 2A, right). Thus, Lys88 of CsPYLI ^5m is more amenable to polar interactions in the pocket of CsPYLI ^5m bound to SB. Taken together these structural insights may explain the enhanced sensitivity of CsPYLI ^5m to SB. The synthetic CsPYLI ^5m receptor displays a similar chain of stabilizing interactions as AtPYLIO along the five mutated residues (Figure 2A, left and center panels). However, it is difficult to foresee the precise contribution of a distant mutation, such as Val168Ala, to reshape the ligand binding pocket to better accommodate SB. Nevertheless, distant mutations from the binding pocket of AtPYRI were required to bind mandipropamid and trigger activation of an engineered receptor. The comparison of the ternary complexes of CsPYLI or CsPYL1^5m with ABA and HAB1 reveals that there is a reduction of the water mediated hydrogen bonds that bind ABA to the receptor in the vicinity of the carboxylate group, which may explain the diminished ABA sensitivity of CsPYLI ^5m with respect to the wild type (Figure 11A-C). Indeed, both HAB1 and ABI1 were less inhibited by ABA in the presence of CsPYLI ^5m than with CsPYLI (Figure 11 B and C).

Next, we generated Arabidopsis genetically altered plants expressing CsPYLI ^5m driven by the 35S promoter and, as a control, we also generated genetically altered plants expressing the wild type version, i.e. 35S:CsPYL1 (Figure 2D). We selected T3 plants expressing similar levels of CsPYLI and CsPYLI ^5m and analysed their sensitivity to ABA and SB (Figure 2D and E). Both 35S:CsPYL1 and 35S:CsPYL1^5m plants showed similar sensitivity to ABA-mediated inhibition of seedling establishment (Figure 2E, right). In accordance with the in vitro IC50 of SB for CsPYLI and CsPYLI ^5m, we used higher concentrations of this compound than ABA in the seedling establishment assay (Figure 2E). Interestingly, 35S:CsPYL1^5m lines were sensitive to 10 μM SB, whereas 35S:CsPYL1 lines were not affected even by 100 μM SB (Figure 2E). These results indicate that structure-guided modifications of CsPYLI ^5m were effective in vivo and suggest increased capability of the mutated receptor to bind SB. However, the dosage of SB required to inhibit seedling establishment of CsPYLI ^5m lines was still 20-fold higher than ABA.

Example 4: Swapping of SO2 and CH2 in SB’s sulfonamide linker leads to iSB derivatives with enhanced activity as ABA agonists

The wealth of crystallographic information on ABA receptors in complex with agonist and antagonist molecules has revealed key structural features of the ligand-receptor coordination that enable ligand improvement. In order to optimize the SB scaffold for enhanced activation of ABA receptors, substitutions in the dihydroquinoline of SB to fill the 3’-tunnel of ABA receptors were first explored and different SB derivatives were synthesized (Figure 9A). The 3’-tunnel is a small solvent-exposed space adjacent to ABA’s 3'-CH that normally interacts with ABA’s 7' methyl group. The 3’-tunnel is formed by five highly conserved hydrophobic residues (in PYR1 : Phe61 , Leu87, Pro88, Phe159 and Vai 163) and can accept alkyl substituents of ABA agonists to form hydrophobic contacts and increase agonist potency. Interestingly, introduction of 1 -ethyl instead of the 1 -methyl group in the dihydroquinoline, i.e. compound SB-01 , led to enhanced inhibitory activity of the agonist with both CsPYLI and CsPYL1^5m (Figure 9A). In the case of CsPYLI ^5m, it was found that SB-01 showed enhanced inhibition of seedling establishment compared to SB in CsPYL1^5m lines (Figure 9B). Introduction of a bulkier hydrophobic substituent as the cyclopropyl group in SB-02 also showed some capability to inhibit HAB1 activity (Figure 9A). Changes in the SB’s benzyl ring were also tested, i.e. SB-03 to SB-06. Introduction of the methyl substituent in SB-03 led to some improvement compared to SB (Figure 9A and B).

Both CsPYL1-SB-HAB1 and CsPYLI ^5m-SB-HAB1 complexes lack an interaction of the agonist with the conserved Lys88 residue of the receptor, rather the structures show that the hydrophobic CH2 of the SB linker faces to a water-filled cavity harboring the side chain NH3+ of Lys88 (Figure 2A, center and right panels). Hence, it was reasoned that swapping the positions of the SO2 and CH2 moieties in the linker might promote the formation of a hydrogen bond between the SO2 moiety of the agonist molecule and the Lys88 side chain, increasing ligand activity. Two new compounds incorporating the above swap were therefore synthesized, iSB07 and iSB09 (“inverted SB”) (Figure 3A). Compared to iSB07, iSB09 contains a larger alkyl group (ethyl versus methyl) at the dihydroquinoline ring that might increase ligand activity through interaction with the 3’- tunnel as described above for SB-01 (Figure 3C). The IC50 values for HAB1 of SB, iSB07 and iSB09 with CsPYLI ^5m were 876, 346 and 316 nM, respectively, which indicates that both iSB07 and iSB09 are improved versions of SB (Figure 2C and 3A). This improvement was also evident when IC50 for HAB1 of iSB07 and iSB09 was obtained with CsPYLI (Figure 2C and 3A), compared to SB. The iSB07/iSB09-dependent PP2C inhibition assay was extended to ABI1 and PP2CA, which are phosphatases very relevant for ABA signaling. Both ABI1 and PP2CA were even more sensitive to iSB09- mediated CsPYLI ^5m-dependent inhibition, particularly ABI1 was 5-fold more sensitive than HAB1 (Figure 3A). Finally, the action of iSB07 and iSB09 with seven ABA receptors that are biologically relevant in Arabidopsis was also tested (Figure 3B). Interestingly, iSB09 was more active towards dimeric receptors than iSB07, and using 1 μM iSB09, 60-70% inhibition of HAB1 was achieved when the dimeric PYR1 , PYL1 and PYL2 as well as monomeric PYL5 were assayed (Figure 3B). Comparison of these results with those obtained for 100 μM SB (Figure 1A) also reveals a dramatic improvement of the agonist potency in iSB compounds, being the ethyl group of iSB09 an additional improvement compared to the methyl group of iSB07 (Figure 3B).

Isothermal titration calorimetry (ITC) experiments were also conducted comparing ABA- CsPYL1-HAB1 and iSB09-CsPYL1^5m-HAB1 binding reactions (Figure 3C and D). These experiments show a similar Kd for ABA and iSB09 binding in these ternary complexes, which suggests that the combination iSB09-CsPYL1^5m-HAB1 matches the affinity of ABA binding in the ABA-CsPYL1-HAB1 complex. Additional experiments to examine ligand binding in ternary complexes were performed by using Native Red Elecrophoresis (NRE) analysis of ligand-induced ternary complexes (Figure 3E and F). In this case ABA- AtPYL5-HAB1 and iSB09-AtPYL5-HAB1 binding reactions were examined by NRE. A dose response NRE analysis followed by quantification of the ternary complex formed versus free ABA or iSB09 concentration also revealed a similar apparent Kd for both ligands.

Therefore, the swapping of SO2 and CH2 in SB’s sulfonamide linker leads to iSB derivatives that have enhanced activity as ABA agonists.

Example 5: Structural insights into iSB-receptor-phosphatase complexes

To gain insight into the structural basis of iSB agonist activity, the crystal structures of CsPYLI -iSB07-HAB1 , CsPYLI -iSB09-HAB1 , CsPYL1^5m-iSB07-HAB1 and CsPYLI ^5m- iSB09-HAB1 were solved (Figure 4A-C). Consistent with the docking predictions, both iSB07 and iSB09 form a hydrogen bond between the oxygen of the SO2 moiety and side chain NH3+ of Lys88, and unexpectedly an additional one to the guanidinium moiety of Arg108 side chain (Figure 4A and C). These additional H-bonds likely explain the enhanced activity of iSB07 and iSB09 with respect to that observed for SB, as SB lacks these interactions (Figure 4A and 1G). Additionally, iSB09 structures reveal that iSB09’s ethyl group, compared to iSB07’s methyl group, increases the number of hydrophobic contacts at the 3’-tunnel without distorting the conformation of the gate (Figure 4B-C). In addition, there are several favorable rearrangements in the conformation of the iSB07, iSB09 and SB molecules in the ternary complex with CsPYL1^5m compared to CsPYLI. Specifically, the structure of the sulfonamide linker displays a more likely and relaxed conformation (in terms of the torsion angles) for agonists in the ligand binding pocket of CsPYLI ^5m than in CsPYLI . Thus, the molecular geometries of the ligands in the ternary complexes with CsPYL1^5m relax, showing torsion values closer to the maximum values observed for the ligands in the unbound form (Figure 4D).

Altogether, the structural data suggest that the differences observed for iSB09, iSB07 and SB potency as agonists may be a consequence of a number of adjustments in the conformation of the ligand as well as interactions in the binding pocket of the receptors, which include the formation of additional hydrogen bonds and favorable hydrophobic contacts for iSB compounds.

Example 6: The iSB09 compound is an efficient ABA agonist that applied to 35S:CsPYL1^5m plants markedly enhances drought tolerance.

The combination of genetic and chemical approaches is a powerful tool to enhance ABA signalling; therefore, we tested application of iSB07 and iSB09 in Arabidopsis genetically altered plants that express either the wild-type CsPYLI or the CsPYL1^5m receptor. We performed different assays in seeds and vegetative tissues to evaluate ABA responses after applying the compounds in these plants. Interestingly, plants expressing CsPYLI ^5m were markedly more sensitive to agonist-mediated inhibition of seed germination compared to plants expressing the wild-type version (Figure 5A).

Moreover, iSB09 was 3-fold more effective than iSB07 to inhibit seed germination in plants expressing CsPYLI ^5m (data in Figure 5A, right). Likewise, inhibition of seedling establishment by iSB07 and iSB09 was markedly enhanced in plants expressing CsPYLI ^5m compared to CsPYLI lines (Figure 5B). Moreover, the iSB09 compound was able to inhibit seedling establishment of Col-0 wild type at 5 μM, whereas this dosage was reduced to 0.5 μM in plants that express CsPYLI ^5m (Figure 5B). Next, we tested iSB07 and iSB09-mediated inhibition of root growth in 5-d-old seedlings of wild-type Col- 0 and CsPYLI ^5m lines (Figure 5C). At 10 μM concentration, iSB07 and iSB09 did not significantly inhibit root growth of wild type plants; however, a dramatic inhibition of CsPYLI ^5m lines was observed. Therefore, we conclude that the combination of iSB07 or iSB09 with CsPYLI ^5m is a powerful tool to activate ABA signalling and promote ABA responses.

Finally, we tested the capability of iSB07 and iSB09 to regulate stomatai aperture in wildtype Col-0 and CsPYLI ^5m lines by whole plant gas exchange analysis of stomatai conductance (Gs) after spraying the compounds. There were no significant differences between the lines in basal pre-treatment Gs (Figure 6A). However, spraying with 5 μM iSB07 or iSB09 had a significant effect on Gs in the genetically altered lines expressing CsPYL1^5m (repeated measures ANOVA, Fig 6A-B). In Col-0, the small effect of 5 μM iSB09 was non-significant, but spraying with 20 μM iSB09 was effective to reduce Gs whereas iSB07 was not effective in Col-0 even at 20 μM (Figure 6C). The dynamic courses of Gs after spraying are presented in Figure 6B and show that the reduction of Gs induced by iSB07 in genetically altered lines was significant but weaker compared to iSB09. In genetically altered lines, reduction of Gs by iSB09 was still significantly evident 24 and 48 hours after spraying, with no signs of recovery (Figure 6D). The Gs of genetically altered lines was lower compared to pre-treatment values 24 and 48 h after spraying with iSB07, however, these differences were non-significant (Figure 6D). Thermal imaging in genetically altered lines revealed increased leaf temperature upon spraying with both iSB07 and iSB09 (Figure 6E and 6F), which also occurred in nontransformed Arabidopsis Col-0 plants (Figure 6F). Finally, thermal imaging of nontransformed Nicotiana benthamiana leaves 24 h after spraying with 50 μM ABA, iSB07 or iSB09 also revealed increased leaf temperature compared to mock-treated (DMSO) plants (Figure 6G and 6H, Jorge methods). Application of the iSB09 compound was the most effective to increase leaf temperature, which indicates that this compound also can promote stomatai closure in wild-type plants, either in Arabidopsis or N. benthamiana (Figure 6F and 6H). Nicotiana benthamiana shows a high biomass production and transpiration, so these results suggest that spraying of iSB09 even might be effective as antitranspirant treatment in those plant species that show high ratios of transpiration. Given that the iSB9 effect on Gs was markedly enhanced in lines expressing CsPYL1^5m, in which lasted at least for 48 h, we decided to test whether spraying of this compound over CsPYL1^5m plants during a drought period was effective to enhance drought resistance, reduce water consumption of the soil (increasing water retention in the soil, ‘water banking’) and promote survival of the plants after rewatering (Figure 7). We devised two drought treatments, either in a plant growth chamber under short day conditions to favor rosette development (Figure 7A-C) or in greenhouse under long day conditions (Figure 7D-F). Figure 7A shows that iSB9 treatment markedly enhanced drought resistance of CsPYL1^5m plants grown under short day conditions compared to mock-treated plants. Water consumption of the soil was measured by gravimetric analysis and we found that iSB09 treatment enhanced water banking in soil (Figure 7B). For example, 21 -d after stopping irrigation circa 70% water had been lost in pots of mock- treated plants whereas only 40% water consumption was recorded in iSB09-treated plants (Figure 7B). In agreement with these data, a high percentage of plants survived after iSB09 treatment (Figure 7C). Next, we performed drought treatments under greenhouse conditions and overall results were similar to those described above. Thus, enhanced resistance to drought was observed in iSB09-treated plants compared to mock-treated plants, as well as dramatic enhancement of plant survival and reduced water consumption (Figures 7D-F). The effect of iSB09 was markedly enhanced when combined with lines expressing CsPYL1^5m; however, the compound was also effective in wild-type Col-0 plants when higher dosage was applied.

To examine iSB09’s effect on ABA-induced transcriptional response, we used Arabidopsis lines where the ABA-responsive MKKK18 promoter is fused to the LUC reporter. At 100 μM iSB9, induction of LUC was approximately 2-fold higher than that achieved with 25 μM ABA, which suggests that iSB09 also promotes ABA transcriptional response through activation of Arabidopsis ABA receptors (Fig 8A-B). iSB07’s effect at 100 μM was approximately 5-fold lower than iSB09 (Fig. 8B). We also compared ABA- and iSB09-upregulation of RAB18/RD29B gene expression in wild-type Col-0 and plants that express CsPYL1^5m (Fig. 10A). Similar expression of RAB18/RD29B was achieved in wild-type plants treated with 5 μM ABA compared to CsPYL1^5m plants treated with 5 μM ABA (Fig. 10A). The effect of iSB09 on RAB18/RD29B upregulation in wild-type plants was markedly lower than in CsPYL1^5m plants, which indicates that the iSB09- CsPYL1^5m combination markedly enhances ABA-like transcriptional response (Figure 10A). In order to obtain a global perspective on the combined iSB09-CsPYL1^5m transcriptional effect, we performed high-throughput RNA sequencing (RNAseq) by using an Illumina HiSeq platform (Figure 8C). We compared the iSB09 effect (versus mock-treatment) in wild type and CsPYL1^5m genetically altered lines (Figure 8C). Gene ontology (GO) analyses indicated that iSB09-upregulated/downregulated genes overlap with ABA-responsive genes. Appreciable induction of ABA-responsive markers by iSB09 in wild type was observed; however, a dramatic upregulation (8 to 10-fold higher) was achieved when the iSB09 ligand was applied to CsPYL1^5m genetically altered background (Figure 8C and E). On the other hand, the transcriptional profile of CsPYL1^5m genetically altered background is similar to wt plants in the absence of agonist treatment (correlation of 0.99 in TPM RNAseq analysis, Figure 8D), which indicates that no constitutive activation of stress response occurs in CsPYL1^5m plants. Finally, to identify the Arabidopsis receptors that mediate iSB09’s effect in wild-type seeds/seedlings, we analyzed agonist-induced inhibition of seedling establishment in wild-type or mutant strains lacking certain Arabidopsis receptors (Fig. 10B). As a result, we found that Arabidopsis mutants lacking functional PYR1 and PYL1 were insensitive to iSB09, which was corroborated in the pyr1 pyl1 pyl2 triple and 112458 sextuple mutants. On the other hand, pyl4 pyl5 and pyl8 pyl9 double mutants were sensitive to iSB09. These results suggest that iSB09’s effect in seeds is mediated mostly by dimeric receptors (subfamily III). Given that iSB09 is also perceived by PYL5 (Figure 3B), a diminished effect of iSB09 might be expected in pyl4 pyl5 double mutant; however, other agonists that also target PYL5, such as QB and AMF4, lack effect in pyr1 pyl 1 double mutant. This suggests that PYL5 effect in seed is likely overtaken by dimeric receptors.

In summary, chemical manipulation of ABA signalling is important for abiotic and biotic stress management in agriculture and together with genetic approaches can provide a powerful tool to dynamically activate the pathway. We have developed the iSB09 compound, which activates ABA signalling in Arabidopsis wild-type Col-0 plants mostly through interaction with the dimeric PYR1 , PYL1 and PYL2 and monomeric PYL5 receptors. iSB09 was also effective to reduce transpiration in Nicotiana benthamiana leaves, which suggests that similar receptors occur in this plant that can be activated by iSB09. To enhance iSB09’ effect on ABA signalling, we further devised the synthetic CsPYL1^5m receptor, and a combined genetic-chemical approach using iSB09 + CsPYL1^5m was very effective to enhance drought resistance in Arabidopsis.

Example 7

To investigate if iSB09 displays activity against other dimeric receptors harboring the CsPYL1_5m mutations, we computed bioinformatic models of Arabidopsis thaliana PYR1_5m, PYL1_5m, PYL2_5m and PYL3_5m and examined whether iSB09 docks into their ABA binding pockets. For this purpose, we employed as a template the crystal structures of the native receptors (PDB codes 3QN1 , 3KDj, 4LA7 and 4DS8). Our results showed that iSB09 maintains all the interactions observed in the experimentally determined crystal structure of CsPYL1^5m-iSB09 complex, thus indicating that the described mutations can be broadly used for other dimeric receptors (Figure 13). The ABA binding pocket is defined as those residues from the wild type protein that interact directly, or through a water molecule, to iSB9 (Table 1).

Table 1. CsPYLI residues interacting with iSB9

Forming strong Hydrogen Bonds (direct or using a water molecule bridge)

Forming weak Hydrogen Bonds

Hydrophobic at short distances (less than 4 angstrom)

191 Y149 1139 L146 F188 V192 V112 A118 H144 V110 F90 F137

Example 8

We are transforming Nicotiana benthamiana plants with the CsPYLI ^5m receptor. Figure 6G and H show that iSB07 and iSB09 are effective in wild-type N. benthamiana plants, so we expect to increase the phenotype in plants transformed with the CsPYLI ^5m receptor. This citrus receptor, together with iSB09, was also effective in Arabidopsis, meaning this combination will be effective in other plants. The procedure for transformation and in vitro regeneration is the leaf-disc method, which was followed with minor modifications as described by Vazquez-Vilar et al. (2021). REFERENCES

Clough, S.J. and Bent,A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.

Deblaere.R., Bytebier, B., De Greve, H., Deboeck.F., Schell, J., Van Montagu, M. and Leemans, J. (1985). Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 13, 4777-4788.

Koilist, T., Moldau,H., Rasulov, B., Oja,V., Ramma.H., Huve,K., Jaspers, P., Kangasjarvi.J. and Koilist, H. (2007). A novel device detects a rapid ozone-induce transient stomatai closure in intact Arabidopsis and its absence in abi2 mutant. Physiol Plant 129, 796-803.

Moreno-Alvero,M., Yunta.C., Gonzalez-Guzman, M., Lozano-Juste, J., Benavente.J.L., Arbona.V., Menendez, M., Martinez-Ripoll.M., Infantes, L., Gomez-Cadenas,A., Rodriguez.P.L. and Albert, A. (2017). Structure of Ligand-Bound Intermediates of Crop ABA Receptors Highlights PP2C as Necessary ABA Co-receptor. Mol. Plant 10, 1250-1253.

Dupeux.F., Antoni, R., Betz,K., Santiago, J., Gonzalez-Guzman, M., Rodriguez.L., Rubio, S., Park.S.Y., Cutler, S.R., Rodriguez.P.L. and Marquez, J.A. (2011). Modulation of Abscisic Acid Signaling in Vivo by an Engineered Receptor-Insensitive Protein Phosphatase Type 2C Allele. Plant Physiol 156, 106-116.

Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., Park, S.Y., Marquez, J.A., Cutler, S.R., and Rodriguez, P.L. (2009b). Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. The Plant journal : for cell and molecular biology 60, 575-588.

Vazquez-Vilar.M., Garcia-Carpintero,V., Selma, S., Bernabe-Orts.J.M., Sanchez-Vicente, J., Salazar-Sarasua.B., Ressa,A., de Paola, C., Ajenjo.M., Quintela.J.C., Fernandez-Del- Carmen, A., Granell,A. and Orzaez.D. (2021). The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants. Front Plant Sci. 12, 689937.

Claims

CLAIMS: 1. A compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof:

wherein R₁ to R₃ are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; R₄ to R₇ are independently selected from a structure according to Formulae 1-1 or 1-2, hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl;

at least one of R₄ to R₇ has a structure according to Formulae 1-1 or 1-2; wherein in Formulae 1-1 and 1-2, R₈ to R₁₁ are independently selected from hydrogen, hydroxy, halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, optionally substituted alkanoyl, optionally substituted amino, optionally substituted aryl and optionally substituted heteroaryl; L1 and L2 are independently selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene and optionally substituted heteroarylene; 12568980-1 a1 and a2 are independently selected from 1 to 6; represents a single or double bond; and represents a connection point to the rest of the compound; wherein the compound is not Compound 1:

.

2. A compound according to claim 1, wherein the compound has a structure according to Formula II:

wherein R₁ to R₁₁, L₁, L₂, a1 and a2 are as defined in claim 1.

3. A compound according to claim 1 or claim 2, wherein R1 is optionally substituted alkyl; preferably wherein R1 is C1-C6 alkyl; more preferably wherein R1 is methyl.

4. A compound according to any one of claims 1 to 3, wherein R3 is selected from optionally substituted alkyl and optionally substituted cycloalkyl; preferably wherein R3 is selected from C1-C6 alkyl and C3-C8 cycloalkyl; more preferably wherein R3 is selected from methyl, ethyl and cyclopropyl; even more preferably wherein R3 is ethyl.

5. A compound according to any one of claims 1 to 4, wherein R6 has a structure according to Formulae 1-1 or 1-2; preferably wherein R6 has a structure according to Formula 1-1.

6. A compound according to any one of claims 1 to 5, wherein L1 and/or L2 is optionally substituted alkylene; preferably wherein L1 and/or L2 is C1-C6 alkylene; more preferably wherein L1 and/or L2 is methylene.

7. A compound according to any one of claims 1 to 6, wherein R9 and/or R11 is optionally substituted aryl; preferably wherein R9 and/or R11 is optionally substituted phenyl; more preferably wherein R9 and/or R11 is selected from phenyl, halogen-substituted phenyl and hydroxy-substituted phenyl.

8. A compound according to any one of claims 1 to 7, wherein a1 and/or a2 is 1.

9. A compound according to claim 1, wherein the compound is selected from any one of Compounds 2 to 9:

10. A composition comprising a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, and an agrochemically acceptable carrier, wherein R1 to R11, L1, L2, a1 and a2 are as defined in any one of claims 1 to 9; preferably wherein the compound is selected from any one of Compounds 1 to 9.

11. Use of a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, or a composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier, in enhancing abiotic stress resistance in a plant, wherein R₁ to R₁₁, L₁, L₂, a1 and a2 are as defined in any one of claims 1 to 9.

12. Use according to claim 11, wherein the plant comprises at least one mutation in at least one pyrabactin resistance (PYR)-like (PYL or PYR) polypeptide, wherein the mutation corresponds to one or more of V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1; or wherein the mutation corresponds to one or more of V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5; or wherein the mutation corresponds to one or more of V87L, F112I, T128I and V145A of SEQ ID NO: 7; or wherein the mutation corresponds to one or more of V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence; or wherein the plant comprises a nucleic acid construct comprising a nucleic acid sequence encoding a PYL1 polypeptide as defined in SEQ ID NO: 2, 11, 12 or 13 or a functional variant thereof.

13. Use according to any one of claims 11 or 12, wherein the compound is selected from any one of Compounds 1 to 9.

14. Use according to any one of claims 11 to 13, wherein the stress is drought.

15. Use according to any one of claims 11 to 14, wherein the plant is a crop plant or biofuel plant, wherein preferably said crop plant is selected from maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

16. A method of enhancing abiotic stress resistance in a plant, comprising applying a compound according to Formula I or a salt, a solvate, a tautomer, a stereoisomer or a deuterated analogue thereof, or a composition comprising the compound according to Formula I or the salt, the solvate, the tautomer, the stereoisomer or the deuterated analogue thereof, and an agrochemically acceptable carrier, wherein R1 to R11, L1, L2, a1 and a2 are as defined in any one of claims 1 to 9.

17. A method according to claim 16, wherein the plant comprises a nucleic acid construct comprising a nucleic acid sequence encoding a PYL1 polypeptide as defined in SEQ ID NO: 2, 11, 12 or 13 or a functional variant thereof.

18. A method according to claim 16 or claim 17, wherein the plant comprises at least one mutation in at least one PYL or PYR polypeptide, wherein the mutation corresponds to one or more of V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1 ; or wherein the mutation corresponds to one or more of V83L, F108I, T106L, T124I and V139A of SEQ ID NO: 5; or wherein the mutation corresponds to one or more of V87L, F112I, T128I and V145A of SEQ ID NO: 7; or wherein the mutation corresponds to one or more of V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence.

19. A method according to any one of claims 16 to 18, wherein the compound is selected from any one of Compounds 1 to 9.

20. A method according to any one of claims 16 to 19, wherein the compound or composition is applied to the plant, a part thereof or a seed thereof, a soil in which plants are growing, or a soil on which seeds are sown.

21. The method according to any one of claims 16 to 20, wherein the stress is drought.

22. The method according to any one of claims 16 to 21 , wherein the plant is a crop plant or biofuel plant, wherein preferably said crop plant is selected from maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.

23. An isolated nucleic acid comprising a nucleic acid sequence encoding a mutant PYL polypeptide, wherein the polypeptide comprises at least one mutation that corresponds to one or more of V112L, F137I, T135L, T153I and V168A of SEQ ID NO: 1 ; or at least one mutation that corresponds to one or more of V83L, F108I, T106L, T 1241 and V139A of SEQ ID NO: 5; or at least one mutation that corresponds to one or more of V87L, F112I, T128I and V145A of SEQ ID NO: 7; or at least one mutation that corresponds to one or more of V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence, wherein preferably the nucleic acid sequence encodes a PYL polypeptide as defined in SEQ ID NO: 2, 11 , 12 or 13 or a functional variant thereof.

24. The isolated nucleic acid according to claim 23, wherein the nucleic acid sequence comprises a PYL sequence as defined in SEQ ID NO: 4 or a functional variant thereof.

25. The isolated nucleic acid according to claim 23 or claim 24, wherein the nucleic acid does not have further mutations in the PYL polypeptide.

26. A nucleic acid construct comprising the isolated nucleic acid according to any one of claims 23 to 25, wherein the nucleic acid is operably linked to a regulatory sequence, wherein preferably said regulatory sequence is a constitutive promoter, a strong promoter, an inducible promoter, a stress inducible promoter or a tissue specific promoter.

27. A host cell comprising the nucleic acid construct according to claim 26.

28. A genetically altered plant or plant part thereof, expressing the isolated nucleic acid according to any one of claims 23 to 25 or the nucleic acid construct according to claim 26.

29. A genetically altered plant or part thereof wherein the plant comprises at least one mutation in at least one PYL or PYR gene, wherein the mutation results in at least one of the following mutations: V112L, F137I, T135L, T153I and V168A of SEQ I D NO: 1 ; or at least one of the following mutations: V83L, F108I , T106L, T124I and V139A of SEQ ID NO: 5; or at least one of the following mutations V87L, F112I, T128I and V145A of SEQ ID NO: 7; or at least one of the following mutations: V107L, F132I, T148I and V168A of SEQ ID NO: 9 or a corresponding position in a homologous/orthologous sequence.

30. The genetically altered plant according to claim 28 or claim 29, wherein the plant is a crop plant or biofuel plant, wherein preferably said crop plant is selected from maize, rice, wheat, oilseed rape, sorghum, soybean, potato, tomato, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar. The genetically altered plant part according to any of claims 28 to 30, wherein the plant part is a seed. A method of inhibiting seed germination/prolonging seed dormancy in a plant or a method of activating ABA signalling and/or activating or increasing an ABA response, the method comprising applying a compound according to any one of claims 1 to 9, or a composition according to claim 10. A method of producing a plant with increased abiotic stress resistance, the method comprising introducing and expressing a nucleic acid according to any one of claims 23 to 25 or a nucleic acid construct according to claim 26 into a plant; or wherein the method comprises introducing at least one mutation into a plant genome, wherein the mutation is the addition of one or more additional copy of a mutated PYR/PYL polypeptide, wherein preferably, the mutated polypeptide comprises SEQ ID NO: 2, 11 , 12 or 13 or a functional variant or fragment thereof, and wherein the one or more additional copy of the mutated PYL/PYR polypeptide is operably linked to a regulatory sequence. A plant obtained or obtainable by the method of claim 33.