agricultural formulation, method of increasing the tolerance of abiotic stress in a plant, method of

专利摘要:
SYNTHETIC COMPOUNDS FOR VEGETABLE TAB RESPONSES. The present invention provides agonist compounds, which activate ABA receptors, and agricultural formulations, which comprise the agonist compounds. Agricultural formulations are useful to induce ABA responses in plant plant tissues, reduce abiotic stress in plants and inhibit plant seed germination. The compounds are also useful for inducing the expression of ABA-responsive genes in cells that express endogenous or heterologous ABA receptors.
公开号:BR112014024379B1
申请号:R112014024379-4
申请日:2013-03-15
公开日:2021-02-09
发明作者:Sean R. Cutler；Masanori Okamoto
申请人:The Regents Of The University Of California；
IPC主号:

专利说明:

CROSS REFERENCES TO RELATED ORDERS
[0001] This application claims priority benefit from US Provisional Patent Application No. 61 / 618,386, filed on March 30, 2012, which is incorporated into this document by reference in its entirety. DECLARATION ON THE RIGHTS TO INVENTIONS MADE UNDER RESEARCH AND DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT
[0002] This invention was made with the support of the Government under Concessions Nos DGE0504249 and IOS0820508, granted by the National Science Foundation. The government has certain rights in this invention. BACKGROUND OF THE INVENTION
[0003] Abscisic acid (ABA) is a plant hormone that regulates signal transduction associated with abiotic stress responses (Cutler et al, 2010, Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology 61: 651 -679). The ABA signaling pathway has been explored to improve the plant's stress response and associated yield traits through various approaches (Yang et al, 2010). Direct application of ABA to plants improves water use efficiency (Raedmacher et al., 1987); for this reason, the discovery of ABA agonists (Park et al., 2009; Melcher et al., 2010, Identification and mechanism of ABA receptor antagonism. Nature Structural & Molecular Biology 17 (9): 1102-1110) has received increasing attention , since such molecules can be beneficial for improving crop yields (Notman et al .., 2009). The first synthetic ABA agonist identified was naphthalene sulfonamide called pirabactin (Park et al., 2009), which efficiently activates ABA signaling in seeds, but limited activity in plant tissues, where the most critical aspects of stress tolerance occur. abiotic. Sulfonamides very similar to pirabactin have been disclosed as ABA agonists (see US Patent Publication No. 20130045952) and abiotic stress modulation compounds (see US Patent Publication No. 20110230350); and non-sulfonamide ABA agonists have also been described (see US Patent Publication Nos. 20130045952 and 20110271408). A complementary approach to activate the ABA pathway involves increasing the plant's sensitivity to ABA through genetic methods. For example, conditional antisense of the beta subunit gene for farnesyl transferase, which increases the plant's ABA sensitivity, improves yield under moderate drought in canola and Arabidopsis (Wang et al., 2005). Thus, manipulation of ABA signaling to improve traits that contribute to performance is now well established.
[0004] Recently, ABA was found to elicit many of its cellular responses by binding to a soluble family of receptors called PYR / PYL proteins. PYR / PYL proteins belong to a broad family of ligand-binding proteins called the START superfamily (Iyer et al., 2001); Ponting et al., 1999). These proteins contain a conserved three-dimensional architecture consisting of seven anti-parallel beta sheets, which surround a central alpha helix to form a "helixgrip" motif; together, these structural elements form a ligand-binding pocket to bind ABA or other agonists. BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides small molecule ABA agonists, that is, compounds that activate PYR / PYL proteins. In one aspect, the present invention provides agricultural formulations comprising the ABA agonists described in this document. In some embodiments, the agricultural formulation comprises a compound of Formula I:
wherein R1 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, R2 is selected from the group consisting of cycloalkyl, heterocycloalkyl, aryl and heteroaryl , each optionally substituted by 1 to 4 groups R2a, each R2a is independently selected from the group consisting of H, halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl , C2-6 alkynyl, -OH, C1-6 alkylhydroxy, -CN, -NO2, -C (O) R2b, -C (O) OR2b, -OC (O) R2b, -C (O) NR2bR2c, -NR2bC (O) R2c, -SO2R2b, -SO2OR2b, - SO2NR2bR2c, and -NR2bSO2R2c, each of R2b and R2c is independently selected from the group consisting of H and C1-6 alkyl, each of R3, R4 and R5 is independently selected from from the group consisting of H and C1-6alkyl, L is a linker selected from the group consisting of a bond and C1-6alkylene, the subscript m is an integer from 0 to 4, the subscript n is an int number 0 to 3, or a salt or isomer thereof.
[0006] In some embodiments, the agricultural formulation additionally comprises an agricultural chemical that is useful for promoting plant growth, reducing weeds, or reducing pests. In some embodiments, the agricultural formulation additionally comprises at least one of a fungicide, a herbicide, a pesticide, a nematicide, an insecticide, a plant activator, a synergistic agent, a herbicide protective agent, a plant growth regulator, a insect repellent, an acaricide, a molluscicide or a fertilizer. In some embodiments, the agricultural formulation additionally comprises a surfactant. In some embodiments, the agricultural formulation additionally comprises a carrier.
[0007] In another aspect, the invention provides methods for increasing the tolerance to abiotic stress in a plant, the method comprising the step of contacting a plant with a sufficient amount of the above formulations to increase the tolerance to abiotic stress in the plant compared to tolerance to abiotic stress in the plant when not in contact with the formulation. In some embodiments, the plant is a monocot. In some embodiments, the plant is a dicot. In some modalities, tolerance to abiotic stress comprises tolerance to drought.
[0008] In another aspect, the invention provides a method of inhibiting seed germination in a plant, the method comprising the step of bringing into contact with a plant, a part of the plant, or a seed of the plant with a sufficient amount of above formulations to inhibit germination.
[0009] In another aspect, the invention provides a plant or part of the plant in contact with the above formulations. In some embodiments, the plant is a seed.
[0010] In another aspect, the invention provides a method of activating a PYR / PYL protein. In some embodiments of the method, the PYR / PYL protein binds to a type 2 protein phosphatase polypeptide (PP2C) when the PYR / PYL protein binds to the LC66C6 agonist compound (also referred to herein as quinabactin). In some embodiments, the method comprises the step of contacting the PYR / PYL protein with any of the compounds described in this document. In some embodiments, the PYR / PYL protein that is activated is substantially identical to any of SEQ ID NOs: 1-119. In some embodiments, the PYR / PYL protein is expressed by a cell. In some embodiments, the PYR / PYL protein is expressed by a plant cell. In some embodiments, the PYR / PYL protein is an endogenous protein. In some embodiments, the PYR / PYL protein is a heterologous protein. In some embodiments, the cell additionally expresses a type 2 protein phosphatase (PP2C). In some embodiments, the type 2 protein phosphatase is HAB1 (Homology for ABI1), ABI1 (Insensitive abscisic acid 1), or ABI2 (Insensitive abscisic acid 2). BRIEF DESCRIPTION OF THE FIGURES
[0011] Figure 1. New ABA agonists bind to multiple PYR / PYL. (A) Naturally occurring chemical structure (+) - ABA, its (-) analog and selected ABA agonists. (B) Agonist assays for two yeast hybrids of PYR / PYL receptor sensitivity to 5 μM of test chemicals. Specific PYR / PYL receptors and PP2C HAB1 are expressed as Gal4 BD or AD fusion proteins respectively, as described in the text.
[0012] Figure 2. New ABA agonists inhibit PPC2 activity through multiple PYR / PYL. (A) Naturally occurring chemical structure (+) - ABA and selected ABA agonists. (B) and (C) PP2C enzymatic activity of HAB1, ABI1, and ABI2 based on ABA agonist assays for multiple receptors in the presence or absence of 10 μM of each test chemical.
[0013] Figure 3. (A) receptor-dependent inhibition of PP2C enzyme activity by ABA agonists and analogs. (B) Observed IC50 values of the compound in ABA agonist enzyme assays based on HAB 1 PP2C.
[0014] Figure 4. Quinabactin activates multiple ABA receptors. (A) Chemical structures of ABA, pirabactin and quinabactin. (B) Chemical dependent inhibition of HAB 1 by ABA receptors. IC 50 values (nM) were determined as described in the methods using 50 nM HAB1, 50 nM and multiple concentrations of compounds; total dose response curves are provided as in Figure 3. (nd) correspond to receptors that were not produced as active proteins. The phylogenetic tree is a Neighbor-Joining tree made using the JTT distance matrix in MEGA5 (Tamura K, et al. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28 (10): 2731-2739).
[0015] Figure 5. New ABA agonists inhibit the germination of Arabidopsis seeds more strongly than pirabactin. (A) and (B) Comparison of inhibition of seed germination by ABA agonists. (C) and (D) the effects of ABA and LC66C6 (also called quinabactin), the deficient mutants of Arabidopsis ABA biosynthesis and signaling (C) and seedling establishment (D). The seeds were sown on a 1 / 2X MS agar plate containing chemicals, and were stored at 4 ° C for 4 days, then transferred at 22 ± 2 ° C. Photographs (A and C) and germination (B) or green cotyledon scores (D) were evaluated after a 4-day incubation under continuous lighting. Panel C shows the germination tests in 5 μM ABA or LC66C6.
[0016] Figure 6. LC66C6 inhibits plant growth. (A) Photographs showing the effect of ABA, Pirabactin and LC66C6 on wild type, abil-1 and PYR / PYL quadruple mutant Arabidopsis genotypes. (B) inhibition of root growth and (C) inhibition of plant growth by ABA, LC66C6 and pyrabactin. Two-day-old seedlings were transferred to 1 / 2X MS plate containing chemicals and phenotypes marked or photographed after a 5-day incubation in test compounds.
[0017] Figure 7. LC66C6 improves drought stress tolerance. LC66C6 suppresses the loss of transpirational water from detached leaves in the wild type (A) and the mutant flap2 genotypes (B). (C) LC66C6 cannot rescue the ABA abil-1 insensitive genotype phenotypes. (D) LC66C6 induces stomatal closure in wild type and aba2, but not the abil- 1 genotypes. (E) Effects of compounds on soil water content during soybean drought treatments. The water content of the soil was measured as described in the examples.
[0018] Figure 8. Quinabactin provides tolerance to drought stress for wild type plants. (A) Effect of quinabactin on Arabidopsis drought tolerance. Two-week-old plants were subjected to drought stress by water retention and were photographed after 12 days. During the dry period, the plants were treated in 3 days with 25 μM of the compound. The plants were rehydrated after a two-week drought treatment; the number of surviving plants (out of the total number tested) for each treatment is shown next to each image. (B) Effects of quinabactin on soy. Two-week-old plants were subjected to drought stress by water retention and were photographed after 8 days of drought treatment. For all drought stress treatments, the compounds (tested at 25 μM for Arabidopsis and 50 μM for soybeans) were applied in solutions containing 0.05% Tween-20 and applied as aerosols every 3 days during the drought regime . Values for all experiments are means ± SEM (n = 6, 3 plants used per experiment).
[0019] Figure 9. LC66C6 induces numerous ABA-responsive genes. (A) Shows chemical-induced levels of mRNA expression of the reporter genes responsive to ABA RD29B and MAPKKK18 in wild type, abil-1, the mutant pyr1 / pyl1 / pyl2 / pyl4 quadruple receptor mutant genotypes of vehicle-treated Arabidopsis seedlings (DMSO), pirabactin, LC66C6, or (+) - ABA. (B) LC66C6 efficiently induces ABA-responsive genes in Arabidopsis seedlings, while pirabactin does not. Ten-day-old seedlings were treated with carrier solvent (DMSO) or 25 μM ABA, pyrabactin or LC66C6 for 8 hours. The total RNA was then prepared labeled and hybridized to ATH1 microarrays. The graphically represented data are average expression values transformed by log2 for ~ 13K probes that were detectable through all experiments. The data shown are averages determined from triplicate biological replications. (C) and (D) show the expression of a reporter gene in different plant tissues after treatment with vehicle (DMSO), pirabactin, LC66C6, or (+) - ABA.
[0020] Figure 10. Expression of the ABA-responsive gene in simple PYR / PYL mutants. The responses of MAPAKK18, RD29A, and RD29B mRNAs responsive to ABA for LC66C6, ABA and pyrabactin were characterized in the Col and Ler ecotypes and in the simple mutant genotypes pyr1pyl1, ply2, pyl3 and pyl4.
[0021] Figure 11. LC66C6 induces the expression of the ABA-responsive gene in wild-type, abil-1 plants and quadruple PYR / PYL mutants. LC66C6 and (+) - ABA induced the expression of ABF3, GBF3, NCED3, and RD29A in a dose-dependent manner in wild type Col plants, whereas pirabactin did not.
[0022] Figure 12. Sensitivity to LC66C6 is not influenced by ABA hydroxylation enzymes, CYP707A. (A) shows photographs and (B) shows the quantification of the primary root length in wild type plants, plants that overexpress CYP707A (CYP707AOX), and plants that are double mutants for cyp707a treated with DMSO, 40 μM of (+) - ABA, and 40 μM LC66C6. (C) shows the fresh weight and (D) shows the percentage of plants with green cotyledons in the plants treated as in (A).
[0023] Figure 13. LC66C6 modulates responses to ABA in several species. Germination inhibition (A) and loss of transpirational water in separate leaves 2 hours after detachment (B) in response to the compounds shown. The expression of marker genes responsive to ABA in Soy (C), Barley (D) and Maize (E) after the application of chemicals. D, P, L and A indicate DMSO, pyrabactin, LC66C6 and (+) - ABA, respectively.
[0024] Figure 14. Chemical structure of ABA and agonists.
[0025] Figure 15. The effect of ABA and agonists in yeast and seed germination tests. (A) shows the test results of two yeast hybrids using PYR / PYL, PYR1, PYL1, PYL2, PYL3 and PYL4 receptors to test the response to each of the agonists shown in Figure 14. (B) shows the results of the tests of the agonists in Figure 14 in the germination of wild type seeds. (C) shows the effects of compounds in an ABA reporter lineage as measured using glucuronidase assays in a transgenic lineage expressing glucuronidase under the control of the ABA MAPKKK18-inducible Arabidopsis gene.
[0026] Figure 16. The application of LC66C6 can rescue growth defects observed in the ABA deficient mutant aba2. The chemical solution (25 μM) was sprayed on the plants for 14 days twice a day for 2 weeks. The image (A) and fresh weight (B) were obtained from plants of 4 weeks.
[0027] Figure 17. The effect of ABA and its agonists on Physcomitrella patens and Chlamydomonas. Images of protonema growth (A) and quantitative analysis (B) of the effects of ABA and agonists on Phsycomitrella patens. The protonema was cultured in 200 μM of specific test chemical for 10 days. The effects of LC66C6 were weak, but significantly inhibited, growth of the protonema. Protonema bleached by Pirabactin. (C) The expression of ABA-responsive genes from Physcomitrella patens. The protonema was treated with 200 μM of chemical solutions for 3 h. (D) Growth of Chlamydomonas colony in the chemical with salinity stress and osmotic stress. There was no effect of ABA and LC66C6 on the growth of Chlamydomonas, with and without stresses. Physcomitrella patens and Chlamydomonas bleached by pirabactin, suggesting that this compound may have toxicity in these species unrelated to its ABA agonist activity.
[0028] Figure 18 shows a summary of the agonist compounds tested for their effect on the inhibition of germination and reporter expression of pMAPKK18: Gus. ++++++ indicates strong activity, whereas a single + indicates weak activity, a dash (-) indicates no activity and n.d. indicates undetermined. DEFINITIONS
[0029] "Agonists" are agents that, for example, induce or activate the expression of a described target protein or bind to, stimulate, increase, open, activate, facilitate, increase activation, sensitize or positively regulate the activity of a or more plant PYR / PYL proteins (or polynucleotide code). Agonists can include synthetic and naturally occurring molecules. In some modalities, agonists are combined with agrochemicals for agricultural production and formulation. Examples of suitable agrochemicals include fungicides, herbicides, pesticides, fertilizers and / or surfactants. Assays to determine whether an agonist "agonizes" or "does not agonize" a PYR / PYL protein include, for example, bringing putative agonists into contact with purified PYR / PYL protein (s) and then determining the functional effects of PYR / protein activity PYL, as described in this document, or put putative agonists in contact with cells expressing PYR / PYL protein (s) and then determine the functional effects of the described target protein activity, as described in this document. A person skilled in the art will be able to determine whether an assay is suitable for determining whether an agonist agonizes or not agonizes a PYR / PYL protein. Samples or assays comprising PYR / PYL proteins that are treated with a putative agonist are compared to samples without the agonist to examine the extent of the effect. Control samples (not treated with agonists) are assigned a relative activity value of 100%. The agonism of the PYR / PYL protein is achieved when the activity value in relation to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500% or 1000 to 3000% or higher.
[0030] The term "PYR / PYL receptor polypeptide" refers to a protein characterized in part by the presence of one or more or all of a polyketide cyclase 2 domain (PF10604), a polyketide cyclase 1 domain (PF03364) , and a Bet VI domain (PF03364), which in the wild type mediates abscisic acid (ABA) and ABA analog signaling. A wide variety of PYR / PYL receptor polypeptide sequences are known in the art. In some embodiments, a PYR / PYL receptor polypeptide comprises a polypeptide that is substantially identical to any of SEQ ID NOs: 1-119. See, for example, Published PCT Application WO2011 / 139798.
[0031] The term "activity assay" refers to any assay that measures or detects the activity of a PYR / PYL receptor polypeptide. An exemplary assay for measuring PYR / PYL receptor activity is an assay of two yeast hybrids that detects the binding of a PYR / PYL polypeptide to a type 2 protein phosphatase (PP2C) polypeptide, as described in the Examples.
[0032] Two nucleic acid or polypeptide sequences are considered "identical", if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percentage of "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a certain percentage of amino acid or nucleotide residues which are the same when compared and aligned for maximum matching in a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When the percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are replaced by other amino acid residues with properties similar chemicals (for example, charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. Where sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. This typically involves scoring a conservative substitution as a partial rather than a total mismatch, thereby increasing the percentage of sequence identity. Thus, for example, where a score of 1 is assigned to an identical amino acid and a score of zero is assigned to a non-conservative substitution, a score between zero and 1 is assigned to a conservative substitution. The score for conservative substitutions is calculated according to, for example, the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, California, USA).
[0033] The phrase "substantially identical", used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 60% sequence identity with a reference sequence. Alternatively, the identity percentage can be any integer from 60% to 100%. Some modalities include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described in this document; preferably BLAST using standard parameters, as described below.
[0034] Modalities of the present invention provide polypeptides, and nucleic acids encoding polypeptides, which are substantially identical to any of SEQ ID NO: 1-119.
[0035] For sequence comparison, normally a sequence acts as a reference sequence, with which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and the parameters of the sequence algorithm program are designated. Standard program parameters can be used, or alternative parameters can be assigned. The sequence comparison algorithm then calculates the percentage of sequence identities for the test sequence in relation to the reference sequence, based on the program parameters.
[0036] A "comparison window", as used in this document, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of 20 to 600, generally about 50 to about 200 , more generally about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are known in the art. The ideal sequence alignment for comparison can be accomplished, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), for the search for a similarity method by Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and inspection visual.
[0037] Algorithms that are suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 33893402, respectively. The software for performing BLAST analyzes is available to the public through the National Center for Biotechnology Information (NCBI) website. The algorithm first involves identifying high-score sequence pairs (HSPs) by identifying short words of length W in the query string, which match or satisfy any threshold score with a positive value T when aligned with a word of the same length in a string database. T is referred to as the next word punctuation limit (Altschul et al, supra). These early next command hits act as seeds to initiate searches to find longer HSPs that contain them. Command hits are then extended in both directions along each sequence as the cumulative alignment score can be increased. Cumulative scores are calculated using nucleotide sequences, the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for misalignment residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the command hits in each direction is interrupted when: the cumulative alignment score falls by the amount X of its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word size (W) of 28, an expectation (E) of 10, M = 1, N = -2 and a comparison of both strains. For amino acid sequences, the BLASTP program defaults to a word size (W) of 3, an expectation (E) of 10 and the BLOSUM62 score matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
[0038] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). A measure of similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences could occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the least probability of sum in a comparison between the test nucleic acid and the reference nucleic acid is less than 0.01, more preferably 10-5, and most preferably less than 10-20.
[0039] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acids do not encode an amino acid sequence, for essentially identical sequences. Because of the degeneration of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such variations of nucleic acids are "silent variations", which are a kind of conservatively modified variations. Each nucleic acid sequence in this document that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine) can be modified to produce a functionally identical molecule. In that sense, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.
[0040] As for amino acid sequences, one of those skilled in the art will recognize that individual substitutions in a nucleic acid, peptide, polypeptide or protein sequence that alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively variant" modified "where the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art.
[0041] The following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, for example, Creighton, Proteins (1984)).
[0042] The term "plant" includes whole plants, vegetative organs and / or sapling structures (for example, leaves, stems and tubers), roots, flowers and floral organs (for example, bracts, sepals, petals, stamens, carpels , anthers), ova (including central cells and ova), seed (including zygote, embryo, endosperm and seed coating), fruit (for example, the mature ovary), seedlings, plant tissue (for example, vascular tissue, tissue of soil, and the like), cells (for example, protective cells, eggs, trichomes and the like), and their progeny. The class of plants that can be used in the methods of the invention includes angiosperms (monocot and dicot plants), gymnosperms, ferns, bryophytes, and multicellular and unicellular algae. This includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and heterozygous.
[0043] As used in this document, the term "transgenic" describes a non-naturally occurring plant that contains a genome modified by man, in which the plant includes in its genome, an exogenous nucleic acid molecule, which can be derived from a species of the same or different plant. The exogenous nucleic acid molecule can be a regulatory element of the gene such as a promoter, enhancer, or other regulatory element, or it can contain a coding sequence, which can be linked to a regulatory element of the heterologous gene. Transgenic plants that arise from sexual interbreeding or through self-pollination are descendants of such a plant and are also considered "transgenic".
[0044] As used in this document, the term "drought resistance" or "drought tolerance," including any of its variations, refers to a plant's ability to recover from periods of drought stress (ie , little or no water for a period of days). Typically, drought stress will be at least 5 days and can be as long as, for example, 18 to 20 days or more (for example, at least 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 days), depending, for example, on plant species.
[0045] As used in this document, the terms "abiotic stress", "stress", or "stress condition" refer to the exposure of a plant, plant cell, or the like, to a non-living chemical or physical agent (" abiotic ") that has an adverse effect on the metabolism, growth, development, propagation, or survival of the plant (collectively," growth "). Stress can be imposed on a plant due, for example, to an environmental factor, such as water (for example, flood, drought or dehydration), anaerobic conditions (for example, a lower level of oxygen or a high level of CO2), abnormal osmotic conditions, salinity, or temperature (for example, hot / heat, cold, freezing, or frost), a deficiency of nutrients or exposure to pollutants, or by a hormone, second messenger, or other molecule. Anaerobic stress, for example, is due to a reduction in oxygen levels (hypoxia or anoxia) sufficient to produce a stress response. Flooding stress can be due to prolonged or transient immersion of a plant, part of a plant, tissue, or isolated cell in a liquid medium such as during monsoons, rainy seasons, floods, or excessive irrigation of plants, or the like. A cold stress or heat stress can occur due to a decrease or increase, respectively, in the ideal temperature range of growth temperatures for a specific plant species. Such ideal growth temperature ranges are readily determined or known to those skilled in the art. Dehydration stress can be induced by water loss, reduced turgor, or reduced water content of a cell, tissue, organ or the entire plant. Drought stress can be induced by or associated with water deprivation or reduced water supply to a cell, tissue, organ or organism. Salinity-induced stress (salt stress) can be associated with or induced by a disturbance in the osmotic potential of a cell's intracellular or extracellular environment. As used in this document, the term "abiotic stress tolerance" or "stress tolerance" refers to the increased resistance or tolerance of the plant to abiotic stress compared to plants under normal conditions and the ability to perform relatively superiorly under abiotic stress conditions.
[0046] A polypeptide sequence is "heterologous" to an organism or a second polypeptide sequence if it originates from a foreign species, or, if from the same species, it is modified from its original form. DETAILED DESCRIPTION OF THE INVENTION I. Introduction
[0047] The present invention is based, in part, on the discovery of selective abscisic acid agonists (ABA). Unlike previous ABA agonists, the agonists described in this document potentially activate the ABA pathway in plant vegetative tissues and induce tolerance to abiotic stress. The new agonists can be used to induce stress tolerance in plant crop species. Agonists can be used to induce stress tolerance in monocotyledonous and dicotyledonous plant species, including but not limited to, broccoli, radish, alfalfa, soy, barley, and corn (common corn).
[0048] Abscisic acid is a multifunctional phytohormone involved in a variety of phytoprotective functions, including bud dormancy, seed dormancy and / or maturation, leaf and fruit abscission, and response to a wide variety of biological stresses (for example, cold, heat, salinity and drought). ABA is also responsible for regulating stomatal closure by a mechanism independent of CO2 concentration. The PYR / PYL family of ABA receptor proteins mediates ABA signaling. The plants examined at the time expressed more than one member of the PYR / PYL receptor protein family, which have at least a little redundant activity. PYR / PYL receptor proteins mediate ABA signaling as a positive regulator in, for example, germination seeds, post-germination growth, stomatal movement and plant tolerance to stress including, but not limited to, drought.
[0049] A wide variety of wild-type (naturally occurring) PYR / PYL polypeptide sequences are known in the art. Although PYR1 was originally identified as an abscisic acid receptor (ABA) in Arabidopsis, in fact, PYR1 is a member of a group of at least 14 proteins (PYR / PYL proteins) from the same protein family in Arabidopsis that also mediates the ABA signaling. This protein family is also present in other plants (see, for example, SEQUENCE LISTING) and is characterized in part by the presence of one or more or all of a polyketide cyclase 2 domain (PF10604), a polyketide cyclase 1 domain (PF03364) and a Bet VI domain (PF03364). The START / Bet VI superfamily domains are described in, for example, Radauer, BMC Evol. Biol. 8: 286 (2008). In some embodiments, a wild-type PYR / PYL receptor polypeptide comprises any of SEQ ID NOs: 1-119. In some embodiments, a wild-type PYR / PYL receptor polypeptide is substantially identical (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95 %, 96%, 97%, 98%, or 99% identical to) any of SEQ ID NOs: 1-119. In some embodiments, a PYR / PYL receptor polypeptide is substantially identical (for example, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96 %, 97%, 98%, or 99% identical a) any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 6162, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 , 117, 118, or 119. II. ABA agonists
[0050] The present invention provides small molecule ABA agonists, that is, compounds that activate PYR / PYL proteins. Exemplary ABA agonists include, for example, a compound selected from the following:
[0051] A compound of Formula I:
wherein R1 is selected from the group consisting of H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, R2 is selected from the group consisting of cycloalkyl, heterocycloalkyl, aryl and heteroaryl , each optionally substituted by 1 to 4 groups R2a, each R2a is independently selected from the group consisting of H, halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl , C2-6 alkynyl, -OH, C1-6 alkylhydroxy, -CN, -NO2, -C (O) R2b, -C (O) OR2b, -OC (O) R2b, -C (O) NR2bR2c, -NR2bC (O) R2c, -SO2R2b, -SO2OR2b, - SO2NR2bR2c, and -NR2bSO2R2c, each of R2b and R2c is independently selected from the group consisting of H and C1-6 alkyl, each of R3, R4 and R5 is independently selected from from the group consisting of H and C1-6alkyl, L is a linker selected from the group consisting of a bond and C1-6alkylene, the subscript m is an integer from 0 to 4, the subscript n is an int number 0 to 3, or a salt or isomer thereof.
[0052] In some embodiments, the compound has the formula (II):

[0053] In some embodiments, the compound has the formula (III):

[0054] In some embodiments, R1is C1-6alkyl, and R2is selected from the group consisting of aryl and heteroaryl, each optionally substituted by 1-4 R2a groups.
[0055] In some embodiments, each R2a is independently selected from the group consisting of H, halogen and C1-6alkyl.
[0056] In some embodiments, R2 is selected from the group consisting of phenyl, naphthyl, thiophene, furan, pyrrole, and pyridyl.
[0057] In some embodiments, R1is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl; R2 is selected from the group consisting of phenyl and thiophene, each optionally substituted by 1 group R2a; each R2a is independently selected from the group consisting of H, F, CI, methyl and ethyl; and L is selected from the group consisting of a bond and methylene.
[0058] In some embodiments, the compound has the formula (IV):

[0059] In some embodiments, the compound has the formula (V):

[0060] In some embodiments, the compound is one of the compounds shown in Figure 8.
[0061] In some embodiments, the compound has the formula (VI):

[0062] The compound having the formula (VI) is also referred to as LC66C6 or quinabactin (1- (4-methylphenyl) -N- (2-oxo-1,2,3,4-tetrahydroquinolin-6-yl) methanesulfonamide ). The compounds described above were identified by selecting a library of structurally diverse compounds acquired from Life Chemicals (Orange, CT).
[0063] In some embodiments, the compound has the formula (VII):

[0064] The compounds described above can be synthesized using methods known in the art. For example, compounds based on the same chemical support were synthesized as described in US Patent No. 5,498,755 and US Patent No. 6,127,382, the contents of which are incorporated herein by reference in their entirety. III. ABA agonist formulations
[0065] The present invention provides agricultural chemical formulations formulated by contacting plants, wherein the formulation comprises an ABA agonist of the present invention. In some embodiments, plants that are in contact with agonists comprise or express an endogenous PYR / PYL polypeptide. In some embodiments, plants that are in contact with agonists do not understand or express a heterologous PYR / PYL polypeptide (for example, plants are not transgenic or are transgenic, but express heterologous proteins other than heterologous PYR / PYL proteins). In some embodiments, plants that are in contact with agonists comprise or express a heterologous PYR / PYL polypeptide as described in this document.
[0066] The formulations may be suitable for the treatment of plants or plant propagating material, such as seeds, in accordance with the present invention, for example, in a carrier. Suitable additives include buffering agents, wetting agents, coating agents, polysaccharides, and abrasion agents. Exemplary carriers include water, aqueous solutions, slurries, solids and dry powders (eg peat, wheat, bran, vermiculite, clay, pasteurized soil, many forms of calcium carbonate, dolomite, various types of plaster, bentonite and others clay minerals, rock phosphates and other compounds of phosphorus, titanium dioxide, humus, talc, alginate and activated carbon. Any agronomically suitable carrier known to a person skilled in the art would be acceptable and is intended for use in the present invention). Optionally, the formulations can also include at least one surfactant, herbicide, fungicide, pesticide or fertilizer.
[0067] In some embodiments, the agricultural chemical formulation comprises at least one of a surfactant, a herbicide, a pesticide, such as, but not limited to, a fungicide, a bactericide, an insecticide, an acaricide, and a nematicide, a plant activator, synergist, herbicide protector, plant growth regulator, insect repellent, or fertilizer.
[0068] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more herbicides selected from the group consisting of: paraquat (592), mesotrione (500), sulcotrione (710), clomazone (159), fentrazamide ( 340), mefenacet (491), oxaziclomefone (583), indanophane (450), glyphosate (407), prosulfocarb (656), molinate (542), triasulfuron (773), halosulfuron-methyl (414) and pretylchlor (632). The active ingredients of the above herbicides are described, for example, in "The Pesticide Manual", Editor C. D. S. Tomlin, 12th Edition, British Crop Protection Council, 2000, under the entry numbers added in parentheses; for example, mesotrione (500) is described in that document under the entry number 500. The above compounds are described, for example, in US 7,338,920, which is incorporated by reference in this document in its entirety.
[0069] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from the group consisting of: silkxane, fludioxonil, penthiopyrad, protioconazole, flutriafol, diphenoconazole, azoxystrobin, captan, cyproconazole, cyprodinil, boscalide, diniconazole, epoxiconazole, fluoxastrobin, trifloxystrobin, metalaxyl, metalaxyl-M (mefenoxam), fluquinconazole, fenarimol, nuarimol, pyrifenox, pyraclostrobin, tiabendazole, tebuconazol, triadimenol, benazxil, benalaxil, benalaxil, benalaxil, benalaxil, benalaxil, benalaxil , miclobutanil, tetraconazole, imazalil, metconazole, bitertanol, cymoxanil, ipconazole, iprodione, prochloraz, pencicuron, propamocarb, siltiofam, take out, triazoxide, triticonazole, tolylfluanide and a manganese compound (such as mannequin). In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more of an insecticide, an acaricide and / or nematicide selected from the group consisting of: thiamethoxam, imidacloprid, clothianidin, lambda-cyhalothrin, teflutrin, beta-cyfluthrin, permethrin, abamectin, fipronil and spinosad. Details (eg structure, chemical name, trade names, etc.) of each of the above pesticides with a common name can be found in e-Pesticide Manual, version 3.1, 13th Edition, Ed. CDC Tomlin, British Crop Protection Council , 2004-05. The above compounds are described, for example, in US 8,124,565, which is incorporated by reference in this document in its entirety.
[0070] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from the group consisting of: Ciprodinil ((4-cyclopropyl-6-methyl-pyrimidin-2-yl) - phenyl-amine) (208), Dodina (289); Chlorothalonil (142); Folet (400); Protioconazole (685); Boscalide (88); Proquinazide (682); Ditianon (279); Fluazinam (363); Ipconazole (468); and Metrafenone. Some of the compounds above are described, for example, in "The Pesticide Manual" [The Pesticide Manual-A World Compendium; Thirteenth Edition; Editor: C. D. S. Tomlin; The British Crop Protection Council, 2003], under the entry numbers added in parentheses. The above compounds are described, for example, in US 8,349,345, which is incorporated by reference in this document in its entirety.
[0071] In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more fungicides selected from the group consisting of: fludioxonil, metalaxyl and a strobilurin fungicide, or a mixture thereof. In some embodiments, the fungicide strobilurin is azoxystrobin, picoxystrobin, kresoxim-methyl or trifloxystorbine. In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more of an insecticide selected from a phenylpyrazole and a neonicotinoid. In some embodiments, phenylpyrazole is fipronil and neonicotinoid is selected from thiamethoxam, imidacloprid, thiacloprid, clothianidin, nitenpiram and acetamipride. The above compounds are described, for example, in US 7,071,188, which is incorporated by reference in this document in its entirety. In some embodiments, the agricultural chemical formulation comprises an effective amount of one or more biological pesticides, including but not limited to, Pasteuria spp., Paeciliomyces, Lyophilized Pochonia, Myrothecium metabolites, Muscodor volatiles, Tagetes spp., Bacillus flrmus, including bacillus firmus CNCM 1-1582. IV. Application to Plants
[0072] ABA agonist formulations and compositions can be applied to plants using a variety of known methods, for example, by spraying, atomizing, dipping, pouring, irrigating, dusting or dispersing the compositions on the propagating material, or brushing or spillage or otherwise by contact of the compositions with the plant or, in the case of seeds, coating, encapsulation, spraying, immersion, immersion of the seeds in a liquid composition, or otherwise by treatment of the seed. Alternatively, to directly treat a plant or seed before planting, the formulations of the invention can also be introduced into the soil or other means in which the seed is to be planted. For example, formulations can be introduced into the soil by spraying, dispersing, pouring, irrigating or otherwise treating the soil. In some embodiments, a carrier is also used in this embodiment. The carrier can be solid or liquid, as noted above. In some embodiments, the peat is suspended in water as a carrier for the ABA agonist, and this mixture is sprayed on the soil or planting media and / or on the seed that is planted.
[0073] The types of plant that can be treated with the ABA agonists described in this document include the two monocotyledonous and dicotyledonous plant species, including cereals, such as barley, rye, sorghum, triticale, oats, rice, wheat, soybeans and corn; beet (for example, sugar beet and fodder beet); pumpkins, including cucumber, lacy melon, cantaloupe, pumpkin and watermelon; Brassica crops including broccoli, cabbage, cauliflower, bok choi (Chinese cabbage), and other green leaves, other vegetables such as tomatoes, pepper, lettuce, beans, peas, onions, garlic and peanuts; oilseeds, including canola, peanuts, sunflower, rapeseed and soybeans; solanaceous, including tobacco; tuber and root crops, including potatoes, yams, radishes, beets, carrots and sweet potatoes; fruits including strawberries; fiber crops, including cotton and hemp; other plants, including coffee, flowering plants, perennials, woody ornamentals, lawn and cut flowers, including carnations and roses; sugar cane; tree harvests in containers; green trees, including fir and pine; woody trees including maple and oak; and fruit and nut trees including cherry, apple, pear, almond, peach, walnut and citrus.
[0074] It will be understood that the ABA agonists described in this document mimic the function of ABA in cells. Thus, it is expected that one or more cellular responses, triggered by the cell's contact with the ABA, will also be triggered by the cell's contact with the ABA agonists described in this document. The ABA agonists described in this document mimic the function of ABA and are provided in a useful formulation.
[0075] In some embodiments, the application of the ABA agonists described in this document increases the resistance to abiotic stress in a plant.
[0076] In some embodiments, the application of the ABA agonists described in this document for seeds inhibits seed germination.
[0077] The present invention also provides plants in contact with the ABA formulations described in this document. The plant in contact with the ABA formulation can include a part of the plant and / or a seed. V. Screening for new ABA agonists and antagonists
[0078] The embodiments of the present invention also provide methods of screening putative chemical agonists to determine whether the putative agonist agonizes a PYR / PYL receptor polypeptide when the putative agonist comes in contact with the PYR / PYL receptor polypeptide. As used herein, an agent "agonizes" a PYR / PYL receptor protein if the presence of the agent results in the activation or positive regulation of receptor activity, for example, to increase signaling downstream of the PYR / PYL receptor. For the present invention, an agent agonizes a PYR / PYL receptor if, when the agent is present in a concentration not exceeding 200 μM, contacting the agent with the PYR / PYL receptor results in the activation or positive regulation of the activity of the PYR / PYL receptor. If an agent does not induce the activation or positive regulation of PYR / PYL receptor protein activity when the agent is present in a concentration not exceeding 200 μM, then the agent will not significantly agonize the PYR / PYL receptor. As used in this document, "activation" requires a minimum activity limit to be induced by the agent. The determination of whether this minimum activity limit has been respected can be carried out, for example, by means of an enzyme phosphatase assay that defines a minimum value for the level of enzyme activity that must be induced, or by means of a phosphatase assay. enzymatic in the presence of a colorimetric detection reagent (for example, para-nitrophenylphosphate) in which the minimum activity limit has been respected a color change is observed.
[0079] The present invention also provides screening methods for ABA agonists and antagonists by screening for the ability of a molecule to induce the binding of PYR / PYL-PP2C in the case of agonists, or to disrupt the ability of ABA and other agonists to promote the binding of PYR / PYL-PP2C, in the case of antagonists. A number of different screening protocols can be used to identify the agents that agonize or antagonize a PYR / PYL polypeptide.
[0080] Screening can take place using isolated, purified or partially purified reagents. In some embodiments, the purified or partially purified PYR / PYL polypeptide can be used.
[0081] Alternatively, cell-based screening methods can be used. For example, cells that naturally express a PYR / PYL polypeptide or that recombinantly express a PYR / PYL polypeptide can be used. In some embodiments, the cells used are plant cells, animal cells, bacterial cells, fungal cells, including but not limited to yeast cells, insect cells, or mammalian cells. In general terms, screening methods involve screening a plurality of agents to identify an agent that modulates the activity of a PYR / PYL polypeptide, for example, by binding to a PYR / PYL polypeptide, or by activating a polypeptide of PYR / PYL or by increasing the expression of a PYR / PYL polypeptide, or by encoding by transcription a PYR / PYL polypeptide. 1. PYR / PYL polypeptide binding assays
[0082] Optionally, preliminary screens can be performed by screening agents capable of binding a PYR / PYL polypeptide, since at least some of the identified agents are likely PYR / PYL polypeptide modulators.
[0083] Binding assays may involve contacting a PYR / PYL polypeptide with one or more test agents and leaving sufficient time for the protein and test agents to form a binding complex. Any bonding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure coprecipitation or comigration in non-denaturing SDS-polyacrylamide gels, and comigration in Western blots (see, for example, Bennet, JP and Yamamura, HI ( 1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in Neurotransmitter Receptor Binding (Yamamura, HI, et al, eds.), Pp. 61-89). Other binding assays involve the use of NMR or mass spectrometry techniques to identify molecules bound to the PYR / PYL polypeptide or displacement of labeled substrates (for example, labeled ABA). The PYR / PYL polypeptide protein used in these assays can be naturally expressed, cloned or synthesized. 2. Activity
[0084] PYR / PYL polypeptide agonists can be identified by screening for agents that activate or increase the activity of a PYR / PYL polypeptide. Antagonists can be identified by reducing activity.
[0085] An activity assay involves testing whether a candidate agonist can induce the binding of a PYR / PYL protein to a polypeptide type 2 protein phosphatase (PP2C) in a specific agonist mode. Approaches from two yeast or mammalian hybrids (see, for example, Bartel, PL et. Al. Methods Enzymol, 254: 241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a cell. In some embodiments, agents that agonize a PYR / PYL polypeptide are identified in a two-hybrid assay between a PYR / PYL polypeptide and a type 2 protein phosphatase (PP2C) polypeptide (eg ABI1 or 2 or orthologists, for example). example, from the PP2C group A subfamily), in which an ABA agonist is identified as an agent that activates or allows the binding of the PYR / PYL polypeptide and the PP2C polypeptide. Thus, the two polypeptides bind in the presence, but not in the absence of the agent. In some embodiments, a chemical compound or agent is identified as an agonist of a PYR / PYL protein if the yeast cell turns blue when testing two yeast hybrids.
[0086] The biochemical function of PYR1, and PYR / PYL proteins, in general, is to inhibit PP2C activity. This can be measured in living cells with the two yeast hybrids or other cell-based methods. This can also be measured in vitro using enzymatic phosphatase assays in the presence of a colorimetric detection reagent (for example, para-nitrophenylphosphate). The yeast-based assay used above provides an indirect indicator of ligand binding. To address this potential limitation, in vitro competition assays, or cell-based assays using other organisms, can be used as alternative approaches to the identification of weakly bound target compounds. 3. Expression Essays
[0087] Screening for a compound that increases the expression of a PYR / PYL polypeptide is also provided. Screening methods generally involve carrying out plant-based or cell-based assays in which test compounds are contacted with one or more cells that express the PYR / PYL polypeptide and then detecting an increase in the expression of PYR / PYL (transcription or translation product). The assays can be performed on cells that naturally express PYR / PYL or on cells recombinantly altered to express PYR / PYL, or on cells recombinantly altered to express a reporter gene under the control of the PYR / PYL promoter.
[0088] Various controls can be carried out to ensure that an observed activity is authentic, including running parallel reactions with cells without the reporter construct or not contacting a cell that houses the reporter construct with the test compound. 4. Validation
[0089] Agents that are initially identified by any of the preceding screening methods can be further tested to validate the apparent activity and / or determine other biological effects of the agent. In some cases, the identified agent is tested for its ability to affect plant stress (eg drought tolerance), seed germination, or other phenotype affected by ABA. A number of such assays and phenotypes are known in the art and can be employed according to the methods of the invention. 5. High-performance soluble and solid-phase tests
[0090] In the high performance tests of the invention, it is possible to track up to several thousand different modulators or ligands in a single day. In particular, each well on a microtiter plate can be used to perform a separate assay against a selected potential modulator, or, if the effects of concentration or incubation time have to be observed, each of the 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can analyze about 100 (for example, 96) modulators. If 1536 well plates are used, then a single plate can easily analyze about 100 to about 1500 different compounds. It is possible to analyze several different plates per day; test screenings for up to about 6,000 to 20,000 or more different compounds are possible using the integrated systems of the invention. In addition, microfluidic approaches to handling the reagent can be used.
[0091] The molecule of interest (for example, PYR / PYL or a cell that expresses a PYR / PYL polypeptide) can be linked to the solid-state component, directly or indirectly, through covalent or non-covalent bonding.
[0092] The invention provides in vitro assays to identify, in a high-throughput format, compounds that can modulate PYR / PYL expression or activity.
[0093] Resistance to abiotic stress can be analyzed according to any of a number of well-known techniques. For example, for drought tolerance, plants can be grown under conditions where less than ideal water is supplied to the plant. Drought resistance can be determined by any of a number of standard measures, including turgor pressure, growth, yield and the like. SAW. Methods of increasing tolerance to abiotic stress in plants
[0094] The present invention also provides methods of tolerating abiotic stress growing in a plant. Thus, in some embodiments, a plant is brought into contact with an ABA agonist described in this document, or an ABA agonist formulation, in an amount sufficient to increase the tolerance to abiotic stress in the plant. The amount of the ABA agonist formulation applied to the plant may be sufficient to increase the tolerance to abiotic stress compared to the non-contact of the plant with the ABA agonist formulation. The plant can come into contact with the ABA formulation using any of the methods described in this document. Increasing tolerance to abiotic stress can improve plant growth and / or survival under conditions of abiotic stress that negatively affect plant growth or survival. Abiotic stress includes physical or chemical conditions described in this document. VII. Methods of inhibiting seed germination in a plant
[0095] The present invention also provides methods of inhibiting seed germination. Thus, in some embodiments, a plant, part of the plant, or a seed is brought into contact with an ABA agonist formulation in an amount sufficient to inhibit seed germination. The seed can come into contact with the ABA formulation using any of the methods described in this document. In some embodiments, the seed is in direct contact with the ABA agonist formulation. In some modalities, the soil or soil is brought into contact with the ABA agonist formulation before or after planting or sowing the seeds. In some modalities, a plant is in contact with the ABA agonist formulation sufficient to inhibit the germination of the seeds that later develop from the plant. VIII. Methods of Activating PYR / PYL Receptor Polypeptides
[0096] The present invention also provides methods of activating a PYR / PYL receptor polypeptide. In some embodiments, a PYR / PYL polypeptide is contacted with a compound described above, and the activated PYR / PYL polypeptide binds to a PP2C polypeptide. In some embodiments, the PYR / PYL polypeptide is capable of being activated by the agonist compound LC66C6. In some embodiments, the PYR / PYL protein that is activated is substantially identical to any of SEQ ID NOs: 1119. Examples of ABA receptor sequences from various plants are provided in US Patent Publication 2011/0271408, which is incorporated by reference in this document in its entirety.
[0097] In some embodiments, the method activates a PYR / PYL receptor in a cell-free in vitro assay. In some embodiments, the method activates a PYR / PYL receptor expressed in a cell. In some embodiments, the cell also expresses a PP2C polypeptide. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an animal or mammalian cell. In some embodiments, the cell expresses an endogenous PYR / PYL protein. In some embodiments, the cell is designed to express a heterologous PYR / PYL polypeptide. In some embodiments, the cell expresses a heterologous PP2C polypeptide. In some embodiments, the cell expresses a PP2C polypeptide selected from HAB 1 (homology to AMI), AMI, or ABI2.
[0098] In some embodiments, the activated PYR / PYL polypeptide induces the expression of heterologous genes. In some embodiments, heterologous genes are responsive ABA genes. In some embodiments, the gene expression induced occurs in cells that express an endogenous PYR / PYL polypeptide. In some embodiments, the induced gene expression occurs in cells that express a heterologous PYR / PYL polypeptide. EXAMPLES Example 1
[0099] This example demonstrates that new ABA agonists described in this document bind to and activate multiple PYR / PYL receptors. Methods Chemical screening
[0100] A two-yeast hybrid system described earlier was used in high-throughput screening (HTS) to identify ABA agonists (see Peterson FC, et al. (2010) Structural basis for selective activation of ABA receptors. Nature Structural & Molecular Biology 17 (9): 1 109-1 11 1). In this system, the receptor interaction promoted by the agonist - PP2C activates the expression of a URA3 or HIS3 reporter gene and rescues the auxotrophy of uracil or histidine from parental strains (Peterson FC, et al. (2010); Vidal M, Brachmann RK , Fattaey A, Harlow E, & Boeke JD (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proceedings of the National Academy of Sciences of the United States of America 93 ( 19): 10315-10320). HTS were conducted using 5 different reporter strains that express the binding domain (BD) fusions for PYR1, PYL1, PYL2, PYL3 or PYL4; these were coexpressed with the fusions of the activation domain (AD) to HAB1 (pACT-HAB 1); the constructs used have been described previously (Park et al. 2009). We use these strains in two separate screens. In the first screening ~ 65,000 compounds obtained from Chembridge (San Diego, USA) were analyzed for agonist activity using a halo assay, essentially as described by Gassner NC, et al. (2007) (Accelerating the discovery of biologically active small molecules using a high-throughput yeast halo assay. Journal of Natural Products 70 (3): 383-390). In this method, the yeast strains are incorporated in selective agar and compounds transferred by needle of 10 mM stock solutions of DMSO in assay plates; the hits are evident by the higher cell density in the vicinity of the active compounds. The experiments using the halo assay used the yeast strain PJ69-4A and media supplemented with 10 mM 3-aminotriazole to improve selections. Halo screens were defined using a Biomek FX equipped with an automated microplate hotel (Thermo Cytomat) and a 384-pin tool (V & P Scientific), which was used to mark the compounds on the assay plates. Before each chemical transfer, the needles were washed in a 1: 1 mixture of DMSO / water followed by a wash with 95% ethanol. After chemical transfer, the plates were incubated at 28 ° C and the candidate agonists were evidenced by manual inspection.
[0101] Although the halo screening method is potent from a productivity perspective, we subsequently employ a more conventional screening method for a second screening of a 12,000-member library obtained by Life Chemicals (Ukraine). This change was motivated by the desire to better control the concentration of the trial. In our second screening, the reporter constructs were expressed in the yeast strain MAV99, which allows selections based on uracil via a URA3 transgene activated by the GAL1 promoter (Peterson FC, et al. (2010)). The screening compounds were added to the selective uracil media "seeded with 96-well plate reporter strains in a final concentration of 25 M; yeast growth was inspected manually after ~ 3 days. The compounds were transferred to the wells of screening 2.5 mM of stock solutions using a Biomek FX liquid handler.
[0102] According to a third screening approach, the Life Chemicals library was also screened for Arabidopsis germination inhibitors on solidified agar medium containing 0.5 X MS of salts, 0.5% sucrose and 25 μM of the compound of test. The germination assay hits were subsequently tested in two assays of two yeast hybrids. The hit compounds were re-stored from their original suppliers and used in secondary screenings and in the characterization of the compound. Quinabactin and its analogs were purchased by Life Chemicals. PP2C activity assay
[0103] The HAB1 and PYL proteins were expressed and purified as described previously (Park SY, et al. (2009) Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR / PYL Family of START Proteins. Science 324 (5930): 1068- 1071), with minor modifications. To obtain the GST-HAB1, -ABI1 and -ABI2 fusion proteins, the HAB1 cDNA was cloned into pGex-2T, while the ABI1 and ABI2 cDNAs were cloned into the pGex-4T-1 vector. Expression was conducted on BL21 [DE3] pLysS host cells. The transformed cells were pre-cultured overnight, transferred to LB medium and cultured at 30 ° C for ~ 0.5 A600 culture. The culture was then cooled on ice and MnC2 was added to 4 mM and IPTG added to 0.3 mM. After 16 hours of incubation at 15 ° C, the cells were harvested and the recombinant proteins were purified on glutathione agarose, as described previously (Park SY, et al. (2009). PYL, the receptor cDNAs for all 13 ABA receptors were cloned into the pET28 vector and expressed and purified as described previously (Mosquna A, et al. (2011) Potent and selective activation of abscisic acid receptors in vivo by mutational stabilization of their agonistbound conformation PNAS 108 (51): 20838-20843); this produces the soluble and functional protein (assessed using receptor-mediated PP2C inhibition assays) for all receptors except PYL7, PYL11 and PYL12. therefore, alternatively expressed as maltose-binding fusion proteins (MBP) using the vector pMAL-c, the expression of these constructs was performed on the host strain BL21 [DE3] pLysS with the same conditions of induction us for GST-HAB1. Recombinant MBP-PYL fusion proteins were purified from sonicated lysates and cleaned using amylose resin (New England Biolab, Inc.) using the manufacturers' purification instructions. This effort produced an active MBP-PYL11 fusion protein, but failed for PYL7 and PYL12.
[0104] Assays for PP2C activity using recombinant receptors and PP2Cs were performed as follows: Purified proteins were pre-incubated in 80 μl of assay buffer containing 10 mM MnCl2, 3 μg of bovine serum albumin and 0, 1% 2-mercaptoethanol with ABA or ABA agonist for 30 minutes at 22 ° C. The reactions were started by adding 20 μl of a reaction solution containing 156 mM Tris-OAc, pH 7.9, 330 mM KOAc and 5 mM 4-methylumbelliferyl phosphate after which fluorescence measurements were immediately collected using a 355 nm excitation filter and a 460 nm emission filter in a Wallac plate reader. The reactions contained 50 nM PP2C and 100 nM PYR / PYL proteins, respectively.
[0105] Figure 1A shows a representative group of ABA agonists. As shown in Figure 1B, multiple PYR / PYL receptors are activated by several LC66C6 agonists, in an assay of two yeast hybrids. This assay reports the physical interaction promoted by the agonist of the PYR / PYL proteins and PP2C clade A proteins when a specific receptor and PP2C are fused to the DNA binding and GAL4 activation domains respectively, as previously described (Park et al. 2009) . These yeast-based assays indicate that LC66C6 is a multiple PYR / PYL receptor agonist, unlike the previously described pyrabactin agonist, which has much higher receptor selectivity than ABA or the new LC66C6 agonist. As previously described, binding promoted by a receptor agonist to a PP2C clade inhibits PP2C phosphatase activity. In Arabidopsis, there are 14 PYR / PYL receptors, 13 of which can mediate ABA responses in a protoplasty-based assay system (Fujii et al. 2009). To examine the selectivity of LC66C6 more closely, we tried to express and purify the recombinant 6X-His-PYR / PYL proteins for all 14 recovered ABA-responsive members and receptors, except PYL7, 12 and 13, which could not be produced in the active forms for technical reasons. This panel of recombinant receptors allows a close complete picture of ABA agonist activity in members of the Arabidopsis PYR / PYL receptor family. As shown in Figure 2, the PPC2 enzyme activity of HBA1, ABI1, and ABI2 is inhibited by> 90% by 10 μM ABA in the presence of all tested ABA receptors (Figure 2B). In response to LC66C6 (Quinabactin),> 70% PP2C inhibition of HBA1, ABI1, and ABI2 was observed with PYR1, PYL1, PYL2, PYL3 and PYL5 receptors.
[0106] To further characterize quinabactin activity and define the selectivity of its receptor, receptor-mediated PP2C inhibition assays were conducted using 10 recombinant receptors in combination with PP2Cs HAB1, ABI1 or ABI2. These experiments showed that quinabactin activates PYR1, PYLs 1-3 and PYL5 with submicromolar IC50 values and exhibits substantially greater activity at dimeric receptor sites (Figures 2, 3 and 4). The results also show that quinabactin is a stronger PYR1 or PYL1 agonist than ABA (Figures 2 and 3). In addition, the maximum inhibition of PP2C observed by quinabactin was greater than that observed with pyrabactin with all tested receptors. Although pirabactin can activate PYL5 with an IC50 of 0.90 μM, it saturates ~ ~ 40%> PP2C inhibition, suggesting that it is an incomplete / partial PYL5 agonist. Thus, this example demonstrates the identification of a new sulfonamide agonist with a broader spectrum activity of the receptor and greater bioactivity in relation to pyrabactin. Example 2
[0107] This example demonstrates that new ABA agonists inhibit plant germination and growth.
[0108] Arabidopsis germination and analysis of hypocotyl growth inhibition
[0109] For Arabidopsis germination and hypocotyl growth inhibition analysis, post-ripened seeds about 4 weeks were sterilized on the surface with a solution containing 5% NaCIO and 0.05% Tween-20 for 10 minutes , and washed with water four times. The sterilized seeds were suspended with 0.1% agar and showed 0.8% solidified agar medium containing 1/2 salts of Murashige and Skoog (MS) (Sigma-Aldrich) in the presence of chemicals and were stored at 4 ° C for 4 days, then transferred to 22 ° C in the dark or light. Germination was determined after a 4-day incubation, while the growth of the hypocotyl was photographed after a 6-day incubation. Plant materials
[0110] The following mutant alleles / strains were used: aba2-1 (Leon-Kloosterziel KM, et al. (1996) Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci. Plant J 10 (4): 655 -661), abil-1 (Umezawa T, et al. (2009) Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 106 (41): 17588-17593), abi3-9, abi4-11 (Nambara E, et al. (2002) A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana. Genetics 161 (3): 1247-1255), and quadruple of pry1pyl1pyl2ply4 (Park SY, et al. (2009) Abscisic Acid Inhibits Type 2C Protein Phosphatases via the PYR PYL Family of START Proteins. Science 324 (5930): 1068-1071); all of these strains are at the foundations of Columbia. The pry1pyl1pyl2ply4 quadruple mutant strain used was backcrossed with Columbia three times. Barley and soybean seeds were purchased from Living Whole Foods, Inc., while corn seeds were obtained from W. Atlee Burpee & Co. Detail, the methods used for physiological experiments using these materials are provided as background information.
[0111] To explore the physiological consequences of the properties of the exclusive LC66C agonist, we characterize its effects on Arabidopsis seeds, seedlings and adult plants. As shown in Figure 5, the ABA agonists described in this document strongly inhibit the germination of Arabidopsis seeds. Figures 5A and 5B show that several agonists, including LC66C6, inhibit seed germination in a dose-dependent manner. In particular, LC66C6 was almost as effective as, on a per mol basis, in inhibiting germination as (+) - ABA, and was more effective than the other tested agonists.
[0112] Figures 5C and 5D show the effect of agonists (+) - ABA and LC66C6 in inhibiting seed germination from several ABA insensitive mutants. As shown in Figure 5C, at a concentration of 5 μM, the LC66C6 showed a similar pattern of inhibiting germination as (+) - ABA did for all mutants except for the PYR / PYL quadruple mutant (pyr1 / pyl1 / pyl2 / pyl4) and single mutant of pyr1. Combined with the IC50 data presented above in Figure 4, these genetic data suggest that the inhibitory activity of LC66C6 germination is largely explained by its ability to agonize PYR1, PYL1 and PYL2. The ability of ABA to inhibit germination in the quadruple mutant is probably explained by its agonist activity in the other receptors. Our genetic data are consistent with the hypothesis that PYR1 plays an important but redundant role in seed germination in response to ABA, as the pyr1 mutant germinates in the presence of both 5 μM LC66C6 and pyrabactin (Park et al. 2009 ).
[0113] As shown in Figure 6, LC66C6 also inhibits plant growth after germination. Figures 6A and 6B show that LC66C6 inhibits root elongation in the wild type, abil1 and quadruple mutant, and is comparable or slightly more effective than (+) - ABA in its inhibitory effects at all concentrations tested. In addition, Figure 6C demonstrates that LC66C6 inhibits the growth of wild-type and mutant plants in a concentration-dependent manner. The inhibition of plant growth by LC66C6 is significantly greater than the inhibition by pirabactin, and comparable to that of (+) - ABA.
[0114] This example demonstrates that LC66C6 is a potent inhibitor of seed germination and growth of wild-type plants and ABA insensitive mutants. Example 3
[0115] This example demonstrates that the LC66C6 agonist induces drought stress tolerance. Physiological tests
[0116] Physiological tests were performed on Arabidopsis plants grown at 22 ± 2 ° C and relative humidity (RH) 45 ± 10% under a 16/8 hour light / dark cycle. For the analysis of water loss by transpiration in Arabidopsis, the plants were pretreated by aerosol spray of 4 ml of solution containing 25 μM of the compound and 0.05% of Tween-20. 12 plants were sprayed at 4 weeks age per compound or control analyzed. After overnight pretreatment with the compounds, the aerial portions were separated from the roots, and their fresh weight measured at 20 min intervals over a 2 hour time period. To measure the opening of the stoma, the plants were pre-treated with the compounds as described above, covered with plastic caps to keep RH high and after pre-treatment overnight, the epidermal impressions of the leaves were obtained using the method of Suzuki Universal Micro Printing (SUMP), using SUMP printing solution with SUMP B plates (SUMP Laboratory). Leaf impressions were analyzed by light microscopy and stoma openings were determined from pore widths using ImageJ 1.43v software (National Institutes of Health, USA). For Arabidopsis drought stress tests, approximately 1.5 ml of 25 μM of chemical solution was aerosolized to the plants at daily intervals over a period of 3 days. The plants were grown in 6 X 6 X 5 cm square pots containing 100 g of soil per pot. The stress tests for soybean drought were performed on plants grown at 25 ± 2 ° C, 65 ± 10% RH under 16/8 hour light / dark cycles. Approximately 20 ml of 50 μM of chemical solution containing 0.05% Tween-20 was sprayed per pot (3 plants per pot) four times every 3 days. The pots used were 250 ml in size, and contained 200 g of soil per pot. The pots were covered in Parafilm so that the measured water loss was mediated by perspiration. The water content of the soil% was determined by measuring the weight of the pot and calculated by removing the weight of the dry soil from the total weight. Analysis of water loss in soybeans, barley and corn.
[0117] For the analysis of water loss using soybeans, barley and corn, 100 μM of chemicals containing 0.05% Tween-20 were sprayed on the aerial parts of the plants. The soybean, barley and corn plants used were approximately 4, 2 and 2 weeks old, respectively. The compounds were applied 16 hours before the water loss tests were conducted. To measure water loss, whole shoots were separated and their fresh weight monitored.
[0118] Figure 7 shows the effect of LC66C6 on various parameters related to drought stress. As shown in Figures 7A and 7B, LC66C6 reduced the amount of water loss from transpiration in detached leaves of wild-type plants and aba2 mutants (ABA-deficient mutant 2). However, as shown in Figure 7C, LC66C6 does not reduce water loss from transpiration in detached leaves of the abil-1 mutant. Figure 7D shows that LC66C6 induces closure of stomata in the wild type and in the aba2 mutant, but not in the abil-1 mutant. Figure 7E shows the effects of agonist compounds on soil water content during the treatment of soybean plant drought.
[0119] Figure 8A shows that the treatment of plants with quinabactin confers tolerance to drought stress in Arabidopsis plants similar to that conferred by treatment with (+) - ABA. In this example, two-week-old plants were subjected to stress by water retention drought and were photographed after 12 days. The plants were rehydrated after a 2-week drought treatment. The number of plants surviving by the total number of plants tested is shown adjacent to the photographs. Figure 8B shows that the treatment of soybean plants with quinabactin gives tolerance to drought stress similar to that conferred by the treatment with (+) - ABA. In this example, two-week-old plants were subjected to drought stress by water retention and photographed after 8 days of drought treatment. For all drought stress treatments, the compounds (tested at 25 μM for Arabidopsis and 50 μM for soybeans) were applied in solutions containing 0.05% Tween-20 and applied as aerosols every 3 days during the drought regime . Values for all experiments are the mean ± SEM (n = 6, 3 plants used per experiment).
[0120] This example shows that LC66C6 induces drought stress tolerance in Arabidopsis wild-type and mutant aba2 plants and in wild-type soy plants similar to that conferred by (+) - ABA. Example 4
[0121] This example demonstrates that LC66C6 induces ABA-responsive genes in a similar way to that induced by (+) - ABA. Microarray analysis
[0122] Total RNA was isolated using the RNAeasy Plant Mini Kit (Qiagen, USA), according to the manufacturer's instructions. The synthesis, labeling and hybridization of cDNA in fragments of ATH1 from Arabidopsis (Affymetrix, USA) were performed by the IIGB Core Instrumentation Facility at the University of California at Riverside using Affymetrix protocols. The biological samples in triplicate were hybridized for the DMSO controls, treatments with ABA, pyrabactin and quinabactin; the compounds were applied in a final concentration of 25 μM and the RNA was prepared from frozen tissue after 6 hours of exposure to the compounds or controls. The expression signals for probe groups were calculated and normalized by the MAS5 Statistical Algorithm (Affymetrix, USA). The experimental filtering of the array data was performed for the presence of the signals in all experiments. The levels of average transcripts in each chemical treatment were purchased from those in the control experiments and used to calculate the amount of the change values. The amount of the change values transformed into Log2 were used to calculate the Person's Correlation Coefficients between the experimental conditions. Quantitative RT-PCR analysis
[0123] Total RNA was isolated using the vegetable RNA purification reagent (Invitrogen, USA), according to the manufacturer's instructions. The cDNA was synthesized from 1 μg of total RNA using the QantiTec reverse transcription kit (Qiagen, USA). Real-time PCR using Maxima® Master Mix SYBR Green / Fluorescein qPCR (Fermentas) was performed with the iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA). The relative amounts of the target mRNAs were determined using the relative standard curve method and were normalized by the relative amount of the internal control mRNA. Biological experiments in triplicate were performed. The primer sequences used in these experiments are shown in Table 1. Table 1. Primer sets for quantitative RT-PCR
ABA-responsive reporter gene assays
[0124] The existing ABA-responsive promoter-GUS mergers are not, in our experience, ideal due to high background levels and relatively low induction levels in response to ABA. MAPKKK18 as a highly inducible ABA gene with low background levels (Matsui A, et al, Plant Cell Physiol 49 (8): 1135-1149 (2008)); MAPKKK18 is also strongly induced by salt and drought stress. Therefore, we characterize the effects of agonists on the MAPKKK18 promoter: transgenic plants reporter GUS. GUS staining was performed in a reaction buffer of the following composition: 50 mM sodium phosphate buffer, pH 7.0, 0.05% Tween-20, 2.5 mM potassium ferrocyanide, 2.5 mM of potassium ferricianide, 1 mM of X-gluc. The reaction buffer was vacuum infiltrated in the test samples for 10 min twice and then incubated at 37 ° C for 5 h. The reaction was stopped by washing the samples with 70% ethanol, and the chlorophyll pigments were discolored by incubation at 65 ° C.
[0125] Figure 9 shows the changes in gene expression induced in response to pirabactin, LC66C6, and (+) - ABA. As shown in Figure 9A, LC66C6 induced the expression of RD29B and MAPKKK18 mRNA in a dose-dependent manner in wild-type plants, while these levels of induction are decreased in abil-1 plants and PYR / PYL quadruple mutants. The induction of gene expression by LC66C6 is similar to that observed with (+) - ABA. In contrast to (+) - ABA and LC66C6, pirabactin does not induce gene expression in wild type plants, although it induces modest ABA-related gene expression in seedlings when higher concentrations are used in the treatment (Park et al, 2009).
[0126] Figure 9B shows the broad genome comparison of ABA and LC66C or the effects of pyrabactin, compared to control treatments, on wild type seedlings, as measured by the hybridization of labeled RNAs to ATH1 microarrays. As shown in Figure 98, LC66C6 induces a similar set of genes to those induced by ABA in a microarray experiment. In contrast, pirabactin does not induce an expression pattern similar to that of ABA.
[0127] Figures 9C and 9D show that LC66C6 induces the expression of reporter genes in the same tissues as (+) - ABA. The expression of reporter genes was observed in protective cells and vascular tissues of leaves and roots, and in root tips of absorbed seeds.
[0128] Figure 10 shows ABA-responsive gene expression in single PYR / PYL mutants. As shown in Figure 10, MAPKKK18, RD29A, and RD29B mRNAs responsive to ABA were induced by LC66C6 and (+) - ABA in the Col and Ler ecotypes and the unique mutant genotypes of pyr1, pyl1, ply2, pyl3 and pyl4. In contrast, pirabactin does not significantly induce the expression of any of the genes evaluated in any of the unique mutants or wild type ecotypes.
[0129] Figure 11 shows ABA-responsive gene expression in wild-type, abil-1 plants and PYR / PYL quadruple mutants. As shown in Figure 11, LC66C6 and (+) - ABA induced the expression of ABF3, GBF3, NCED3, and RD29A in a dose-dependent manner in wild type Col plants, while induction levels are decreased in abil-1 plants and PYR / PYL quadruple mutants. Consistent with the above results, pirabactin does not induce significant expression of any genes analyzed in wild type plants. Example 5
[0130] This example demonstrates that the key enzymes for ABA's catabolism do not affect the responses induced by LC66C6.
[0131] As shown in Figure 12, inhibition of plant growth and germination by ABA is increased in plants that are double mutants for cyp707a, a key enzyme for ABA catabolism, but reduced in plants that overexpress cyp707A (CYP707AOX; see Figures 12A-D). In contrast, the effects on plant growth and germination by LC66C6 are not significantly different in plants that are double mutants for cyp707a, wild-type plants, or in plants that overexpress CYP707AOX (see Figures 12A-D).
[0132] This example shows that the enzymes that are involved in the decomposition of ABA do not influence the phenotypes regulated by LC66C6. Example 6
[0133] This example shows that LC66C6 is bioactive in several plant species, including monocots and dicots.
[0134] Figure 13A shows that LC66C6 inhibits germination of broccoli, radish, alfalfa, soybean seeds, sparingly, wheat, sorghum and corn. The level of inhibition of germination by LC66C6 is higher than that of pirabactin. As shown in Figure 13B, LC66C6 reduces water loss from transpiration over a period of 2 hours on detached leaves of the above species. In addition, LC66C6 strongly induces the expression of ABA-responsive genes, GmNAC4 and GmbZIP1, in soybeans (Figure 13C), moderately induces the expression of ABA-responsive genes, HVA1 and HvDRF1, in barley (Figure 13D), and weakly induces expression of genes responsive to ABA, ZmRab17 and ZmLEA, in corn (Figure 13E).
[0135] This example demonstrates that LC66C6 inhibits germination and reduces water loss through transpiration in a diverse group of agriculturally important species, indicating that LC66C6 is useful in reducing drought stress in several species. Example 7
[0136] This example shows the chemical structures of ABA and agonists described in this document, and the effect of agonists in vitro and in vivo.
[0137] Figures 14 and 18 show the chemical structures of ABA and the tested agonists. Figure 15A shows the test results of two yeast hybrids using PYR / PYL, PYR1, PYL1, PYL2, PYL3 and PYL4 receptors to test the response to each of the agonists shown in Figure 14. Figure 15B shows the test results of the agonists in Figure 14 in the germination of wild type seeds, and demonstrates that LC66C6 is one of the most effective agonists, after (+) - ABA, in inhibiting the germination of wild type seeds. Figure 15C shows the effects of compounds in an ABA reporter strain as measured using glucuronidase assays in a transgenic strain expressing glucuronidase under the control of the ABD MAPKKK18-inducible Arabidopsis gene.
[0138] This example demonstrates that LC66C6 is one of the most effective agonists tested in vitro and in vivo. Example 8
[0139] This example shows that LC66C6 can increase the size of ABA deficient mutant plants.
[0140] In this example, 14-day-old wild-type or aba2 mutant plants were sprayed with a solution containing 25 μM of the agonist twice daily for two weeks. Images and fresh weight were obtained from 4-week-old plants. As shown in Figure 16, the application of LC66C6 for flap2 mutant plants significantly increased the size of the mutant plants compared to control plants treated with the DMSO carrier only.
[0141] This example demonstrates that LC66C6 can complement the growth phenotype observed in the aba2 mutation in a similar way to that of (+) - ABA. Example 9
[0142] This example shows that LC66C6 can weakly inhibit the growth of the protonema in moss, but has no effect on the growth of the single-celled green alga Chlamydomonas.
[0143] As shown in Figures 17A and 17B, LC66C6 showed a weak but significant inhibition of the protease growth of the moss Physcomitrella patens. Pirabactin has bleached the protonema, suggesting that it may be toxic to this species.
[0144] Figure 17C shows that LC66C6 can induce the expression of ABA-responsive genes in moss. However, these levels of induction were weaker than those of ABA.
[0145] As shown in Figure 17D, (+) - ABA and LC66C6 had no effect on the growth of Chlamydomonas with and without salinity and osmotic stress. Again, pirabactin has bleached Chlamydomonas, suggesting that it can be toxic to this species as well.
[0146] This example shows that LC66C6 can weakly inhibit the growth of the protonema and weakly induce ABA-responsive gene expression in the Physcomitrella patens moss, but does not affect the growth of the Chlamydomonas unicellular alga.
[0147] It is understood that the examples and modalities described in this document are for illustrative purposes only and that several modifications or alterations in the light of the same will be suggested to those skilled in the art and should be included in the spirit and competence of this request and in the scope of the claims attached. All publications, sequence accession numbers, patents and patent applications cited in this document are incorporated in their entirety through this document by reference for all purposes.

权利要求:
Claims (29)
[0001]
1. Agricultural formulation characterized by comprising a compound of Formula I:
[0002]
2. Formulation, according to claim 1, characterized by the fact that the compound has the formula:
[0003]
3. Formulation, according to claim 2, characterized by the fact that the compound has the formula:
[0004]
4. Formulation according to claim 2, characterized by the fact that R1 is C1-6alkyl.
[0005]
5. Formulation according to claim 4, characterized by the fact that R2a is independently selected from the group consisting of H, halogen and C1-6alkyl.
[0006]
6. Formulation according to claim 4, characterized by the fact that R2 is selected from the group consisting of phenyl, naphthyl, thiophene, furan, pyrrole and pyridyl.
[0007]
7. Formulation according to claim 4, characterized by the fact that R1 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl ; R2 is selected from the group consisting of phenyl and thiophene, each optionally substituted by 1 group R2a; each R2a is independently selected from the group consisting of H, F, CI, methyl and ethyl; and L is selected from the group consisting of a bond and methylene.
[0008]
8. Formulation according to claim 7, characterized by the fact that the compound has the formula:
[0009]
9. Formulation according to claim 7, characterized by the fact that the compound has the formula:
[0010]
10. Formulation according to claim 1, characterized by the fact that the compound is one of the compounds below:
[0011]
11. Formulation according to claim 1, characterized by the fact that the compound is
[0012]
Formulation according to claim 1, characterized in that it further comprises at least one of a fungicide, a herbicide, a pesticide, a nematicide, an insecticide, a plant activator, a synergistic agent, a herbicide protective agent, a plant growth regulator, an insect repellent, an acaricide, a molluscicide or a fertilizer.
[0013]
Formulation according to claim 1, characterized in that it further comprises a surfactant.
[0014]
14. Formulation according to claim 1, characterized in that it also comprises a carrier.
[0015]
15. Method of increasing the tolerance of abiotic stress in a plant, the method characterized by understanding the contact of a plant with a sufficient amount of the formulation, as defined in any one of claims 1 to 14, to increase the tolerance to abiotic stress in the compared to the non-contact of the plant with the formulation.
[0016]
16. Method, according to claim 15, characterized by the fact that the plant is a monocot.
[0017]
17. Method, according to claim 15, characterized by the fact that the plant is a dicot.
[0018]
18. Method, according to claim 15, characterized by the fact that tolerance to abiotic stress comprises drought tolerance.
[0019]
19. Method, according to claim 15, characterized by the fact that the contact stage comprises the distribution of the formulation to the plant by airplanes or by irrigation.
[0020]
20. Method of inhibiting seed germination in a plant, the method characterized by comprising contacting a seed with a sufficient amount of the formulation, as defined in any one of claims 1 to 14, to inhibit germination.
[0021]
21. Method for producing a treated plant characterized by comprising the contact of a plant with the formulation, as defined in any one of claims 1 to 14.
[0022]
22. Method according to claim 21, characterized by the fact that the plant is a seed.
[0023]
23. Method of activation of a PYR / PYL protein, the method characterized by comprising the contact of the PYR / PYL protein with the compound, as defined in any one of claims 1 to 14.
[0024]
24. Method according to claim 23, characterized by the fact that the PYR / PYL protein is expressed by a cell.
[0025]
25. Method according to claim 24, characterized by the fact that the cell is a plant cell.
[0026]
26. Method according to claim 24, characterized by the fact that the PYR / PYL protein is an endogenous protein.
[0027]
27. Method according to claim 24, characterized in that the PYR / PYL protein is a heterologous protein.
[0028]
28. Method according to claim 24, characterized by the fact that the cell still expresses a type 2 protein phosphatase (PP2C).
[0029]
29. Method according to claim 28, characterized by the fact that the protein phosphatase type 2 is HAB1 (homology to ABI1), ABI1 (insensitive to abscisic acid 1), or ABI2 (insensitive to abscisic acid 2).

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CA2868803A1|2013-10-03|

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EA025432B1|2016-12-30|

---

MX348287B|2017-06-02|

---

KR102059948B1|2019-12-27|

---

CN104363760A|2015-02-18|

---

JP6166349B2|2017-07-19|

---

WO2013148339A1|2013-10-03|

---

ES2709025T3|2019-04-12|

---

AU2013240193A1|2014-10-23|

---

UA115237C2|2017-10-10|

---

EP2830422B1|2018-12-26|

---

MX2014011723A|2015-01-22|

---

JP2015512915A|2015-04-30|

---

EA201401067A1|2015-01-30|

---

KR20140144216A|2014-12-18|

---

US20150047073A1|2015-02-12|

---

AU2013240193B2|2016-09-01|

---

CN104363760B|2017-03-01|

---

EP2830422A1|2015-02-04|

---

ZA201407226B|2016-05-25|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US5498755A|1994-08-23|1996-03-12|Chandraratna; Roshantha A.|Disubstituted aryl and heteroaryl imines having retinoid-like biological activity|

---

US6127382A|1999-08-16|2000-10-03|Allergan Sales, Inc.|Amines substituted with a tetrahydroquinolinyl group an aryl or heteroaryl group and an alkyl group, having retinoid-like biological activity|

---

US7071188B2|2001-06-14|2006-07-04|Syngenta Crop Protection, Inc.|Composition and method for improving plant growth|

---

CO5380003A1|2001-10-05|2004-03-31|Syngenta Participations Ag|SYNERGIC HERBICIDE COMPOSITION INCLUDING PRETILACHLOR AND PROSULFOCARB|

---

ES2365592T3|2005-09-29|2011-10-07|Syngenta Participations Ag|FUNGUICIDE COMPOSITIONS.|

---

US8124565B2|2006-02-09|2012-02-28|Syngenta Crop Protection, Inc.|Method of protecting a plant propagation material, a plant, and/or plant organs|

---

CA2750991C|2009-02-13|2019-09-10|The Regents Of The University Of California|Control of plant stress tolerance, water use efficiency and gene expression using novel aba receptor proteins and synthetic agonists|

---

JP2013522274A|2010-03-18|2013-06-13|バイエル・インテレクチユアル・プロパテイー・ゲー・エム・ベー・ハー|Arylsulfonamides and hetarylsulfonamides as activators against abiotic plant stress|

---

AR081242A1|2010-04-28|2012-07-18|Univ California|MODIFIED PYR / PYL RECEPTORS ACTIVATED BY ORTOGONAL LIGANDS|

---

US20130045952A1|2011-08-19|2013-02-21|Van Andel Research Institute|ABA Receptor Agonists for Increased Plant Stress Resistance|

---

JP2016531091A|2013-06-28|2016-10-06|ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア|Compounds that induce ABA responses|FR2655191A1|1989-11-28|1991-05-31|Genral Electric Cgr Sa|ANODE FOR X-RAY TUBE.|

---

DE10049567B4|1999-10-08|2017-12-14|Toyota Jidosha Kabushiki Kaisha|Vehicle control unit for controlling a four-wheel drive motor vehicle|

---

CN104170823A|2013-05-23|2014-12-03|中国科学院上海生命科学研究院|Small molecule compound for enhancing plant stress resistance|

---

JP2016531091A|2013-06-28|2016-10-06|ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア|Compounds that induce ABA responses|

---

AR099828A1|2014-04-10|2016-08-24|Bayer Cropscience Ag|USE OF SUBSTITUTED OXOTETHERROCHINOLINYLSULPHONAMIDS OR THEIR SALTS TO INCREASE STRESS TOLERANCE IN PLANTS|

---

WO2016022915A1|2014-08-08|2016-02-11|The Regent Of The University Of California|6-sulfonylamino quinoline compounds as plant growth regulators|

---

KR20170066330A|2014-08-08|2017-06-14|더 리전트 오브 더 유니버시티 오브 캘리포니아|2-oxo-3,4-dihydroquinolin-6-yl sulphonamide cpds and their use as plant growth regulators|

---

WO2016069637A1|2014-10-28|2016-05-06|The Regents Of The University Of California|Sulfonamides that activate aba receptors|

---

CN104365605A|2014-10-29|2015-02-25|上海绿盎生物科技有限公司|Preparation method and application of water suspension agent of abscisic acid receptor agonist|

---

CN104277000A|2014-10-29|2015-01-14|上海绿盎生物科技有限公司|Abscisic acid receptor stimulant raw drug and preparation method thereof|

---

AU2016218073B2|2015-02-09|2019-12-19|Syngenta Participations Ag|2-oxo-3,4-dihydroquinoline compounds as plant growth regulators|

---

AR103543A1|2015-02-13|2017-05-17|Bayer Cropscience Ag|1-CICLOALQUIL-2-OXOTETRAHIDROQUINOLINA-6-ILSULFONAMIDA REPLACED OR A SALT OF THE SAME AND ITS USE TO INCREASE STRESS TOLERANCE IN PLANTS|

---

CN104788553A|2015-04-29|2015-07-22|中国农业科学院作物科学研究所|Application of stress-tolerant related protein R10 to regulating plant adverse resistance|

---

JP2018527318A|2015-07-15|2018-09-20|バイエル・クロップサイエンス・アクチェンゲゼルシャフト|Substituted oxotetrahydroquinolinylphosphinic acids and phosphinic acid amides or their salts and their use to enhance plant stress tolerance|

---

EP3341361A1|2015-08-27|2018-07-04|The Regents of The University of California|Derivatives of halo quinabactin|

---

US11076597B2|2015-12-28|2021-08-03|Cas Center For Excellence In Molecular Plant Sciences|High stress resistant plant growth regulator and preparation method and use thereof|

---

CN106749044B|2015-12-28|2020-04-21|中国科学院上海生命科学研究院|ABA analogue for enhancing plant stress resistance|

---

GB201611717D0|2016-07-05|2016-08-17|Syngenta Participations Ag|Plant growth regulator compounds|

---

WO2018017490A1|2016-07-18|2018-01-25|The Regents Of The University Of California|Carbamate quinabactin|

---

CN106719660B|2016-11-09|2021-06-25|中国农业大学|Application of naphthalenesulfonamide compounds in regulating plant growth activity|

---

WO2019025153A1|2017-07-31|2019-02-07|Bayer Cropscience Aktiengesellschaft|Use of substituted n-sulfonyl-n'-aryl diaminoalkanes and n-sulfonyl-n'-heteroaryl diaminoalkanes or salts thereof for increasing the stress tolerance in plants|

---

GB201820729D0|2018-12-19|2019-01-30|Syngenta Participations Ag|Plant growth regulator compositions|

法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

---

2020-08-25| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-12-08| B09A| Decision: intention to grant|

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2021-02-09| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/03/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261618386P| true| 2012-03-30|2012-03-30|

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US61/618,386|2012-03-30|

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PCT/US2013/032281|WO2013148339A1|2012-03-30|2013-03-15|Synthetic compounds for vegetative aba responses|

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