Plant stomatal opening regulator

Abstract

Provided is a plant stomatal opening regulator. The plant stomatal opening regulator comprises a CLE5 peptide and / or an expression cassette thereof.

Description

Plant stomatal opening regulator

[0001] This invention relates to a plant stomatal opening regulator, etc.

[0002] Droughts hinder plant growth and limit crop yields worldwide. To cope with this, terrestrial plants have evolved sophisticated strategies through a number of morphological, physiological, and biochemical adaptations. Stomata are specialized pores on the surface of leaves that control the evaporation of water absorbed from the soil through transpiration. Therefore, closing stomata is the most effective strategy for terrestrial plants to prevent water loss due to drought stress.

[0003] Decades of research have revealed the central importance of abscisic acid (ABA), a stress hormone that actively controls stomatal closure due to drought (Non-Patent Literature 1). Synthesized ABA is captured by PYRABACTIN RESISTANCE 1 (PYR) / PYR1-LIKE / REGULATORY COMPONENT OF ABA RECEPTOR, binds to and inhibits clade A protein phosphatease type 2Cs (PP2C), and releases SNF1-related protein kinase 2s (SnRK2s). Activation of subclass III SnRK2s such as SRK2E / OPEN STOMATA1 / SnRK2.6 (SRK2E) and SRK2D / SnRK2.2 (SRK2D) via phosphorylation plays a crucial role in regulating stomatal movement and ABA signaling to counter dehydration. However, accumulating evidence suggests that stomatal closure due to drought stress occurs before endogenous ABA levels in leaves gradually rise. This suggests the existence of an unknown signaling pathway that activates an early response to drought stress without stimulating the orthodox ABA pathway.

[0004] Brodribb, TJ & McAdam, SA Passive origins of stomatal control in vascular plants. Science 331, 582-585 (2011).

[0005] The object of this invention is to provide a plant stomatal opening regulator.

[0006] In view of the above problems, the inventors diligently conducted research and found that a plant stomatal opening regulator containing CLE5 peptide and / or its expression cassette can solve the above problems. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A plant stomatal opening regulator containing CLE5 peptide and / or its expression cassette.

[0008] Item 1A. Use of CLE5 peptide and / or its expression cassette as a plant stomatal opening regulator.

[0009] Item 1B. Use of CLE5 peptide and / or its expression cassette for the manufacture of plant stomatal opening regulators.

[0010] Item 1C. CLE5 peptide and / or expression cassette thereof for use as a plant stomatal opening regulator.

[0011] Item 1D. A method for regulating the opening of a plant mechanism, comprising applying a CLE5 peptide and / or its expression cassette to a plant.

[0012] Item 2. The plant stomatal opening regulator according to Item 1, wherein the CLE5 peptide is a peptide having the ability to suppress plant stomatal opening, comprising: (a) an amino acid sequence a shown in Sequence ID No. 1, or (b) an amino acid sequence b in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence a, and having the ability to suppress plant stomatal opening.

[0013] Item 3. The plant stomatal opening regulator according to item 1 or 2, wherein the CLE5 peptide is a glycosylated peptide.

[0014] Item 4. The plant stomatal opening regulator according to item 3, wherein the sugar chain is a sugar chain containing arabinose as a constituent monosaccharide.

[0015] Item 5. The plant stomatal opening regulator described in Item 4, wherein the sugar chain (arabinose) is composed of 2 to 5 linked sugar chains.

[0016] Item 6. The plant stomatal opening regulator according to Item 2, wherein the CLE5 peptide is a peptide modified with a sugar chain consisting of 2 to 5 arabinose molecules linked together, the 7th amino acid residue from the N-terminus of amino acid sequence a is a hydroxyproline residue, and the modification site of the sugar chain is the 7th hydroxyproline residue from the N-terminus of amino acid sequence a or the hydroxyproline residue at the corresponding position in amino acid sequence b.

[0017] Item 7. A plant stomatal opening inhibitor, as described in any of items 1 to 6.

[0018] Item 8. A drought tolerance enhancer containing a plant stomatal opening regulator as described in any of Items 1 to 7.

[0019] Item 8A. Use of CLE5 peptide and / or its expression cassette as a desiccation tolerance enhancer.

[0020] Item 8B. Use of CLE5 peptide and / or its expression cassette for the manufacture of desiccation tolerance enhancers.

[0021] Item 8C. CLE5 peptide and / or expression cassette thereof for use as a desiccation tolerance enhancer.

[0022] Item 9. A method for improving drought tolerance, comprising applying a plant stomatal opening regulator described in any of Items 1 to 8 to a plant.

[0023] Item 10. The method for improving drought tolerance according to item 9, wherein the CLE5 peptide is applied so as to come into contact with the stomata of a plant.

[0024] Item 11. A screening method for plant stomatal opening regulators, using at least one indicator selected from the group consisting of binding ability to BAM1 and / or GHR1 and phosphorylation-promoting ability of SRK2D and / or SRK2E.

[0025] Item 12. A plant stomatal opening regulator containing a substance selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote the phosphorylation of SRK2D and / or SRK2E.

[0026] Item 12A. Use of a substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote phosphorylation of SRK2D and / or SRK2E as a plant stomatal aperture regulator.

[0027] Item 12B. Use for the production of a plant stomatal aperture regulator of a substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote phosphorylation of SRK2D and / or SRK2E.

[0028] Item 12C. A substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote phosphorylation of SRK2D and / or SRK2E for use as a plant stomatal aperture regulator.

[0029] Item 12D. A method for regulating the opening of plant organs, comprising applying to a plant a substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote phosphorylation of SRK2D and / or SRK2E.

[0030] Item 13. The plant stomatal aperture regulator according to Item 12, wherein the substance is a peptide.

[0031] According to the present invention, a plant stomatal aperture regulator and a drying tolerance improver can be provided.

[0032] This figure shows that shoot-derived CLE5 affects gene expression induced by dehydration without promoting ABA accumulation. a Leaves of wild-type, cle5-1, and pCLE5::CLE5 / cle5-1 were detached for 1.5 hours. Scale bar: 1 cm. b Water loss at each time point when leaves of wild-type, cle5-1, and pCLE5::CLE5 / cle5-1 were detached. c GUS (β-glucuronidase) staining of pCLE5::GUS in leaves (stomata) of 8-day-old seedlings before (left) and after (right) 30 minutes of dehydration stress. Scale bar: 10 μm. e RNA-seq analysis of transcriptome changes in shoots of CLE5ox induced by β-estradiol for 2.5 hours. Data in the volcano plot represent differentially expressed genes (DEGs): red and blue dots indicate genes with significantly increased and decreased expression, respectively. g. qRT-PCR analysis of NCED3 expression in CLE5ox seedlings treated with mock or 1 μM β-estradiol (Est) for 2.5 hours. h. Endogenous ABA levels when 8-day-old CLE5ox shoots were treated with mock or 1 μM β-estradiol (Est) for 2.5 hours. i. Time-resolved stomatal conductance analysis of the ABA (2 μM) response in wild-type, cle5-1, pCLE5::CLE5 / cle5-1, and pGC1 (stomatal-specific promoter)::CLE5 / cle5-1 mutants. Data represent mean ± sd. DW: dry weight. P-values ​​shown in b, g, and h were calculated using two-sided unpaired t-tests. The adjusted P-value (Padj) in d was analyzed by Tukey Honest Significant Differences test after one-way ANOVA. Experiments a and b were repeated four times with similar results. This figure shows that CLE5p induces stomatal closure in relation to BAM1 and GHR1 LRR-RK. a, b The isothermal titration calorimetry (ITC) profiles of the interaction between CLE5p and the extracellular domains of BAM1 (a) and GHR1 (b) are shown. Dissociation constant (Kd) ± fitting error, binding stoichiometry (N), and thermal parameters (ΔG Gibbs free energy, ΔH enthalpy, -TΔS entropy) are shown.c Left: Representative images of stomata after treatment of wild-type and bam1ghr1 leaves with Mock or 1 μM [Ara3]CLE5p for 1 hour. Right: Statistics of stomatal openings measured as the ratio of stomatal width to length. d: qRT-PCR analysis of RD29B (ABA response) and NCED3 (ABA biosynthesis) expression after treatment of wild-type and bam1ghr1 seedlings with Mock or 1 μM [Ara3]CLE5p for 1 hour. This figure shows that the BAM1-GHR1-CLE5p module is required for SRK2E activation via phosphorylation of its activation site. a, b Short-term administration of [Ara3]CLE5p is sufficient to phosphorylate SRK2E-GFP (P-SRK2E; a) and SRK2D-GFP (P-SRK2D; b) in vivo. Wild-type and bam1ghr1 protoplasts expressing 35S::SRK2E-GFP (a) or 35S::SRK2D-GFP (b) were treated with 1 μM [Ara3]CLE5p or without CLE5p for 30 minutes. Untreated protoplasts (-) or 1 μM ABA-treated protoplasts (ABA) were used as negative or positive controls, respectively. Protoplasts expressing SRK2E-GFP (a) or SRK2D-GFP (b) were first treated with 1 μM [Ara3]CLE5p for 30 minutes, and then incubated with 1 μM λ-phosphatase (λ-PPase) for 30 minutes. SRK2E-GFP (a) and SRK2D-GFP (b) proteins were detected by anti-GFP antibody (αGFP). The phosphorylation site of SRK2E catalyzed by the BAM1 kinase domain was identified from LC-MS / MS spectra. Top: Shows the structure and main phosphorylation sites of SRK2E. The region within the activation domain (T-loop) phosphorylated by the BAM1 kinase domain is highlighted in red. Bottom: Shows the fragmentation spectrum of the MBP-SRK2E phosphorylation site map. The MS / MS spectrum of the phosphorylated 175-STVGTPAYIAPEVLLKK (SEQ ID NO: 9)-190 fragment is shown.Recombinant GST-BAM1 kinase domain (GST-BAM1(KD)), MBP-SRK2E(D140A), MBP-SRK2E(G33R / D140A), and MBP-SRK2E(S175A / T176A / T179A, Triple A) proteins were tested. e Kinase activity of GST-BAM1(KD) against MBP-SRK2E(D140A, G33R / D140A, Triple A). f, g Time-resolved stomatal conductance analysis of ABA (2 μM) response in wild-type, bam1, ghr1, bam1ghr1(f) or srk2e, 35S::SRK2E(Triple A) / srk2e, and 35S::SRK2E / srk2e leaves(g) is shown. Coomassie brilliant blue (CBB) staining in a, b, d, and e was used as a loading control; experiments a, b, d, and e were repeated at least twice to obtain similar results. This figure shows the function of the BAM1-GHR1-CLE5p module in the drought response. a Images of leaves of wild-type, bam1, ghr1, bam1ghr1, and bam1ghr1cle5-1 after being dehydrated for 1.5 hours. b Endogenous ABA levels in the aerial parts after 2.5 hours of dehydration stress in wild-type, cle5-1, and bam1ghr1. Different letters above the column represent the Tukey Honest Significant Differences test following two-way ANOVA, indicating a statistically significant difference between the control group and the dehydrated group at a level of P < 0.05. d Leaves of wild-type, srk2e, and nced3 were briefly coated with 1 μM [Ara3]CLE5p for 30 minutes. Left: Representative image of stomata. The scale bar is 5 μm. This shows qRT-PCR analysis of RD29B and RAB18 (drought stress response genes) expression after treatment of wild-type and ABA-insensitive (abi1-1) seedlings with Mock or 1 μM [Ara3]CLE5p for 1 hour. Under drought stress, CLE5p is captured by the BAM1-GHR1 receptor complex on the cell membrane of guard cells. This module phosphorylates SRK2E and SRK2D kinases to induce stomatal closure and activates the expression of drought stress-responsive genes without stimulating the normal ABA pathway. P-values ​​for c and e were calculated using two-tailed unpaired t-tests. This figure shows the complementarity of cle5 mutants.a. Structure of the CLE5 gene. The blue rectangle represents the CLE domain (208-246 bp). The T-DNA insertion site (white triangle) and the primer pairs used for q-PCR (#3 black arrow and #4 red arrow) are shown. The yellow box highlights the site of a single nucleotide insertion in the CRISPR / Cas-9 (CRISPR-cle5) edited CLE5 gene of Arabidopsis thaliana. Confirmation of the inserted nucleotide is shown in the sequence readout. b. qRT-PCR analysis of CLE5 expression in seedlings of wild-type and cle5 T-DNA insertion mutant (cle5-1) using primer sets #3 and #4 (a; N ≥ 4). c. Representative photographs of leaves of wild-type, cle5-1, and CRISPR-cle5 at 2 weeks of age are shown. d shows the stomatal index (n = 21, N = 2) and e shows the stomatal density (n = 15, N = 2) of 2-week-old wild-type, cle5-1, and CRISPR-cle5. P-values ​​were calculated using a two-tailed unpaired t-test. Experiment c was repeated three times with similar results. This figure shows the structural analysis and physiological activity evaluation of CLE5p. a shows the structural analysis of CLE5. Top: Mature CLE5 peptide (CLE5p) secreted from the 48-hour induced CLE5ox strain was collected and analyzed using a nanoLC-MS / MS separation system. The triarabinosylation site within mature CLE5p was identified. Bottom: shows the structure of triarabinosylated CLE5p, named [Ara3]CLE5p. Left: representative stomatal image after treatment of [Ara3]CLE5p or CLE5p at the indicated concentration for 30 minutes. Scale bar is 10 μm. Right: Stomatal bioassay (indicator concentration) of occlusion induced by [Ara3]CLE5p or CLE5p. Stomatal opening statistics were measured as the ratio of stomatal width to length (n = 15, N = 4). c Stomatal bioassay of 1 μM [Ara3]CLE5p-induced occlusion in cle5-1 and CRISPR-cle5 mutants. Stomatal opening statistics were measured as the ratio of stomatal width to length (n = 15, N = 4). d Time-resolved stomatal conductance analysis of ABA (2 μM) response in leaves of wild-type, cle5-1, and pCLE5::CLE5 / cle-1. Data were calculated using a two-tailed unpaired t-test, with P values ​​representing mean ± sd from N ≥ 3 biological replicas; ns are not significant.

[0033] 1. Definition In this specification, all notations of amino acid sequences are represented in one-letter notation.

[0034] In this specification, the expressions "containing" and "comprising" include the concepts of "containing", "comprising", "consisting essentially of", and "consisting only of".

[0035] The "identity" of an amino acid sequence refers to the degree of coincidence of the amino acid sequences of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the consistency of two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity is determined, for example, using the FASTA sequence analysis tool with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes "Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on such a BLAST algorithm, has been developed. The specific methods of these analysis methods are known, and reference may be made to the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). Also, the "identity" of a nucleotide sequence is defined according to the above. [[ID=⑨]] [[ID=⑩]]

[0036] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0037] In this specification, "nucleic acids" and "polynucleotides" are not particularly limited and include both natural and artificial ones. Specifically, in addition to DNA, RNA, etc., known chemically modified nucleotides may also be used, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide can be replaced with chemically modified phosphate residues such as phosphorothioates (PS), methylphosphonates, or phosphorodithionates. Furthermore, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). In addition, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. In addition, BNA (LNA), in which the conformation of the sugar portion of the nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be used.

[0038] 2. Plant Stomatal Opening Regulator The present invention relates, in one embodiment, to a plant stomatal opening regulator (which may also be referred to herein as "the plant stomatal opening regulator of the present invention") containing a CLE5 peptide and / or its expression cassette.

[0039] The CLE5 peptide is a peptide consisting of a partial sequence located in the C-terminal region of the gene product (protein) of the CLE5 (CLAVATA3 / ESR-RELATED 5) gene (Arabidopsis Information Resource (TAIR): AT2G31083) or its ortholog gene in Arabidopsis thaliana, and is secreted extracellularly. This subsequence is conserved across various species, and for example, CLE5 ortholog proteins in plants such as macadamia, Arabis alba, rapeseed, kale, cauliflower, cabbage, kohlrabi, broccoli, Chinese broccoli, ornamental cabbage, mizuna, turnip, Nozawana, komatsuna, Chinese cabbage, bok choy, long-headed jasmine, rubella, cardamine amara, clementine, garland laurel, saltrick mustard, rubber tree, rose of Sharon, calendula, wild mustard, indigo, radish, and cacao tree contain 100% identical amino acid sequences to the CLE5 peptide of Arabidopsis thaliana.

[0040] Examples of CLE5 peptide amino acid sequences include the partial amino acid sequence encoded by the Arabidopsis thaliana CLE5 gene (RVSPGGPDPQHH (SEQ ID NO: 2)) and the amino acid sequence of the major CLE5 peptide secreted from Arabidopsis thaliana CLE5 protein-expressing cells (RVSX 1 GGX 2 DPQHH(X 1 and X 2 (where is a hydroxyproline residue) (SEQ ID NO: 3) is an example.

[0041] The CLE5 peptide is preferably (a) or (b) below: (a) an amino acid sequence a shown in SEQ ID NO: 1, or (b) an amino acid sequence b in which one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence a, and is a peptide having the ability to suppress plant stomatal opening.

[0042] Sequence ID 1 is RVSX 3 GGX 4 DX 5 QHH(X 3 , X4 and X 5 is the same as or different from, and represents a proline residue or a hydroxyproline residue). In SEQ ID NO: 1, X 3 is preferably a hydroxyproline residue, and X 4 is preferably a hydroxyproline residue, and X 5 is preferably a proline residue.

[0043] The amino acid mutation in amino acid sequence b with respect to amino acid sequence a is preferably a substitution, more preferably a conservative substitution. From the viewpoint of retaining more activity, the position of the amino acid mutation is preferably any one of the second, fourth, and sixth to ninth positions from the N-terminus of amino acid sequence a (particularly, the second position from the N-terminus). The number of amino acid mutations is preferably 1 to 4, more preferably 1 to 3, still more preferably 1 to 2, and particularly preferably 1.

[0044] The ability to inhibit plant stoma opening is measured according to the method described in "(1-16) Stoma Opening Measurement" in the Examples described below. When treated with a test peptide solution at any concentration (for example, any concentration from 1 to 1000 μM), if the stoma opening rate is decreased compared to Mock, it is determined that the test peptide has the ability to inhibit plant stoma opening.

[0045] The CLE5 peptide may have other amino acid sequences added thereto other than those described above. The other amino acid sequences can be designed so as not to impair the binding between the CLE5 peptide and its receptor BAM1 / GHR1. Thereby, the ability of the CLE5 peptide to inhibit plant stoma opening can be ensured.

[0046] The number of amino acid residues of the CLE5 peptide is preferably 10 to 50, more preferably 10 to 30, still more preferably 10 to 20, even more preferably 10 to 15, particularly preferably 11 to 13, and particularly preferably 12.

[0047] The CLE5 peptide may be glycosylated as long as it has the ability to inhibit plant stoma opening.

[0048] In a preferred embodiment of the present invention, the CLE5 peptide is a glycosylated peptide. From the viewpoint of inhibiting plant stomatal opening, a glycosylation containing arabinose as a constituent monosaccharide is preferred. The number of constituent monosaccharides in the glycosylation is preferably 2 to 10, more preferably 2 to 5, even more preferably 2 to 4, and particularly preferably 3. The glycosylation is particularly preferably a glycosylation in which 2 to 5 arabinose molecules (even more preferably 2 to 4, and particularly preferably 3) are linked together. The number of glycosylation molecules is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1.

[0049] The glycosylation modification site is preferably in the central region of the CLE peptide (for example, the central amino acid residue of amino acid sequence a or b, or a total of 2 to 5 amino acid residues centered on the peptide bond). Furthermore, the glycosylation modification site is preferably a hydroxyproline residue. In a particularly preferred embodiment of the present invention in which the CLE5 peptide comprises amino acid sequence a or b, the 7th amino acid residue from the N-terminus of amino acid sequence a is preferably a hydroxyproline residue, and the glycosylation modification site is preferably the 7th hydroxyproline residue from the N-terminus of amino acid sequence a or the hydroxyproline residue at the corresponding position in amino acid sequence b. The "hydroxyproline residue at the corresponding position in amino acid sequence b" refers to the hydroxyproline residue in amino acid sequence b that is located opposite the 7th amino acid residue from the N-terminus of amino acid sequence a when amino acid sequence a and amino acid sequence b are compared using Blast's default parameters.

[0050] The CLE5 peptide may be chemically modified, insofar as it retains the ability to inhibit plant stomatal opening.

[0051] CLE5 peptide has a carboxyl group (-COOH) at the C-terminus, and a carboxylate (-COO ‐ It may be any of the following: amide (-CONH2) or ester (-COOR).

[0052] Here, R in esters can be C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3－8 Cycloalkyl groups; for example, phenyl, α-naphthyl, etc. 6－12 Aryl group; for example, phenyl-C such as benzyl and phenethyl. 1－2 Alkyl groups; such as α-naphthylmethyl and α-naphthyl-C 1－2 C such as alkyl groups 7－14 Aralkyl groups and pivaloyloxymethyl groups are used.

[0053] Furthermore, CLE5 peptides have a protecting group (for example, a formyl group, an acetyl group, etc.) at the amino group of the N-terminal amino acid residue. 1－6 C such as Alkanoyl 1－6 This also includes oligopeptides protected by acyl groups, etc.

[0054] CLE5 peptide may also be in the form of an agronomically acceptable salt with an acid or a base. The salt is not particularly limited as long as it is an agronomically acceptable salt, and either an acidic salt or a basic salt can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate. Examples of basic salts include alkali metal salts such as sodium and potassium salts; as well as alkaline earth metal salts such as calcium and magnesium salts; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0055] The CLE5 peptide may also be in the form of a solvate. The solvent is not particularly limited as long as it is agriculturally acceptable, and examples include water, ethanol, glycerol, and acetic acid.

[0056] CLE5 peptides can be prepared according to known peptide synthesis methods, depending on their amino acid sequence.

[0057] The expression cassette for the CLE5 peptide is not particularly limited as long as it can produce the CLE5 peptide in plant cells, and can be, for example, a polynucleotide containing the CLE5 peptide coding sequence, or a polynucleotide containing the coding sequence of a protein containing the amino acid sequence of the CLE5 peptide (e.g., Arabidopsis thaliana CLE5 protein or its orthologous protein).

[0058] The protein containing the amino acid sequence of the CLE5 peptide is preferably at least one selected from the group consisting of protein (A) and protein (B): (A) a protein containing amino acid sequence A shown in SEQ ID NO: 4, and (B) a protein containing amino acid sequence B which has 80% or more identity with amino acid sequence A and which generates the CLE5 peptide in plant cells.

[0059] In (B) above, the identity is more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0060] The expression cassette preferably contains a promoter sequence and a peptide / protein coding sequence (and optionally, a transcription termination signal sequence). The expression cassette may also be in the form of a vector.

[0061] The promoter is not particularly limited and examples include the RPS5A promoter, UBQ promoter, CaMV35S promoter, NOS promoter, etc. Furthermore, promoters of genes expressed in a tissue-specific and / or time-specific manner can also be used. Specifically, guard cell-specific promoters such as the GC1 promoter are preferably used.

[0062] The expression cassette for the CLE5 peptide can be manufactured using genetic engineering techniques according to known methods.

[0063] The plant stomatal opening regulator of the present invention contains a CLE5 peptide and / or its expression cassette. Furthermore, by regulating stomatal opening, photosynthesis can be regulated, and even plant growth can be controlled. Therefore, the CLE5 peptide and / or its expression cassette can be used as an active ingredient in photosynthesis regulators, plant growth regulators, and the like.

[0064] In particular, CLE5 peptide and / or its expression cassette have the effect of suppressing stomatal opening in plants (especially in response to light or drugs (such as fusicoccin)) and inducing stomatal closure in plants. Furthermore, it is known that suppressing stomatal opening in plants reduces transpiration and maintains the moisture content within the plant. Therefore, CLE5 peptide and / or its expression cassette can be used as active ingredients in plant stomatal opening inhibitors, drought tolerance enhancers, plant stomatal closure inducers, and freshness preservatives. In addition, it is known that pathogenic microorganisms enter through the openings of plant stomata. Therefore, CLE5 peptide and / or its expression cassette can be used as active ingredients in disease resistance enhancers and the like.

[0065] The plants to which the plant stomatal opening regulator of the present invention can be applied are not particularly limited, as long as they are plants that have stomata. For example, it can be broadly applied to plants such as angiosperms (dicotyledonous plants, monocotyledonous plants, etc.), gymnosperms, and ferns. Specific examples include tomatoes, bell peppers, chili peppers, eggplants, cucumbers, pumpkins, melons, watermelons, vegetables such as cabbage, broccoli, and Chinese cabbage, raw vegetables or spices such as celery, parsley, and lettuce, onions, garlic, and other alliums, beans such as soybeans, peanuts, green beans, peas, and adzuki beans, other fruit vegetables such as strawberries, taproots such as radishes, turnips, carrots, and burdock, tubers such as taro, cassava, potatoes, sweet potatoes, and yams, asparagus, spinach, and Japanese parsley. Examples include soft vegetables, flowers such as lisianthus, stock, carnation, and chrysanthemum, grains such as rice, wheat, barley, oats, and corn, grasses such as bentgrass and Korean lawn grass, oil crops such as rapeseed and peanuts, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, fodder crops such as clover, sorghum, and dent corn, deciduous fruit trees such as apples, pears, grapes, and peaches, citrus fruits such as Satsuma mandarins, lemons, and grapefruits, and woody plants such as azaleas, rhododendrons, and cedars.

[0066] The plant stomatal opening regulator of the present invention may consist only of the above-mentioned agent, but may also contain various additives in addition to the above-mentioned agent, depending on the dosage form, application method, etc., as described later. The content ratio of the above-mentioned agent in the plant stomatal opening regulator can be appropriately determined depending on the dosage form, application method, etc., as described later, but for example, a range of 0.0001 to 100% by mass can be exemplified. As a more specific example, when the plant stomatal opening regulator of the present invention, which is a liquid formulation, is brought into contact with the stomata, the content ratio of the above-mentioned agent is exemplified as 1 to 500 μM, preferably 10 to 200 μM, and more preferably about 50 to 150 μM.

[0067] The dosage form of the plant stomatal opening regulator of the present invention is not particularly limited as long as it is an agriculturally acceptable dosage form. Examples include liquid formulations, solid formulations, powder formulations, granular formulations, granular formulations, wettable powder formulations, flowable formulations, emulsion formulations, paste formulations, dispersants, and the like.

[0068] The additives are not particularly limited as long as they are agriculturally acceptable. Examples include carriers, surfactants, spreading agents, spray adjuvants, thickeners, bulking agents, binders, vitamins, antioxidants, pH adjusters, volatilization inhibitors, and pigments.

[0069] The application methods of the plant stomatal opening regulator of the present invention are not particularly limited, as long as they are known methods of using pesticides (or methods that may be developed in the future). Examples include spraying, dropping, coating, mixing or dissolving in the plant growth environment (in soil, water, solid culture medium, liquid culture medium, etc.). Since the target of the plant stomatal opening regulator of the present invention is the stomata, it is preferable to apply the plant stomatal opening regulator of the present invention by bringing it into contact with the stomata of the plant.

[0070] 3. Screening Method In one embodiment, the present invention relates to a screening method for plant stomatal opening regulators (which may be referred to herein as "the screening method of the present invention") using at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote phosphorylation of SRK2D and / or SRK2E as an indicator.

[0071] BAM1 is the gene product (protein) of the BAM1 (BARELY ANY MERISTEM 1) gene of Arabidopsis thaliana (Arabidopsis Gene Database (TAIR: The Arabidopsis Information Resource): AT3G23920) or its orthologous gene.

[0072] BAM1 is preferably at least one selected from the group consisting of protein (C) and protein (D): (C) a protein containing amino acid sequence C shown in Sequence ID No. 5, and (D) a protein containing amino acid sequence D having 80% or more identity with amino acid sequence C and having CLE5 peptide binding ability.

[0073] GHR1 is the gene product (protein) of the GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) gene (Arabidopsis thaliana gene database (TAIR: The Arabidopsis Information Resource): AT4G20940) or its orthologous gene in Arabidopsis thaliana.

[0074] GHR1 is preferably at least one selected from the group consisting of protein (E) and protein (F): (E) a protein containing the amino acid sequence E shown in Sequence ID No. 6, and (F) a protein containing an amino acid sequence F having 80% or more identity with the amino acid sequence E and having CLE5 peptide binding ability.

[0075] The CLE5 peptide binding capacity is measured according to the method described in "(1-18) Thermal Shift Assay (TSA)" in the Examples below.

[0076] SRK2D is the gene product (protein) of the SRK2D (SNF1-RELATED PROTEIN KINASE 2-2) gene (Arabidopsis Information Resource (TAIR): AT3G50500) of Arabidopsis thaliana, or its orthologous gene.

[0077] SRK2D is preferably at least one selected from the group consisting of protein (G) and protein (H): (G) a protein containing amino acid sequence G shown in Sequence ID No. 7, and (H) a protein containing amino acid sequence H having 80% or more identity with amino acid sequence G and having kinase activity.

[0078] SRK2E is the gene product (protein) of the SRK2E (SNF1-RELATED PROTEIN KINASE 2.6) gene (Arabidopsis Information Resource (TAIR): AT4G33950) of Arabidopsis thaliana, or its orthologous gene.

[0079] SRK2E is preferably at least one selected from the group consisting of protein (I) and protein (J): (I) a protein containing amino acid sequence I shown in SEQ ID NO: 8, and (J) a protein containing amino acid sequence J having 80% or more identity with amino acid sequence I and having kinase activity.

[0080] Kinase activity is measured according to the test method shown in Figure 3e of the examples described later.

[0081] In (D), (F), (H), and (J) above, identity is more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more.

[0082] The type of test substance is not particularly limited as long as it can act as a receptor ligand. Examples include proteins, peptides, non-peptide compounds (nucleotides, amines, carbohydrates, lipids, etc.), small organic molecules, and inorganic compounds.

[0083] The test substance can be selected as a plant stomatal opening regulator or a candidate substance for a plant stomatal opening regulator if at least one of the following conditions is met: the test substance has the ability to bind to BAM1 and / or GHR1, and the phosphorylation of SRK2D and / or SRK2E (particularly within the T-loop) is promoted when the test substance is brought into contact with plant cells (particularly guard cells).

[0084] The binding ability of the test substance to BAM1 and / or GHR1 is not particularly limited and can be evaluated according to known methods. For example, it can be evaluated according to "(1-18) Thermal Shift Assay (TSA)" or "(1-20) Isothermal Titration Calorimetry (ITC) Assay" in the examples below.

[0085] The phosphorylation-promoting ability of the test substance to SRK2D and / or SRK2E is not particularly limited and can be evaluated according to known methods. For example, it can be evaluated according to the test method shown in Figure 3a / b of the Examples described below.

[0086] In one embodiment, the present invention relates to a plant stomatal opening regulator containing a substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote the phosphorylation of SRK2D and / or SRK2E. The plant stomatal opening regulator can be a test substance in the screening method of the present invention described above, and is preferably a peptide, more preferably (a) or (b) below: (a) an amino acid sequence a represented by SEQ ID NO: 1, or (b) a peptide containing an amino acid sequence b in which one or more amino acids are substituted, deleted, added or inserted to the amino acid sequence a. The composition of the peptide and the plant stomatal opening regulator can be based on the GLE5 peptide described above, or the composition of all or any part of the plant stomatal opening regulator of the present invention.

[0087] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0088] (1) Methods (1-1) Plant materials and growth conditions Except for stomatal conductance measurements (Figures 1i, 3f, 3g, 6d), Arabidopsis thaliana was grown under long-day conditions (16 / 8-hour photoperiod) at 22°C. Nicotiana benthamiana was grown under short-day conditions (10 / 14-hour photoperiod) at 22°C and used for BiFC analysis and co-immunoprecipitation assay (see below). For analysis of intact leaf gas exchange using Arabidopsis thaliana leaves, the plants were grown under short-day conditions (10 / 14-hour photoperiod) at 22°C. All seeds were sterilized in bleach solution (10% commercial bleach, 0.02% Triton X-100) for 10 minutes, washed three times with sterile water, and then first left to stand at low temperature in the dark at 4°C for 3 days. All knockdown mutants were obtained from the Arabidopsis Biological Resource Center (http: / / www.arabidopsis.org) or GABI Kat (https: / / www.gabi-kat.de). The rbohDrbohF double mutant was provided by K. Kuchitsu of Tokyo University of Science. The bam1ghr1 and bam1bam3 double mutants were created by crossbreeding. For CLE5 induction experiments and RNA-seq analysis, 8-dag (several days after germination) seedlings containing the pMDC7-CLE5 construct were placed on 1 / 2 Murashige Skoog (1 / 2 MS) agarose plates containing either 1 μM β-estradiol (Fujifilm) or a solvent as a sample or mock, respectively. In the peptide application test, Arabidopsis thaliana seedlings were transferred to 1 / 2 MS plates containing either 1 μM CLE5 peptide (CLE5p) or 0.1 μM [Ara3]CLE5 peptide ([Ara3]CLE5p) for the instructed time.

[0089] (1-2) PCR fragments containing plasmid DNA constructs and transformation-functional CLE5, BAM1, BAM3, and GHR1 coding sequences (amino acid sequences: CLE5: SEQ ID NO: 4, BAM1: SEQ ID NO: 5, GHR1: SEQ ID NO: 6) and their promoter regions were designed based on previously reported information. For CLE5 complementarity testing, a 4kb genomic region spanning 3.2kb upstream of the translation start site and the 3' UTR (referred to as the full-length CLE5 genomic region) was cloned by PCR and used to construct the pCLE5::CLE5 transgene. To create complementary plants that specifically express CLE5 in stomatal guard cells, a 1.73kb promoter region of GC1 was amplified by PCR and inserted in frame with the CLE5 coding region into the pGWB504 vector (pGWB504::pGC1). All coding sequence PCR fragments were inserted into the pENTR / D-TOPO vector according to the manufacturer's instructions (Invitrogen). After sequencing, each clone was identified as pMDC163 (GUS reporter vector), pBGYN (EYFP+NLS tag fusion vector), R4pGWB501 (complementarity test), pGWB11 (C-terminal FLAG tag fusion vector), pGWB20 (C-terminal 10×Myc tag fusion vector), pGWB504:pGC1 (stomatal-specific expression vector), and pMDC7 (β-estradiol-inducible overexpression vector). CRISPR-cle5 mutants were generated using the CRISPR / Cas9 system as described in other previously published reports. Target sequences were determined using CRISPRdirect software. Cloning was performed according to previously published reports. Three types of 35S::SRK2-GFP constructs (35S::SRK2D (amino acid sequence is SEQ ID NO: 7)-GFP, 35S::SRK2E (amino acid sequence is SEQ ID NO: 8)-GFP, and 35S::SRK2G-GFP) were generated according to previously published reports. All constructs were confirmed by sequencing. Arabidopsis thaliana plants were transformed by flower immersion using Agrobacterium tumefaciens (C58MP90 strain).

[0090] (1-3) Site-directed mutagenesis and recombinant protein purification For in vitro protein purification, pMALc4x-SRK2E was constructed by inserting the coding region of SRK2E. Site-directed mutagenesis was performed by inverse PCR to obtain pMALc4x-SRK2E (G33R), pMALc4x-SRK2E (D140A), pMALc4x-SRK2E (G33R / D140A), and pMALc4x-SRK2E (S175A / T176A / T179A, Triple A). The kinase domain sequence of BAM1 was amplified and cloned into the pDONR221 vector using BP Clonase (Invitrogen), and then inserted into the pDEST15 binary vector using LR Clonase (Invitrogen). All recombinant proteins were batch purified according to the manufacturer's protocol (GST protein from Cytiva, MBP protein from New England Biolabs).

[0091] (1-4) In vitro phosphorylation assay and gel kinase assay. The phosphorylation assay was performed according to previously reported procedures. Kinase (0.5 μg) and substrate (1.5 μg) were mixed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 2.5 mM CaCl2, 1× protease inhibitor cocktail (Roche), 10 mM NaF, 5 mM β-glycerophosphate, and 20 μCi [γ- 32The reaction was incubated in 12.5 μl of 1× kinase buffer containing [P]ATP for 30 minutes at room temperature. The reaction was stopped by adding 4× SDS-PAGE loading buffer and heating at 95°C for 5 minutes. The protein was separated by SDS-PAGE, and phosphorylation of the substrate was detected by applying a fluorescence imaging screen to the dried gel using a Typhoon FLA9000 Phosphor Imager (GE Healthcare). In the in-gel assay, total protein was extracted from 10-day-old Arabidopsis thaliana seedlings and subjected to either no treatment (Mock) or dehydration stress (50-55% humidity) for 20 minutes. The seedlings were then finely ground in liquid nitrogen and extracted using the following buffer: 100 mM HEPES-HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 0.5% Triton X-100, 150 mM NaCl, 0.5 mM NaCl, 0.5 mM DTT, 10 mM NaF, 5 mM Na VO2 34 5 mM β-glycerophosphate and a complete ULTRA protease inhibitor cocktail (Sigma). The resulting crude extract was centrifuged at 15,300 g at 4°C for 30 minutes to remove residue. The supernatant was collected and 20 μg per sample was separated onto an SDS-PAGE polyacrylamide gel containing 200 ng of histone III (Sigma). For recombinant MBP-SRK2E protein, 0.2 μg of purified protein was loaded. The gel was incubated overnight at 4°C in buffer (25 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg / ml BSA, 0.1% (v / v) Triton X-100), and then resaturated buffer (25 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF). In-gel assays were performed according to previously reported procedures. The resulting gel was dried and analyzed using a Typhoon FLA9000 Phosphor Imager.

[0092] (1-5) Nano LC-MS / MS and Data Analysis Full-length recombinant maltose-binding protein (MBP)-SRK2E protein purified from E. coli was subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS). The in vitro phosphorylation reaction was carried out in the same kinase reaction buffer as above, except that 100 μM cold ATP was used. The samples were separated by SDS-PAGE, selected protein bands were excised from the gel, and then digested in the gel. The resulting peptides were analyzed by nano LC-MS / MS using an UltiMate 3000 Nano LC system (ThermoFisher Scientific) coupled with a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific) equipped with a nano electrospray ionization source. After injection, the peptides were trapped in a 5 × 0.3 mm inner diameter trap column packed with 5 μm C18 resin and separated over 100 minutes using a 5–40% buffer B gradient on a NANO-HPLC capillary column C18 (0.1 × 125 mm, Nikkyo Technos) at a flow rate of 500 nl / min. LC buffer A consisted of 0.5% (v / v) acetic acid in water, and LC buffer B consisted of 80% (v / v) acetonitrile and 0.5% (v / v) acetic acid. Full-scan MS spectra were collected using Orbitrap from 350 to 1800 m / z with a resolution of 70,000 and an AGC target of 3E6. In the MS / MS experiment, the 10 most intense double-charged precursors (z≧2) were accumulated to a 1E5 target value and fragmented within the collision cell by high-energy collision dissociation (HCD). The separation width of the precursors was 2.0 m / z. The normalized collision energy of the HCD was 27%. The obtained MS and MS / MS datasets were analyzed using Proteome Discoverer 2.4.1.15 (ThermoFisher Scientific).The following parameters were selected: Databases: TAIR10 Arabidopsis protein database, SRK2E protein sequence, and cRAP for contaminants (http: / / www.thegpm.org / crap / ); Enzyme: Trypsin; Max Missed Cleavage Sites: 2; Minimum Peptide Length: 6; Max Peptide Length: 144; Variable Modifications: Phosphorylation (S, T, Y); Oxidation (M); Carbamide-methyl (C); Precursor Mass Tolerance: ± 10 ppm; Fragment Mass Tolerance: ± 0.02 Da; and Precursor Abundance Based On: Intensity. Peptide identification was performed based on significant Xcorr values ​​(high-reliability filter). The probability of modification sites was calculated using the IMP-ptmRS node. The peptide identification and modification information returned from SEQUEST was filtered using the Percolator node of Proteome Discoverer with a false detection rate of 1% to obtain a list of confirmed peptide identifications and modifications from HCD MS / MS.

[0093] (1-6) Stomatal Conductance Measurement Gas exchange measurements were performed using the LI-6800 instrument (LI-COR). Sample preparation was as previously reported. Separated leaves were placed in the gas exchange chamber and equilibrated until the stomatal conductance value stabilized. The LED light source was 200 μmol m -2 s -1 (90% red and 10% blue), the gas exchange temperature was 22°C, the relative humidity in the chamber was 75%, the airflow was 200 rpm, and the CO2 gas concentration was 400 ppm. ABA treatment was performed 10 minutes after the start of recording. All data were expressed as n≧3 for each genotype and normalized by the average gsw value for the first 10 minutes.

[0094] (1-7) RNA preparation and transcriptome analysis Total RNA was isolated from Arabidopsis thaliana seedlings using the RNeasy Plant Mini kit (QIAGEN) according to the manufacturer's instructions. A cDNA library was prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. RNA-seq was performed using the Illumina NextSeq500 platform (Illumina) in single-ended mode with a read length of 75 bp. Subsequent data analysis was performed using previously reported pipelines, except for the Bowtie version (0.12.9). In this analysis, the thresholds were set to |log2FC (fold change)| ≥ 1 and padj (justified P value) < 0.05, respectively. Gene ontology enrichment analysis was performed using DAVID (https: / / david.ncifcrf.gov). For quantitative real-time PCR (RT-qPCR) analysis, 1 μg of total RNA was reverse transcribed using oligo-dT primers with Superscript III (Invitrogen), and then amplified using LightCycler SYBR Green Master (Roche) according to the manufacturer's guidelines. Expression data were normalized to the mean expression level of the reference gene UBQ10 (AT4G05320). Each assay was performed using at least three biological triplicates, yielding similar results. Error bars represent the standard deviation (sd) of replicas. All RT-qPCR primer information is shown in Supplementary Table 5c.

[0095] (1-8) Bimolecule Fluorescence Complement (BiFC) The functional coding sequences of BAM1, BAM3, and GHR1 were amplified by PCR, cloned into pENTR, and recombined into vectors pBGYN and pBGCN (Funakoshi) using Gateway LR (ThermoFisher Scientific) to create C-terminal fusion YFP constructs of each protein. These proteins were transiently expressed in N. benthamiana epidermal cells under short-day conditions (8 / 16 hours at 22°C). Agrobacterium tumefaciens (C58MP90), which had been transformed with appropriate plant expression vectors beforehand, were infiltrated into leaves according to established protocols. After incubation at 22°C for 2 days, leaf discs were excised from the infiltrated leaves and observed with an FV1200 confocal microscope (Olympus). Fluorescence signals for YFP (excitation 514 nm, emission 517-569 nm) and PI (excitation 536 nm, emission 617 nm) were detected. The BiFC assay was repeated twice on four or more plants using independent transfected strains, and similar expression patterns were obtained.

[0096] (1-9) Histochemical GUS staining GUS staining and fixation were performed according to previously reported procedures. For the dehydration assay, 10-dag seedlings were transferred to Whatman filter paper (GE Healthcare) and incubated for the indicated time. Immediately afterward, they were fixed in cold 90% acetone for 1 hour, rinsed with water, and immersed in the staining solution [1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide), 0.5 mM K3 Fe(CN)6, 0.5 mM K2 Fe(CN)6, 1% Triton X-100, 10 mM EDTA, and 100 mM NaPO4 (pH 7.2)] under vacuum for 10 minutes, followed by incubation at 37°C. Photographs were taken with Nomarski Optics (BX53; Olympus).

[0097] (1-10) Peptide structure analysis CLE5 cDNA was obtained by RT-qPCR using total RNA isolated from Arabidopsis thaliana roots and specific primers. The amplified fragment was cloned into pMDC7 using Gateway LR Clonase II (Invitrogen). This construct was introduced into wild-type Col-0 by Agrobacterium-mediated transformation. Arabidopsis thaliana seeds (~100 seeds) containing the β-estradiol-inducible CLE5 gene were directly seeded into 100 ml of B5 liquid medium containing 1% sucrose and cultured at 22°C under continuous light irradiation without shaking. After 14 days of culture, expression of the transgene was induced by adding 75 μM β-estradiol for 24 hours. Peptides were extracted from the culture medium using o-chlorophenol and precipitated with acetone as previously reported. The fraction containing all secretory peptides was prepared to 500 μl with 0.1% trifluoroacetic acid. NanoLC-MS analysis was performed using a DiNa-M splitless nanoHPLC system (KYA Technologies) connected to an LTQ Orbitrap XL mass spectrometer (ThermoFisher Scientific). 5 μl aliquots of the peptide were used. 18 The peptide was loaded onto a trap column (0.5 mm inner diameter × 1 mm cartridge; KYA Technologies) and washed with 10 μl of 0.1% formic acid. Subsequently, the peptide was eluted from this pre-column and eluted with MonoCap C 18 Separation was performed for 30 minutes at a flow rate of 500 nl / min using a gradient of 2-50% acetonitrile containing 0.1% formic acid in a fast-flow nano-column (100 μm id × 150 mm; GL Sciences). Tandem mass spectra were obtained by scanning the mass range from m / z 350 to m / z 1,500 using a data-dependent acquisition method with HCD fragmentation. Data were analyzed using the SEQUEST search engine (ThermoFisher Scientific) in Proteome Discoverer 1.3 software.

[0098] (1-11) Synthesis of [Ara3]CLE5 peptide Asp(tBu)-Pro-Gln(Trt)-His(Trt)-PEG-resin was prepared using an automated peptide synthesizer (Initiator+Alstra). N,N-diisopropylethylamine dissolved in Fmoc-[AcAra3]Hyp-OH (6.0 mg, 5.8 μmol), 1-hydroxybenzotriazole (2.7 mg, 20 μmol), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.6 mg, 20 μmol), and dried N-methylpyrrolidone (200 μl) was added to peptide-resin (25 μmol) that had been pre-swollen with dried N-methylpyrrolidone (50 μl). The mixture was stirred at room temperature for 2 hours. The peptide resin was filtered and recovered, and the remaining N-terminal amino acids were added to the peptide using the peptide synthesizer. The synthesized peptide was deprotected and cleaved from the resin using trifluoroacetic acid / water (95:5v / v) (1 ml) for 30 minutes, and precipitated with cold ether (10 ml) at -20°C for 5 minutes. The precipitate was washed twice with cold ether, dissolved in water, and lyophilized. The crude peptide was dissolved in dry methanol (3 ml) and treated with sodium methoxide (28% solution, 60 μl) at room temperature for 1 hour. HPLC purification using an amide column (TSK-gel amide-80; TOSOH) yielded analytically pure [Ara3]CLE5 peptide (3.7 mg).

[0099] (1-12) Co-immunoprecipitation experiment in N. benthamiana: Fully unfolded 6-week-old N. benthamiana leaves were co-infiltrated with Agrobacterium cultures containing appropriate combinations of 35S::GHR1-FLAG, 35S::BAM1-10xMyc, and 35S::BAM3-10xMyc plasmids. After 2 days, the leaves were harvested and homogenized with extraction buffer [20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, 2.5 mM EDTA, 0.1% Nonidet P-40, 1 mM PMSF, protease inhibitor cocktail (Sigma)]. Crude extracts were clarified by centrifugation at 15,300 g for 10 minutes at 4°C. 1 ml of the supernatant was incubated with 50 μl of anti-FLAG agarose beads (#A2220; Sigma) and rotated at 4°C for 3 hours. The immunoprecipitate was mixed with 25 μl of 4× SDS-PAGE loading dye and eluted by heating at 95°C for 5 minutes. Anti-FLAG antibody (#F1804; Sigma) and anti-cMyc antibody (#M4439; Sigma) were used for immunodetection, and HRP-conjugated anti-mouse antibody (#W4021; Promega) was used as the secondary antibody. Three independent biological replicates were performed, yielding similar results.

[0100] (1-13) As a method for LRR-RLK screening receptor discovery, the sensitivity of roots treated with a high concentration of CLE5 peptide (CLE5p) was first examined, and their growth was inhibited. LRR-RLK mutants were germinated on 1 / 2 MS agar plates for 3 days, then transferred to 1 / 2 MS agar plates containing 0.75 μM CLE5p, and the length of the primary root of each mutant was measured 5 days after peptide treatment. To compare the biological activity of two types of CLE5 peptide (CLE5p and [Ara3]CLE5p), 4-day-old wild-type seedlings were transplanted onto 1 / 2 MS agar plates containing the indicated amount of CLE5p and grown for a further 5 days. The length of the primary root was measured using ImageJ software (https: / / imagej.nih.gov / ij / ). From the candidates obtained in this initial screening, we investigated the expression patterns at the cellular level using GUS staining and eFP Browser 2.0 (https: / / bar.utoronto.ca / efp2 / Arabidopsis / Arabidopsis_eFPBrowser2.html) to narrow down the receptors expressed in stomatal guard cells, and finally targeted receptor mutants that did not cause stomatal closure upon CLE5 peptide treatment.

[0101] (1-14) Protein extraction and Western blotting of Arabidopsis thaliana. Preparation and transfection of Arabidopsis thaliana leaf protoplasts have been previously described (see http: / / genetics.mgh.harvard.edu / sheenweb / ). Protoplasts (6 × 10 4The protoplasts were transfected with 10 μg of plasmid DNA and incubated in W5 solution under continuous illumination at 21°C for 18 hours. Arabidopsis thaliana leaf protoplasts expressing 35S::SnRK2s-GFP (SRK2D, SRK2E, SRK2G) were treated with 1 μM [Ara3]CLE5p or 1 μM ABA for the time indicated, immediately frozen in liquid nitrogen, pulverized using a multi-bead shocker (QIAGEN), and dissolved in protein extraction buffer [50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.1% Triton X-100, 1 mM DTT, 10% glycerol, protease inhibitor cocktail (Merck)]. In the phosphatase treatment, proteins were extracted with extraction buffer [100 mM HEPES-KOH, pH 7.5, 10% glycerol, 0.5% PVP, protease inhibitor cocktail (Merck)] and incubated with λ-phosphatase (New England Biolabs) in and out of a phosphatase inhibitor cocktail (50 mM NaF and 20 mM NaVO3) at 30°C for 30 minutes. The resulting homogenized cell lysates were centrifuged at 15,300 g for 10 minutes at 4°C, separated using a Phos-Tag SDS-PAGE system (Fujifilm), transferred to a PVDF membrane (Bio-Rad), and blotting was performed according to the manufacturer's instructions. Western blotting was performed using anti-GFP antibody (Abcam) as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG antibody (Santa Cruz) as the secondary antibody. Immunodetection was performed using Pierce ECL detection reagent (ThermoFisher Scientific). PVDF membranes were stained with Coomassie Brilliant Blue and used as a loading control.

[0102] (1-15) Dehydration Assay and Water Loss Measurement To measure the water loss rate, the aerial parts of 3-week-old plants were detached, dried on Whatman filter paper (GE Healthcare) at 22°C under normal light conditions, and then weighed. The experiment was repeated three times independently. In the dehydration leaf assay, fully developed true leaves were detached and placed on Whatman filter paper at room temperature (50-55% humidity) for up to 2.5 hours. In the short-term dehydration stress treatment, 12-dag seedlings grown on 1 / 2 MS plates supplemented with 1% (w / v) sucrose were placed on Parafilm for 1.5 hours, and total RNA was extracted for RT-qPCR analysis. Three independent experiments were performed, and similar results were obtained.

[0103] (1-16) Stomatal opening measurement: The axial side of leaves from 3-4 week old Arabidopsis thaliana plants of each genotype was manually peeled off, and the leaves were suspended in stomatal opening buffer (5 mM mesbistrispropane, pH 6.5, 50 mM KCl, 0.1 mM CaCl2) for 2 hours under normal light conditions to induce stomatal opening. Subsequently, the samples were treated with 2 μM ABA or 1 μM [Ara3]CLE5p for 30-60 minutes. After recording the stomatal openings with an optical microscope (BX53; Olympus), the width and length of the stomata were measured using ImageJ software, and the opening ratio (width / length) was calculated. Similar results were obtained in at least two independent experiments (n > 30 / time point) for each genotype.

[0104] (1-17) Quantification of ABA Extraction and semi-purification of ABA were performed using a biological triplicate for each sample. ABA was measured using an ODS column (AQUITY UPLC BEH C 18 The quantification was performed using a 1.7 μm, 2.1 × 100 mm (Waters) sample and a superhigh performance liquid chromatography (UHPLC)-electrospray interface-quadrupole orbit trap mass spectrometer (UHPLC / Q-Exactive; ThermoFisher Scientific) as described above.

[0105] (1-18) Thermal Shift Assay (TSA) The LRR repeat coding sequences of BAM1 and GHR1 were cloned into a pET42b expression vector designed to produce an N-terminal GST fusion. The purification step followed the manufacturer's protocol (Cytiba). TSA was performed using the CFX Connect real-time detection system (Bio-Rad). Protein unfolding was monitored by measuring the fluorescence of SYPRO Orange dye (Bio-Rad). Samples contained 30–50 μM of GST fusion protein, with CLE5p concentrations varying from 0–0.5 μM, and the total volume was 20 μl. A 96-well PCR plate was used for the assay. The buffer was 137 mM NaCl, 10 mM NaH2PO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4. Each reaction was performed in double or triple layers and repeated on at least two different plates. The data was analyzed using CFX Maestro Software, and the melting temperature (Tm) value was calculated according to the manufacturer's protocol (Bio-Rad). ΔTm was obtained from the first derivative of the melting curve with and without CLE5p.

[0106] (1-19) Measurement of reactive oxygen species Histochemical detection of H2O2 in situ was performed by DAB staining as previously reported. Specifically, rosette leaves of 4-week-old Arabidopsis thaliana were treated with 100 μM CLE5p or 1 μM ABA for 2 hours and then immersed in a freshly prepared 100 μg / ml 3,3'-diaminobenzine (DAB, Sigma-Aldrich) solution in the dark for 8 hours. The stained leaves were fixed, bleached with ethanol / acetic acid / glycerol (3:1:1) solution, rinsed with distilled water, and photographed against a white background. Reactive oxygen species in stomatal guard cells were detected using H2DCF-DA (Sigma-Aldrich) as previously reported. Briefly, leaf epidermis was collected from 4-week-old Arabidopsis thaliana plants, incubated in 30 mM KCl, 10 mM MES-KOH, pH 6.15 for 3 hours, then 20 μM H2DCF-DA was added and incubated for another 30 minutes. Excess dye was removed by washing three times with distilled water. The pre-treated epidermal strips were incubated for 30 minutes with or without 1 μM [Ara3]CLE5p. Fluorescence was measured using a confocal microscope (Zeiss LSM 800 confocal laser scanning microscope) at an excitation wavelength of 488 nm and manipulated with Zen software (version 13). Fluorescence intensity of guard cells was quantified using ImageJ.

[0107] (1-20) Isothermal titration calorimetry (ITC) assay experiments were performed at 37°C using a MicroCal ITC200 calorimeter (Malvern Instruments, UK). Using a syringe, 2 μl of 1 mM CLE5p was sequentially injected into a 200 μl titration cell containing the protein sample. To ensure rapid mixing, injections were performed with a 180-second time interval between injections while maintaining a baseline output of 5 μcal / s, and the solution in the sample cell was continuously stirred at 800 rpm. Recombinant GST-BAM1 and GST-GHR1 proteins, prepared as described in the TSA, were subjected to gel filtration in titration buffer (20 mM Tris-HCl pH 7.5, 25 mM NaCl, and 5% glycerol). Changes in binding stoichiometry (N), Gibbs free energy (ΔG), enthalpy (ΔH), entropy (-TΔS), and dissociation constant (Kd) were calculated from titrations of 0.1 mM GST-BAM1 and 0.1 mM GST-GHR1 in titration buffer with 1 mM CLE5p. Only GST proteins were used as a negative control. Data were analyzed using One Set of Sites Model Fitting with the manufacturer-provided Origin software package (MicroCal ITC program).

[0108] (1-21) Docking Simulation Structural models of the leucine-rich repeat (LRR) domains of GHR1 and BAM1 were constructed using the SWISS-MODEL homology modeling server. Blind docking (without explicitly describing binding site residues) was performed using HPEPDOCK to identify the peptide binding sites for each receptor. Given a slightly modified CLE5p peptide sequence (standard proline instead of hydroxyproline at residues 4 and 7) and the LRR domain models of BAM1 and GHR1 obtained above, 1000 peptide conformations generated from the MODPEP program were rigidly docked on the receptor surface. The binding region between the modified CLE5p and the BAM1 or GHR1 LRR domain was found to be close to the homologous TDR / TDIF peptide complex interface. In the case of GHR1, more variability was observed, but some poses still indicated conserved interaction regions.

[0109] Considering the results of blind docking and the possibility that CLE peptide receptors share a conserved ligand recognition mechanism, template-based modeling was performed starting from the structure of TDR bound to TDIF peptide (TDIFp) (PDB ID: 5GIJ) to obtain a more detailed structural model of CLE5p bound to BAM1 and GHR. First, BAM1 and GHR1 were aligned to form the TDR-TDIFp complex, and the CLE5p sequence was passed through TDIFp. By placing the amino acid sequence of CLE5p in the coordinates of TDIFp, the overall structure of the peptide and its orientation to the homologous receptor binding site were maintained. Such an approach has been previously reported to identify peptides that bind to histone deacetylase (HDAC8). These initial coarse-grained CLE5p-LRR domain models were energy-minimized using the Rosetta FlexPepDock refinement protocol and purified as designed when a coarse-grained model of the complex was available. In this process, the peptide backbone of the receptor protein and its rigid orientation were optimized along with the orientation of the peptide and receptor side chains. Minimization of the receptor backbone was also performed during the refinement of the peptide-receptor interface. This procedure began with low-resolution refinement (centroid mode), generating 500 unique models. Subsequently, the top models were further refined at higher resolution, creating another 500 models. All refinements were performed using the Rosetta Energy Function from the Rosetta modelling suite (v3.13).

[0110] To construct a BAM1-GHR1 complex model, we performed blind docking (without explicitly describing receptor binding site residues) of BAM1 and GHR1 using HDOCK, and optimized the complex using FlexPepDock. The BAM1-GHR1 complex protein-protein docking demonstrated that the peptide-binding region of BAM1 forms a protein-protein interface with GHR1. Therefore, to obtain a model of the BAM1-GHR1 complex bound to CLE5p, we first aligned BAM1 (complexed with GHR1) to a TDR bound to TDIFp (PDB ID: 5GIJ). The CLE5p sequence was passed through the TDIFp coordinate system, and then the energy of the side-chain positions of the interface residues between the BAM1-GHR1 complex and CLE5p was minimized. Optimization was performed using the FlexPepDock protocol. For each FlexPepDock refinement protocol, the models were ranked using Rosetta Energy Unit (REU) interface scores. This score is calculated by subtracting the monomer energy from the complex energy. A lower interface score indicates a better and more stable peptide receptor complex.

[0111] (1-22) Statistical Analysis and Reproducibility The center line of the box plot superimposed on the dot plot indicates the median of the sample, minimum value, maximum value, first quartile (25 th Percentile) and third quartile (75 th Percentiles are also shown. Standard error bars are shown in the violin plot overlaid on the dot plot. Statistical significance was calculated using R or Microsoft Excel 2016 with a two-tailed Student's t-test, or a Tukey Honest Significant Differences test following one-way or two-way ANOVA analysis. P-values, adjusted P-values ​​(Padj), or differences are indicated in the figures. All samples were randomly assigned to experimental groups, and all experiments were blinded during data acquisition and analysis.

[0112] (1-23) Data and material acquisition: Sequence data are listed in the GenBank / EMBL data library under the following accession numbers: NCED3, AT3G1440; RD29B, AT5G52300; RAB18, AT5G66400; ABI1, AT4G26080; CLE5, AT2G31083; BAM1, AT3G23920; BAM3, AT4G17090; GHR1, AT4G20940; SRK2D / SnRK2.2, AT3G50500; SRK2E / SnRK2.6 / OST1, AT4G33950; SRK2G / SnRK2.1, AT5G08590. Data other than RNA-seq and LC-MS / MS data are included in the main text or supplementary materials. RNA-seq data were deposited in DDBJ under accession number DRA011039. LC-MS / MS data created in this study were deposited in the jPOST repository database (Project ID, JPST002362, Accession ID, PXD046831).

[0113] (2) Results (2-1) CLE5 is a peptide signaling module necessary for rapid drought response and is widely used for signal transduction in animals. In plants, the CLAVATA3 / ENDOSPERM SURROUNDING REGION (CLE) peptide and its LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASE (LRR-RLK) receptor were originally identified as key players in development. Recently, it was discovered that the root-derived CLE peptide CLE25p is transported from the roots to the leaves and promotes drought stress resistance by stimulating ABA biosynthesis. To investigate whether the CLE peptide is involved in rapid drought response, various CLE-deficient mutants were subjected to short-term dehydration stress and their susceptibility was evaluated. Of these lines, the leaves of the CLE5-transformed DNA insertion type (cle5-1) and its CRISPR / CAS-9 (CRISPR-cle5) loss-of-function mutant showed significant water loss within 1 hour, but stomatal density and stomatal index were comparable to those of wild-type leaves (Figure 1a,b, and Figure 5). CLE5 was widely expressed in the leaves, and its expression rapidly increased in guard cells within 30 minutes after dehydration stress (Figure 1c,d). The drought-sensitive phenotype shown in the cle5 mutant was completely complemented by introducing a CLE5 genome fragment (Figure 1a,b).

[0114] To explore the key gene regulatory network underlying drought resistance mediated by CLE5, we constructed a CLE5 overexpression system (CLE5ox) that could be induced using a β-estradiol promoter and performed RNA-sequencing (RNA-seq) analysis. CLE5 induction significantly upregulated the expression of 143 genes (including CLE5), and these genes were enriched in the ABA response and water depletion categories of Gene Ontology (Figure 1e,f). Both RNA-seq and quantitative real-time PCR (qRT-PCR) analysis showed that CLE5 induction upregulated the expression of drought-inducible ABA-responsive genes, including RESPONSIVE TO DESICCATION 29B (RD29B) and RESPONSIVE TO ABA 18 (RAB18; Figure 1e). Interestingly, genes involved in ABA biosynthesis (9-CIS-EPOXYCAROTENOID DIOXYGENASE 3; NCED3 and NGATHA1), genes involved in sensation (ABA-INSENSITIVE 1; ABI1), and other stress-induced ABA response genes (SALT OVERLY SENSITIVE 1; SOS1 and SALT OVERLY SENSITIVE 2; SOS2 (salt stress), (DEHYDRATION RESPONSE ELEMENT B1A; DREB1A (osmotic and cold stress)) were largely unaffected by CLE5 overexpression (Figure 1g). Consistent with these transcriptome data, endogenous ABA levels in the induced CLE5ox leaves were similar to those in the mock control (Figure 1h).

[0115] (2-2) CLE5p acts as a local signal and induces stomatal closure. Since CLE5 expression is upregulated in guard cells by dehydration stress, we investigated whether stomatal movement is affected by CLE5p. To elucidate CLE5p-mediated signaling in leaves, we clarified the characteristics of the mature form of CLE5p in plants. Structural analysis of secreted CLE5p using nanoscale liquid chromatography and tandem mass spectrometry (nano LC-MS / MS) revealed that the highest elution peak was observed at 798.4 m / z, suggesting that the CLE5 peptide ([Ara3]CLE5p) consisting of 12 amino acids (SEQ ID NO: 3) triarabinosylated at the 7th hydroxyproline residue is the major form (Figure 6a). Since triarabinosylated CLAVATA3 peptide (CLV3p), the closest paralog of CLE5p, exhibits higher biological activity than the non-arabinosylated form, we evaluated the activity of CLE5p and [Ara3]CLE5p by comparing stomatal responses to the application of these peptides. [Ara3]CLE5p required approximately 100 times lower concentrations than CLE5p to close stomata, indicating that in nature, [Ara3]CLE5p has higher biological activity in inducing stomatal closure (Figure 6b). Furthermore, application of [Ara3]CLE5p to wild-type and cle5 mutant leaves induced stomatal closure (Figure 6c). Since ABA has been shown to induce rapid stomatal closure, we investigated stomatal conductance upon ABA treatment in cle5-1 and tissue-specific CLE5-expressing leaves (Figures 1i and 6d). Wild-type and complementary pCLE5::CLE5 / cle5-1 leaves began to close their stomata rapidly after ABA treatment, while cle5-1 mutant leaves were insensitive to ABA treatment (Figure 1i). Interestingly, stomatal-specific induction of CLE5 (pGC1::CLE5 / cle5-1) was sufficient to induce ABA-induced stomatal closure (Figure 1i).

[0116] Combined with previous RNA-seq analyses, this suggests that CLE5p acts as a local signaler, inducing stomatal closure without stimulating ABA biosynthesis or ABA sensation, and altering the expression of drought-induced ABA-responsive genes. This mechanism differs from that of root-derived CLE25p, which induces stomatal closure by regulating ABA accumulation.

[0117] (2-3) Stomatal closure mediated by CLE5p is independent of reactive oxygen species production. Overexpression of CLE5 also increased the expression of genes that respond to oxidative stress (Figure 1f), and since reactive oxygen species (ROS) are important signals involved in the control of stomatal movement, we investigated whether stomatal closure by CLE5p requires a ROS-related pathway. First, when detached leaves treated with CLE5p were stained with 3,3′-diaminobenzidine (DAB), no accumulation of hydrogen peroxide (H2O2) was observed, but ABA treatment induced the accumulation of H2O2. Next, the endogenous oxidizing agent levels in living guard cells were quantified using a dye called CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate). No significant difference in fluorescence intensity was observed between wild-type and cle5-1 mutant cells, or during stomatal closure induced by CLE5p treatment, suggesting that CLE5p does not stimulate reactive oxygen species levels in guard cells. Furthermore, stomatal closure was also induced by CLE5p treatment in leaves of the RESPIRATORY BURST OXIDASE HOMOLOG D and F (rbohDrbohF) double mutant, which is known to be deficient in NADPH oxidase in guard cells. Taken together, these results indicate that CLE5p-mediated stomatal closure does not involve reactive oxygen species production in guard cells.

[0118] (2-4) BAM1-GHR1 is the CLE5p receptor for guard cells. To identify the receptor that senses CLE5p in guard cells, the LRR-RLK mutant pool was screened by exogenously applying CLE5p to mutant seedlings. Since most CLE peptides have been shown to induce root growth inhibition, mutants that are not sensitive to growth inhibition by prolonged exposure to CLE5p were first looked for. Furthermore, insensitive mutants with functional deficiencies in the BARELY ANY MERISTEM 1 (BAM1) and GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1) genes were selected based on the expression of these genes in stomatal cells. Neither single-gene mutants of bam1 and ghr1 nor double mutants of bam1ghr1 showed growth impairment upon prolonged treatment with CLE5p.

[0119] Since LRR-RLK activation typically relies on ligand-induced heterodimerization with shape-complementary coreceptors via its ectodomain, we investigated whether CLE5p, BAM1, and GHR1 form a complex. First, co-immunoprecipitation and bimolecular fluorescence complementation assays were performed using full-length BAM1 and GHR1 proteins. The results revealed that in plants, GHR1 interacts with BAM1 but not with BAM3. This molecular analysis indicated that bam3 seedlings were sensitive to long-term treatment with CLE5p, similar to the wild type.

[0120] Next, a thermal shift assay (TSA) was performed to investigate the interaction between CLE5p and BAM1 or GHR1. Compared to a GST (glutathione-S-transferase) control, the melting temperatures of the BAM1-CLE5p and GHR1-CLE5p combinations shifted significantly in a dose-dependent manner for CLE5p, with a higher shift observed for the BAM1-CLE5p combination. Furthermore, isothermal titration calorimetry (ITC) assays and computational modeling were used to investigate the binding affinity of CLE5p to the BAM1 and GHR1 receptors. ITC analysis showed that CLE5p binds to both receptors, with dissociation constants (Kd) values ​​of 2.52 μM for BAM1 and 7.39 μM for GHR1 (Figure 2a, b). The thermodynamic parameters, the enthalpy factor (ΔH) and entropy factor (-TΔS), were similar between BAM1 and GHR1, suggesting a similar binding mechanism despite differences in affinity. Computer docking results showed that the interface score of CLE5p was lower when it complexed with BAM1 (-60.5 REU) than when it complexed with GHR1 (-45.2 REU). Combined with the TSA results, these data suggest that CLE5p has a high binding affinity to BAM1.

[0121] Next, we extrapolated [Ara3]CLE5p to the leaves of wild-type and bam1ghr1 mutants to evaluate whether the BAM1-GHR1-CLE5p signaling pathway is necessary for stomatal closure. When [Ara3]CLE5p was applied, stomatal closure was induced in wild-type leaves and the expression level of RD29B increased, but this did not occur in bam1ghr1 leaves (Figure 2c,d). As previously mentioned, overexpression of CLE5 did not alter the expression of genes involved in ABA biosynthesis or the endogenous ABA content of the leaves (Figure 1e-h). Consistent with this, the expression level of NCED3 was not affected by [Ara3]CLE5p treatment (Figure 2d). From these results, it became clear that the BAM1-GHR1-CLE5p signaling pathway plays a role in activating stomatal closure without promoting ABA biosynthesis.

[0122] (2-5) The BAM1-GHR1-CLE5p module activates SRK2E. To further elucidate the CLE5p signaling pathway, we searched for downstream components that could be phosphorylated by the BAM1-GHR1-CLE5p complex. Unlike other catalytic LRR-RLKs (including BAM1), GHR1 is a pseudokinase because it lacks a C-terminal cytoplasmic kinase domain. We hypothesized that the stomatal BAM1-GHR1-CLE5p complex phosphorylates SnRK2 kinase. To investigate this, we treated Arabidopsis thaliana leaf protoplasts transfected with 35S::SRK2E-GFP and 35S::SRK2D-GFP, which are involved in desiccation-induced ABA signaling, with [Ara3]CLE5p for up to 30 minutes and performed a protein mobility shift assay. In both cases, a clear band shift was detected in [Ara3]CLE5p or ABA-treated wild-type protoplasts, and this band was reduced by phosphatase treatment (Figure 3a,b). On the other hand, no such mobility shift was observed in bam1ghr1 protoplasts (Figure 3a,b). These results indicate that the BAM1-GHR1-CLE5p module mediates the phosphorylation of SRK2D and SRK2E, which are important regulators involved in drought-induced ABA signaling.

[0123] Since phosphorylation of SnRK2 kinase within the T-loop is known to be essential for its activation, LC-MS / MS analysis using recombinant BAM1 and GST protein was performed to identify the phosphorylation site of SRK2E targeted by the BAM1-GHR1-CLE5p module. The GST fusion kinase domain of BAM1 (hereinafter, GST-BAM1(KD)) phosphorylated residues S175, T176, and T179 located within the T-loop of SRK2E (from D160 to E184; Figure 3c). Previous studies have shown that the ATP-γ-phosphate proton receptor site of Asp140 is extremely important for SRK2E activity, but the Gly-to-Arg G33R mutation suppresses its activity to the level of kinase death. As shown in Figure 3d, GST-BAM1(KD) effectively phosphorylated these SRK2E kinase death mutants (D140A and G33R / D140A), but the phosphorylation signal was reduced when an SRK2E mutant on the S175A / T176A / T179A site (hereinafter referred to as SRK2E(Triple A)) was used. Furthermore, when the kinase activity of BAM1 was examined using an intragel kinase assay, GST-BAM1(KD) significantly enhanced the activity of maltose-binding protein (MBP)-SRK2E, but MBP-SRK2E(Triple A) showed no detectable activity, similar to inactive SRK2E(D140A) (Figure 3e). Taken together, these findings suggest that the cytoplasmic kinase domain of the BAM1 protein directly activates the SRK2E kinase by phosphorylating three residues (S175 / T176 / T179) within the activated T-loop.

[0124] To clarify the in vivo physiological significance of direct phosphorylation of SRK2E by BAM1, we next investigated whether phosphorylation of SRK2E by BAM1-GHR1-CLE5p is necessary for rapid stomatal closure in vivo. First, stomatal conductance was monitored in leaves of bam1, ghr1, and bam1ghr1 mutants treated with 2 μM ABA (Figure 3f). In contrast to the wild type, and similar to the cle5 mutant (Figure 6d), all mutant leaves tested were insensitive to the decrease in stomatal conductance caused by ABA, suggesting that the BAM1-GHR1-CLE5p module is involved in ABA-induced stomatal closure (Figure 3f). Next, stomatal conductance was monitored in leaves of 35S::SRK2E / srk2e and 35S::SRK2E(Triple A) / srk2e after treatment with 2 μM ABA (Figure 3g). Compared to the steady-state stomatal conductance of ABA-treated loss-of-function srk2e leaves, the stomatal conductance of 35S::SRK2E / srk2e leaves decreased rapidly. This indicates that the expression of 35S::SRK2E in srk2e leaves completely complements the function of SRK2E in mediating stomatal movement. On the other hand, the stomatal conductance of 35S::SRK2E(TripleA) / srk2e leaves was insensitive to ABA treatment, suggesting that BAM1-catalyzed phosphorylation is important for the function of SRK2E in rapid stomatal closure induced by ABA.

[0125] (2-6) The BAM1-GHR1-CLE5p / SRK2E module controls rapid drought response. We showed that CLE5p is required for the leaf drought stress response, and that the BAM1-GHR1-CLE5p module induces stomatal closure without altering the expression of major genes involved in ABA biosynthesis (Figures 1g and 2d). Furthermore, in the response to dehydration, stomatal closure is achieved before ABA accumulation is promoted. Therefore, we investigated whether ABA and orthodox ABA signaling are required for stomatal closure mediated by BAM1-GHR1-CLE5p / SRK2E.

[0126] First, dehydration assays were performed on the leaves of bam1, ghr1, bam1ghr1, and bam1ghr1cle5-1 mutants. The leaves of these mutants showed significantly greater water loss than the wild type, similar to the leaves of cle5 (Figure 1a,b) (Figure 4a). Next, changes in ABA levels during drought were measured in the shoots of cle5-1 and bam1ghr1. Endogenous ABA content in the receptor mutants was similar to that of the wild type and cle5-1 (Figure 4b). Exposure of these plants to drought significantly induced both ABA accumulation and the expression of the ABA biosynthesis gene (NCED3) (Figure 4b). However, induction of dehydration stress marker genes RD29B and RAB18 was observed only in wild-type leaves. Furthermore, administration of ABA to the leaves of cle5-1 and bam1ghr1 mutants did not induce stomatal closure. These results suggest that ABA accumulation cannot mask the BAM1-GHR1-CLE5p-mediated drought response.

[0127] Consistent with previous findings that the srk2e mutant cannot withstand a rapid decrease in humidity, SRK2E was found to be activated even under short-term dehydration (<30 minutes), where ABA accumulation is thought to be insufficient (Figure 4c). Therefore, we investigated whether SRK2E or ABA biosynthesis is necessary for the drought response acting on the CLE5p signal. We found that stomatal closure occurred within 30 minutes of spraying [Ara3]CLE5p in the leaves of wild-type and nced3 mutants, but not in the leaves of the srk2e mutant (Figure 4d). These results suggest that SRK2E activity, rather than ABA biosynthesis, is necessary for stomatal closure triggered by the CLE5p signal.

[0128] In the normal ABA pathway, PP2C members of group A physically interact with SnRK2 to inactivate its kinase activity and suppress the downstream ABA response. This suppression is counteracted by ABA-responsive PYR / PYL / RCAR ABA receptors. However, ABA accumulation and ABA biosynthesis gene expression are not stimulated by CLE5p (Figure 1g, 1h, 2d, 4b), and the BAM1-GHR1-CLE5p module activated the expression of SRK2E and a subset of drought-responsive genes (Figure 2d, 3a,c,e). Therefore, we evaluated whether ABA recognition is necessary for the action of the BAM1-GHR1-CLE5p module by applying [Ara3]CLE5p to the leaves of the abi1-1 mutant, in which SnRK2s are constitutively inactivated by the G180D mutation in the ABI1 protein. The results showed that [Ara3]CLE5p treatment partially masked the dominant-negative role of the abi1-1 protein in ABA perception and activated drought response gene expression (Figure 4e). This suggests that the BAM1-GHR1-CLE5p module induces the drought response by bypassing ABI1-mediated ABA perception (Figure 4f).

Claims

1. A plant stomatal opening regulator containing CLE5 peptide and / or its expression cassette.

2. The plant stomatal opening regulator according to claim 1, wherein the CLE5 peptide comprises (a) or (b) below: (a) an amino acid sequence a shown in Sequence ID No. 1, or (b) an amino acid sequence b in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence a, and is a peptide having the ability to suppress plant stomatal opening.

3. The plant stomatal opening regulator according to claim 1, wherein the CLE5 peptide is a glycosylated peptide.

4. The plant stomatal opening regulator according to claim 3, wherein the sugar chain is a sugar chain containing arabinose as a constituent monosaccharide.

5. The plant stomatal opening regulator according to claim 4, wherein the sugar chain (arabinose) is formed by linking together 2 to 5 units.

6. The plant stomatal opening regulator according to claim 2, wherein the CLE5 peptide is a peptide modified with a sugar chain consisting of 2 to 5 arabinose molecules linked together, the 7th amino acid residue from the N-terminus of amino acid sequence a is a hydroxyproline residue, and the modification site of the sugar chain is the 7th hydroxyproline residue from the N-terminus of amino acid sequence a or the hydroxyproline residue at the corresponding position in amino acid sequence b.

7. A plant stomatal opening regulator according to any one of claims 1 to 6, which is a plant stomatal opening inhibitor.

8. A drought-tolerance enhancer containing a plant stomatal opening regulator according to any one of claims 1 to 6.

9. A method for improving drought tolerance, comprising applying a plant stomatal opening regulator according to any one of claims 1 to 6 to a plant.

10. The method for improving drought tolerance according to claim 9, wherein the CLE5 peptide is applied so as to come into contact with the stomata of a plant.

11. A screening method for plant stomatal opening regulators, using at least one indicator selected from the group consisting of binding ability to BAM1 and / or GHR1 and phosphorylation-promoting ability of SRK2D and / or SRK2E.

12. A plant stomatal opening regulator containing a substance having at least one selected from the group consisting of the ability to bind to BAM1 and / or GHR1 and the ability to promote the phosphorylation of SRK2D and / or SRK2E.

13. The plant stomatal opening regulator according to claim 12, wherein the substance is a peptide.

Images