UV-B activated uvr8-pyr1 receptor elements improve plant drought resistance and its application

Abstract

This invention provides a UV-B-activated UVR8-PYR1 receptor element for enhancing plant drought resistance and its application. It reveals a novel photosignal element involved in plant drought resistance: the UVR8-PYR1 photosignal element, in which UVR8 and PYR1 can interact; wherein UVR8-PYR1 is a positive regulator of drought resistance, and overexpression of genes in UVR8-PYR1 significantly improves plant drought resistance. The technical solution of this invention has significant implications for the genetic improvement of plant stress resistance traits.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany; more specifically, this invention relates to UV-B activation of UVR8-PYR1 receptor elements to improve plant drought resistance and its application. Background Technology

[0002] Optogenetics refers to the use of optical and genetic techniques, utilizing molecular biology, genetic engineering, electrophysiology, and other disciplines to express light-sensitive proteins in specific cells. These proteins sense light stimuli and thus control cellular responses. Plant photoreceptors are a class of light-sensitive proteins that sense and transmit light signals. Photoreceptors absorb and sense light of specific wavelengths through chromophores, causing changes in their conformation and cellular spatial positioning. They then regulate the function of their interacting proteins and gene expression, thereby transmitting light signals and mediating plant growth and development. Using plant photoreceptors as a medium, leveraging their light-sensing properties, and identifying photoreceptor interacting proteins are highly efficient strategies for manipulating cellular functions through optogenetics.

[0003] Optogenetic tools constructed using phytochromes (PHY) and cryptochromes (CRY) have been reported and widely applied. For example, in yeast cells, a light-controlled switch for regulating the transcription of specific genes has been constructed by utilizing the characteristic that red light excites the interaction between PHY and the transcription factor PIF3 (phytochrome interacting factor 3), and far-red light reverses this interaction. In mouse fibroblasts, the interaction between PHY and PIF3 enables light-controlled translocation of specific proteins, thereby affecting mammalian cell morphology. An optogenetic tool constructed using the blue light-excited interaction between cryptochrome CRY2 and CIB1 (cry2-interacting bHLH1) has applications in green algae photosynthetic hydrogen release and the study of synaptic transmission. Light-controlled RNA splicing and the regulation of plant flowering time have been achieved by utilizing the blue light-dependent interaction between CRY2 and the RNA-binding protein CIS1. A "light switch" involving the LOV2-Jα "light switch" in oat (Avena sativa) phot1 and the virus K... + The artificially synthesized blue light-activated ion channel BLINK1 was constructed by fusing Kcv phases. Its introduction into guard cells increased photo-driven Kcv activity. + Electrical conductivity promotes stomatal opening and closing under light and increases biomass under fluctuating light conditions. This study utilizes the light-responsive properties of blue light receptors and ion channels to realize the potential of optogenetics in manipulating ion flux in plant cells, effectively improving both photosynthetic and water use efficiency, and providing important theoretical and technical support for crop genetics and breeding.

[0004] Unlike PHY and CRY photoreceptors, UVR8, as a UV-B receptor, does not require an additional chromophore to sense UV-B. The dimer UVR8 relies on the tryptophan group on its protein to sense UV-B, subsequently depolymerizing to form an active monomer. This monomeric UVR8 accumulates in the cell nucleus and mediates plant growth, development, and stress adaptation under UV-B by regulating the functions of key proteins in the UV-B signaling pathway, such as COP1, HY5, transcription factors BES1, BIM1, MYB73 / 77, and WRKY36. Optogenetics tools based on the structural and localization changes of UVR8 in response to UV-B and the UV-B-dependent UVR8-COP1 interaction mechanism have been successfully applied in mammalian and yeast cells to regulate gene expression, protein localization and secretion, and chromosome loops. Utilizing the interaction between UVR8 and the brassinosteroid signaling pathway transcription factor BES1 and the characteristic of BUV inhibiting BES1 gene transcription, the regulation and application of the brassinosteroid signaling pathway in plant flavonoid synthesis and plant resistance to ultraviolet radiation have been achieved.

[0005] Although optogenetic methods utilizing far-red, red, and blue light receptors are widely used, optogenetic tools based on UV-B or UVR8 are still underdeveloped and have the following limitations: most UV-B-activated UVR8 interacting proteins are transcription factors, thus limiting the application of UV-B-UVR8 to transcriptional regulation; different wavelengths and intensities of UV-B have different effects on plants and animals; and UV-B light has weak tissue penetration, further limiting its in vivo application. Therefore, exploring the effects of UV-B on plant environmental adaptation and its application value, identifying new regulatory factors of UVR8 interaction, and constructing optogenetic tools for UV-B-UVR8-new interacting proteins are crucial to realizing the application value of UV-B. Summary of the Invention

[0006] The purpose of this invention is to provide a UV-B activated UVR8-PYR1 receptor element to improve plant drought resistance and its application.

[0007] In a first aspect of the invention, a method for improving plant drought resistance is provided, comprising enhancing the function of a plant UVR8-PYR1 photoelectric signaling element, wherein the UVR8-PYR1 photoelectric signaling element responds to a UV-B signal and activates PYR1.

[0008] In one or more embodiments, the UVR8 includes its homologs.

[0009] In one or more embodiments, the PYR1 includes its homologs.

[0010] In one or more embodiments, the drought resistance includes enhanced adaptability to drought (stress) or enhanced drought tolerance.

[0011] In one or more embodiments, under UV-B activation, in the UVR8-PYR1 optical signaling element, UVR8 and PYR1 interact to form a complex; preferably, the complex promotes the interaction between PYR1 and ABI1 and inhibits ABI1 phosphatase activity; more preferably, a region containing at least 27 aa of the C-terminus of the UVR8 protein (e.g., based on the sequence shown in SEQ ID NO:1, including 397-423 aa or 397-440 aa) promotes the interaction between PYR1 and ABI1.

[0012] In one or more embodiments, under UV-B activation, the UVR8-PYR1 photosignaling element activates SnRK2s kinase (increasing its phosphorylation level); preferably, the UVR8-PYR1 photosignaling element activates SnRK2s kinase by inhibiting the phosphatase activity of ABI1 and relieving the inhibition of SnRK2s kinase by ABI1; preferably, the activation of SnRK2s kinase promotes the expression of drought-responsive genes, promotes stomatal closure, and improves plant drought resistance; preferably, the drought-responsive genes include: DREB2A and ABF4.

[0013] In one or more embodiments, after the UVR8 interacts with PYR1 to form a complex, it aggregates in the cell nucleus.

[0014] In one or more embodiments, the UV-B (Ultraviolet B, narrowband UV-B, 311-313nm) is a narrowband UV-B; preferably 1-10 μmol nm. –2 s –1 Low-intensity UV-B.

[0015] In one or more embodiments, within a low-intensity UV-B range, the amount of UV-B is manually adjusted to regulate the functional intensity of the UVR8-PYR1 optical signal element; more preferably, the adjustment of the amount of UV-B includes: manually increasing the amount of UV-B to enhance the functional intensity of the UVR8-PYR1 optical signal element, or manually decreasing the amount of UV-B to control (weaken) the functional intensity of the UVR8-PYR1 optical signal element (to keep it within the required or optimal intensity range).

[0016] In one or more embodiments, the enhancement of the plant's UVR8-PYR1 photoelectrochemical signaling element function includes upregulating the expression or activity of UVR8 and / or PYR1, or promoting the interaction between UVR8 and PYR1 to form a complex; preferably, the upregulation of UVR8 and / or PYR1 includes: introducing the coding gene of UVR8 and / or PYR1 or an expression construct or vector containing the coding gene into the plant; performing gain-of-function mutations on UVR8 and / or PYR1, preferably, obtaining functional UVR8 and / or PYR1 by gene modification or mutation in plants with lost or reduced UVR8 and / or PYR1 function; preferably, the introduction of the coding gene of UVR8 and / or PYR1 into the plant includes introduction based on transgenic technology, or introduction based on hybridization technology.

[0017] In one or more embodiments, the introduction of hybridization technology includes: hybridizing a plant highly expressing the UVR8 and / or PYR1 genes as a parent with another plant parent; selecting offspring that highly express the UVR8 and / or PYR1 genes from the hybrid offspring, and these offspring exhibiting improved drought resistance. Preferably, the other plant parent exhibits one or more desirable traits, so that when selecting offspring, offspring exhibiting the desirable traits of the parent and showing improved drought resistance are preferred.

[0018] In another aspect of the invention, there is a use of a UVR8-PYR1 photosignaling element or its upregulated molecule for: improving plant drought resistance; wherein the UVR8-PYR1 photosignaling element responds to UV-B signals and activates PYR1; or, promotes the interaction between PYR1 and ABI1 and inhibits the phosphatase activity of ABI1; wherein, under UV-B activation, UVR8 and PYR1 interact to form a complex and exert the promoting effect; or, activates SnRK2s kinase; wherein, under UV-B activation, the UVR8-PYR1 photosignaling element activates SnRK2s kinase by inhibiting the phosphatase activity of ABI1 and relieving the inhibition of SnRK2s kinase by ABI1; preferably, the activation of SnRK2s kinase promotes the expression of drought-responsive genes and promotes stomatal closure, thereby improving plant drought resistance; preferably, the drought-responsive genes include: DREB2A and ABF4.

[0019] In one or more embodiments, the upregulated molecules of UVR8-PYR1 include (but are not limited to): exogenous UVR8 and / or PYR1 encoding genes or expression constructs or vectors containing such encoding genes; reagents for gain-of-function point mutations of UVR8 and / or PYR1; or reagents for obtaining functional UVR8 and / or PYR1 by gene modification or mutation in plants with lost or reduced UVR8 and / or PYR1 function (such as gene editing reagents, gene recombination reagents, site-directed mutagenesis reagents).

[0020] In one or more embodiments, the UVR8 protein is: (a) a protein with the amino acid sequence shown in SEQ ID NO:1; or (b) a protein derived from (a) having the function of (a) formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:1; or (c) a protein having the function of (a) with an amino acid sequence that is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; such as 98% or more or 99% or more) identical to the amino acid sequence defined in (a); or (d) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence defined in (a), or by adding a signal peptide sequence to its N end.

[0021] In one or more embodiments, the PYR1 protein is: (a) a protein with the amino acid sequence shown in SEQ ID NO:2; or (b) a protein derived from (a) having the function of (a) formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-10; more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; or (c) a protein having the function of (a) with an amino acid sequence that is 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more; e.g., 98% or more or 99% or more) identical to the amino acid sequence defined in (a); or (d) a protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence defined in (a), or by adding a signal peptide sequence to its N end.

[0022] In one or more embodiments, the ABI1 phosphatase has a GenBank accession number of CAA54383.1, and also includes its homologs.

[0023] In one or more embodiments, the SnRK2s kinase includes SnRK2.6 kinase, which is responsible for regulating stomatal closure and drought response in SnRK2s kinase subgroup II, with GenBank accession number AJ316009.1, and also includes its homologs.

[0024] In one or more embodiments, the plant is a plant containing UVR8-PYR1 (or their homologs) photosignaling elements, preferably also including ABI1 and SnRK2s kinases; preferably, the plant includes: dicotyledonous plants, monocotyledonous plants or gymnosperms; preferably, it includes (but is not limited to): cruciferous plants, grasses, mallows, solanaceous plants (etc.); or, the plant includes (but is not limited to): crops, flowering plants or forestry plants (etc.).

[0025] In one or more embodiments, the terms "upregulation," "promotion," "enhancement," or "boost" indicate a significant upregulation, promotion, enhancement, or boost, such as an upregulation, promotion, enhancement, or boost of 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90%, or higher.

[0026] In one or more embodiments, the reduction, lowering, weakening, weakening, inhibiting or downregulating means a significant reduction, lowering, weakening, weakening, inhibiting or downregulating, such as a reduction, lowering, weakening, weakening, inhibiting or downregulating by 5%, 10%, 15%, 20%, 40%, 60%, 80%, 90% or lower.

[0027] In another aspect of the invention, the use of the UVR8-PYR1 optical signaling element is provided for use as a biomarker for identifying plant drought resistance, or for targeted selection or identification of plants.

[0028] In one or more embodiments, the method for targeted selection or identification of plants includes: identifying the expression or activity of UVR8 and / or PYR1 in test plants, or analyzing the interaction between UVR8 and PYR1 under UV-B activation: if the expression of UVR8 and / or PYR1 in the test plant is higher than (significantly higher than) the average expression value of UVR8 and / or PYR1 in this type of plant, or the interaction between UVR8 and PYR1 under UV-B activation is higher than (significantly higher than) the average level of interaction in this type of plant, then it is a plant with improved drought resistance.

[0029] In another aspect of the present invention, a method for screening substances that enhance plant drought resistance is provided, comprising: (1) adding a candidate substance to a system containing UVR8 and / or PYR1; preferably, the system is under drought (stress) conditions; (2) detecting the system, observing the expression or activity of UVR8 and / or PYR1 in the system of (1), or analyzing the interaction between UVR8 and PYR1; if the candidate substance upregulates the expression or activity of UVR8 and / or PYR1, or the interaction between UVR8 and PYR1 is significantly enhanced, then the candidate substance is a substance that enhances plant drought resistance.

[0030] In one or more embodiments, the system further includes ABI1, and the method further includes: analyzing the phosphatase activity of ABI1; if its phosphatase activity decreases, it indicates that the candidate substance is a substance that can improve plant drought resistance.

[0031] In one or more embodiments, the system further includes SnRK2s kinase, and the method further includes: analyzing the activity of SnRK2s kinase (including phosphorylation level), and if its kinase activity is increased (phosphorylation level is increased), it indicates that the candidate substance is a substance that can improve plant drought resistance.

[0032] In one or more embodiments, the system further includes drought-responsive genes, and the method further includes: analyzing the drought-responsive genes; if the expression of the drought-responsive genes is promoted, it indicates that the candidate substance is a substance that improves plant drought resistance; preferably, the drought-responsive genes include: DREB2A and ABF4.

[0033] In another aspect of the invention, a separated complex is provided, the complex being a composite of UVR8 and PYR, constituting a UVR8-PYR1 optical signaling element; the complex is formed under UV-B activation; preferably, the complex further includes ABI1; the complex is used as a regulatory target for improving plant drought resistance, to prepare regulatory molecules (upregulatory molecules) for plant drought resistance, as a biomarker for identifying plant drought resistance, and as a screening target or tool for improving plant drought resistance.

[0034] In one or more embodiments, the screening method described above further includes setting up a control group and a test group to observe the differences between the candidate substances in the test group and the control group.

[0035] In one or more embodiments, the candidate substances include (but are not limited to): overexpression molecules, regulatory molecules, binding molecules (such as antibodies or ligands), small molecule compounds (such as hormones) designed for UVR8 and / or PYR1, or their upstream or downstream proteins or genes.

[0036] In one or more embodiments, the system is selected from: cell system (cell culture system), subcellular system, solution system, plant tissue system, and plant organ system.

[0037] In one or more embodiments, the method further includes: conducting further cell experiments, in vivo plant experiments, and / or transgenic experiments on the obtained potential substances to further identify substances from the candidate substances that have excellent effects in regulating plant drought resistance.

[0038] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0039] Figure 1 UV-B promotes drought resistance and stomatal closure in Arabidopsis thaliana in dependence of UVR8. (AB) UV-B promotes drought resistance in Arabidopsis thaliana via UVR8. Col-0, uvr8, UVR8-FLAG and pyrpyl1124 plants were compared with and without UV-B treatment (±UV-B, 1 μmol / m³). –2 s –1 Under short-day conditions (8h white light / 16h darkness), the plants were treated for 2 hours. Watering continued until the seedlings were 21 days old, followed by a 21-day drought treatment. After the drought treatment, the plants were rehydrated. Survival rates and phenotypic data were collected and photographed three days after rehydration. The scale bar in the figure is 3cm. Figure B shows the survival rate statistics of each genetic material after the drought-rehydration treatment in Figure A. The letters "ab" indicate significant differences. (CD) UV-B-induced stomatal closure depends on the function of UV-B-activated intranuclear UVR8. Leaves from plants grown under short-day conditions for three weeks were examined with and without UV-B (±UV-B, 5μmol / m²). –2 s –1 Three hours after treatment, stomatal opening and closing phenotypes were observed under an optical microscope. The scale bar in the figure is 26 μm. Figure D is a quantitative statistical diagram of the stomatal opening and closing phenotypes of each genetic material in Figure C. Length: width of stomata serves as an indicator of stomatal diameter; a larger value indicates that the stomata are in a closed state. The letter "ae" indicates a significant difference. (EF) ABA receptor PYR1 / PYLs positively regulate UV-B to promote stomatal closure. The scale bar in the figure is 26 μm. Figure F is a quantitative statistical diagram of the stomatal opening and closing phenotypes of each genetic material in Figure E. The letter "ad" indicates a significant difference.

[0040] Figure 2UVR8 interacts with PYR1, and UV-B promotes the accumulation of the UVR8-PYR1 complex in the cell nucleus. (A) Yeast two-hybrid assays revealed that different forms of UVR8 interact with multiple ABA receptors, including PYR1, PYL1, PYL2, PYL5, PYL8, and PYL9. (B) In vitro pull-down assays showed that HIS-UVR8 directly interacts with MBP-PYR1. (C) BiFC assays showed that UV-B promotes the interaction between UVR8 and PYR1. The UV-B treatment intensity was 3 μmol / m³. –2 s –1 The scale bar is 50 μm. (DE)BiLC experiments show that the continuously activated form of UVR8 W285A With the continuously inactivated form of UVR8 W285F Both interact with PYR1. (F) The IP experiment on nuclear enrichment showed that UV-B promoted the accumulation of the UVR8-PYR1 complex in the cell nucleus. The UV-B intensity used in the IP experiment was 3 μmol / m². –2 s –1 .

[0041] Figure 3 UV-B and UVR8 promote drought resistance and stomatal closure in Arabidopsis thaliana in a manner dependent on ABA receptors PYR1 / PYLs. (A) UV-B and UVR8 promote stomatal closure in a manner dependent on ABA receptors PYR1 / PYLs. Leaves from plants grown under short-day conditions for three weeks were subjected to UV-B exposure (±UV-B, 5 μmol / m²) and UV-B exposure. –2 s –1 Three hours after treatment, stomatal opening and closing phenotypes were observed under an optical microscope. The scale bar in the figure is 26 μm. (BC) UV-B and UVR8 promote drought resistance in Arabidopsis thaliana in a manner dependent on the ABA receptors PYR1 / PYLs. Plants of different genotypes were shown with or without UV-B (±UV-B, 1 μmol / m²). –2 s –1 Under short-day conditions (8 hours of white light / 16 hours of darkness), the plants were treated for 2 hours. Watering continued until the seedlings were 21 days old, followed by a 21-day drought treatment. After the drought treatment, the plants were rehydrated. Survival rates and phenotypic data were collected and photographed three days after rehydration. The scale bar in the figure is 3 cm. Figure C is a statistical graph showing the survival rates of each genetic material after the drought-rehydration treatment in Figure B. The letter "ac" indicates significant differences.

[0042] Figure 4UV-B and UVR8 activate ABA-responsive gene expression. (A) Venn plot results show that 462 genes are co-regulated by UV-B and ABA in a UVR8-dependent manner. The Col_UV vs uvr8_UV gene set represents UV-B response genes dependent on UVR8. The Col_WL vs Col_UV gene set represents UV-B response genes. The Col_Mock vs Col_ABA gene set represents ABA response genes. (B) Heatmap analysis shows that UV-B and ABA co-regulate the expression of 462 genes in the same way. (C) Analysis of the proportion of 462 genes upregulated or downregulated by UV-B and ABA. (D) GO analysis results show that genes upregulated by UV-B and ABA are mainly involved in regulating plant responses to abiotic stresses. (EF) qRT-PCR detection of 10-day-old seedlings treated with or without UV-B for 3 hours (±UV-B, 5 μmol m –2 s –1 Quantitative plots of DREB2A expression (E) and ABF4 expression (F) after 0.5. The letter "af" indicates a significant difference.

[0043] Figure 5 UV-B promotes PYR1-ABI1 interaction in a UVR8-dependent manner. (A) Yeast three-hybrid experiments show that UVR8 C27 Or UVR8 C44 Promotes PYR1-ABI1 interaction. (B) Yeast three-hybrid experiments showed that after treatment with MET methionine, UVR8... C27 and UVR8 C44 The tendency to promote PYR1-ABI1 interaction was significantly reduced. (CD)Co-IP experiments showed that UV-B promotes PYR1-ABI1 interaction in a UVR8-dependent manner. The black arrows indicate the PYR1 protein. The UV-B treatment intensity was 3 μmol / m³. –2 s –1 .

[0044] Figure 6 UV-B inhibits ABI1 phosphatase activity and promotes SnRK2s protein phosphorylation through the UVR8-PYR1 element. (A) UVR8 promotes the inhibitory effect of PYR1 on ABI1 phosphatase. The absorbance at 630 nm is used as an indicator of ABI1 phosphatase activity; the higher the value, the higher the ABI1 phosphatase activity. (B) UV-B promotes SnRK2s kinase phosphorylation through UVR8. OST1-FLAG seedlings grown for ten days were used as material. After 24 hours of UV-B treatment with and without UV-B, in vivo kinase IP extraction experiments were performed. The UV-B treatment intensity was 3 μmol / m³. –2 s –1Anti-pSnRK2s 171 With anti-pSnRK2s 175 Phosphorylation antibodies can recognize SnRK2s kinases phosphorylated at sites 171 and 175, and can be used to detect phosphorylation levels. (CE) mass spectrometry results showed that UV-B treatment promoted phosphorylation modification at site 171 of SnRK2E (OST1) protein. Using 10-day-old OST1-FLAG seedlings as material, in vivo kinase phosphorylation proteins were extracted after UV-B treatment with and without UV-B treatment, and the samples were then analyzed by mass spectrometry.

[0045] Figure 7 1. UV-B activates UVR8-PYR1 receptor elements to enhance plant drought resistance and its applications. (A) UV-B activates UVR8-PYR1 receptor elements to promote PYR1-ABI1 interaction, increase SnRK2s kinase phosphorylation levels, promote the expression of ABA-responsive genes such as DREB2A and ABF4, activate the ABA response, establish a defense mechanism for plants in the face of drought stress, and improve plant drought resistance. (B) UV-B-UVR8-PYR1 optogenetic tools respond to UV-B to achieve cytoplasmic-nucleus shuttle. (C) The C-terminal domain of the UVR8 protein promotes ABI1-PYR1 interaction in yeast cells to regulate reporter gene expression. GBD refers to the binding domain of the GAL4 transcription factor, and GAD refers to the activating domain of the GAL4 transcription factor. Detailed Implementation

[0046] This invention is the first to study and reveal a novel photoelectric signaling element involved in plant drought resistance: the UVR8-PYR1 photoelectric signaling element, in which UVR8 and PYR1 can interact; wherein UVR8-PYR1 is a positive regulator of drought resistance, and overexpression of genes in UVR8-PYR1 significantly improves plant drought resistance. The technical solution of this invention is of great significance for the genetic improvement of plant stress resistance traits.

[0047] the term

[0048] As used herein, the term “drought resistance” may be used interchangeably with terms such as “drought tolerance,” “drought resistance capacity,” and “adaptability to drought (stress).”

[0049] As used herein, the terms "optical signal element," "control element / path," and "signal element / path" are interchangeable.

[0050] As used herein, "plant" refers to a plant whose genome contains the UVR8-PYR1 photosignal element of this invention or a homolog (homologous gene / homologous polypeptide (protein)) of a gene / protein involved in the photosignal element. Typically, the plant also includes ABI1 and SnRK2s kinases. The plant may include monocotyledonous or polycotyledonous plants, such as cruciferous plants, grasses, and legumes; specifically, examples include grain crops such as Arabidopsis thaliana, cabbage, millet, wheat, corn, sorghum, and soybeans, and agricultural crops such as vegetables, fruits, flowers, and forage grasses. In some preferred embodiments, the plant is a crop, such as a cereal crop. Those skilled in the art should understand that the plants applicable to the technical solutions of this invention are not limited to those listed above, and suitable plants can be determined by identifying the presence of the UVR8-PYR1 photosignal element (preferably also including ABI1 and SnRK2s kinases).

[0051] As used herein, “homology” refers to a gene or protein derived from a species other than Arabidopsis thaliana that has sequence identity (such as 60-99%, for example, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%) with the UVR8 or PYR1 of the present invention.

[0052] As used herein, “upregulation” includes: promotion, overexpression, enhancement, etc., which are statistically significant or marked upregulation, promotion, enhancement, or enhancement, such as upregulation, promotion, enhancement, or enhancement of 20%, 40%, 60%, 80%, 90%, or higher.

[0053] As used herein, “downregulation” includes: weakening, reducing, lowering, inhibiting; indicating significant downregulation, weakening, lowering, inhibiting, such as downregulation, weakening, lowering, inhibiting or downregulating by 20%, 40%, 60%, 80%, 90% or lower.

[0054] As used herein, "drought resistance" refers to a plant's ability to tolerate drought conditions. Typically, the drought resistance of a test plant is determined by comparing it with a control plant under the same humidity conditions. A test plant is generally considered to have better drought resistance if it has a higher survival rate and can survive for a longer period under drought conditions.

[0055] As used herein, “high (enhanced) drought resistance” refers to a statistically significant increase in drought resistance of a plant (e.g., a modified plant) compared to the drought resistance of similar or identical plants, such as an increase in survival rate of 5%, 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0056] As used herein, “high expression or high activity” means that the expression or activity of a target gene / protein in a specific plant (e.g., a modified plant) is statistically significantly increased compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0057] As used herein, “low expression or low activity” means that the expression or activity of a target gene / protein in a particular plant (e.g., a modified plant) is statistically significantly reduced compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or less.

[0058] As used herein, “gain-of-function mutations” include: enabling the normal expression of a target protein that was previously restricted or not expressed; and in some ways, reverting bases in the target protein coding gene (e.g., reverting to a sequence identical or degenerate to the wild type) to enable the target protein to be expressed again.

[0059] As used herein and as will be understood by those skilled in the art, selecting an appropriate “control plant” is a routine part of experimental design and may include a corresponding wild-type plant or a transgenic plant without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants may also be individuals from which the transgenic plant has been lost due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0060] UVR8-PYR1 optical signal element

[0061] In this invention, the "optical signal element" is a signaling system formed by the mutual restraint or interaction of proteins or genes, which generally leads to the occurrence of certain cellular events. The UVR8-PYR1 optical signal element includes (but is not limited to): the UVR8 gene and / or its encoded protein, the PYR1 gene and / or its encoded protein.

[0062] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0063] The variant forms of the optical signaling element proteins (UVR8, PYR1) described above are also included in this invention, including (but not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the described polypeptide (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:4; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the polypeptide is also included in this invention. Peptides derived from species other than Arabidopsis thaliana that share high homology with the peptide sequence or play the same or similar role in the same or similar regulatory pathways are also included in this invention.

[0064] Homologs of the aforementioned optical signaling element genes / proteins (UVR8, PYR1) are also included in this invention. It should be understood that while this invention preferably studies the corresponding optical signaling element genes / proteins obtained from the specific species Arabidopsis thaliana, other polypeptides or genes obtained from other species that are homologous to the aforementioned optical signaling element genes / proteins (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0065] The polynucleotide (gene) encoding the polypeptide can be a natural gene from a plant, or it can be a degenerate sequence or a codon-optimized sequence.

[0066] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0067] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, including leaf disc transformation and embryonic transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0068] application

[0069] In this invention, the term "plant" refers to a plant containing UVR8-PYR1 (or their homologs) optical signaling elements. According to knowledge in the art, plants containing UVR8-PYR1 (or their homologs) optical signaling elements inherently possess the mechanism of action claimed in this invention, and can achieve the technical effects claimed in this invention.

[0070] This invention proposes that UV-B enhances plant drought resistance through the UVR8-PYR1 receptor element. This is a novel physiological phenotype of UV-B signaling regulating plant growth and development, which has promising prospects and value for practical agricultural applications.

[0071] This invention discovers that, in addition to being activated by ABA as an ABA receptor, PYR1 can be directly activated by UV-B-UVR8.

[0072] This invention discovers that the novel regulatory factor for UVR8 interaction, the ABA receptor PYR1, is different from previous transcription factors. The UVR8-PYR1 dual-receptor interaction element is an efficient strategy for the synergistic regulation of plant growth, development, and environmental adaptation by light and hormone signals.

[0073] This invention discovers that the UV-B-UVR8-PYR1 element can respond to UV-B, thereby promoting PYR1-ABI1 interaction, regulating protein activity, gene transcription, and plant adaptability to drought environments. Constructing a UV-B-UVR8-PYR1 optogenetic tool can broaden the practical application of UV-B optogenetics in plants and enhance the application value of UV-B light signals in regulating crop environmental adaptability and in actual production.

[0074] In a specific embodiment of the present invention, in order to explore the role of UV-B in plant environmental adaptation, to find new regulatory factors that interact with UVR8, to construct new UV-B optogenetic tools, it was found that low-intensity UV-B promotes plant adaptation to drought environment and improves plant drought resistance. Furthermore, using the UV-B receptor UVR8 as a bait protein, the ABA receptor PYL8 was screened through the yeast two-hybrid system, and it was found that UVR8 interacts with the ABA receptor.

[0075] UV-B activated UVR8 interacts with PYR1, promoting PYR1-ABI1 interaction and phosphorylation of downstream SnRK2s kinases, thereby activating the ABA response, promoting stomatal closure, and improving plant drought adaptability. The inventors discovered that PYR1 is not only an ABA receptor but can also be activated by UV-B. The interaction mode of the photoreceptor UVR8 and hormone receptor PYR1 represents a novel model of synergistic regulation of plant environmental adaptation by light and hormone signals. UV-B activated UVR8-PYR1 receptor elements can rapidly activate the ABA response, providing a defense mechanism for plants to resist drought stress. The construction of a UV-B-UVR8-PYR1 optogenetic tool shows promise for applications in gene expression regulation, protein activity regulation, and agricultural production.

[0076] Low-intensity UV-B regulates plant elongation, while high-intensity UV-B causes stress damage to plants and animals. This invention discovers that low-intensity narrowband UV-B improves plant drought resistance through the UVR8-PYR1 element. This is a novel physiological phenotype of UV-B regulation, different from the previous phenotypes of hypocotyl and lateral root elongation. The physiological phenotype of UV-B improving drought adaptability has higher biological significance and agricultural application value.

[0077] In this invention, PYR1, which interacts with UVR8, is identified as one of the ABA hormone receptors. The UVR8-PYR1 receptor element can respond to UV-B, thereby directly activating PYR1 by UV-B, realizing the direct activation of hormone receptors by light signals, and achieving rapid and efficient signal transmission.

[0078] The PYR1 screened in this invention is a hormone receptor protein. UV-B activates the hormone receptor PYR1, thereby activating the ABA response, so that the application of UV-B-UVR8 is not limited to transcriptional regulation.

[0079] The UV-B-UVR8-PYR1 optogenetic tool shows promise in regulating protein activity, gene transcription, and plant environmental adaptation, thus broadening the application areas of UV-B optogenetics.

[0080] UV-B light-controlled switches can be designed using UVR8-PYR1 elements to improve the adaptability of crops to drought environments in agricultural production and to efficiently utilize UV-B light signals to cultivate drought-resistant crop varieties. Preferably, UV-B light-controlled switches are designed in specific cells or tissues using UVR8-PYR1 interactions, and UV-B signals are applied to regulate stomatal closure and drought adaptation.

[0081] UV-B light-controlled switches can be designed using UVR8-PYR1 interactions to leverage receptor signal transduction functions and identify novel proteins that can be regulated by UVR8-PYR1, such as ion channels or genes that respond to abiotic stresses.

[0082] Photosensitive switches can be designed using homologous proteins of UVR8 and PYR1 in other species to realize the practical application value of UV-B signals in multiple species.

[0083] The interaction between UVR8 and PYR1 can be used to explore the changes in the conformation of the PYR1 protein caused by the C-terminus of UV-B and UVR8.

[0084] By leveraging the characteristic of UVR8-PYR1 interaction to promote PYR1-ABI1 interaction, and by controlling ABI1 phosphatase activity and SnRK2s kinase activity, target proteins and genes responding to UV-B and abiotic stresses can be identified.

[0085] UV-B light-controlled switches can be designed using UVR8-PYR1 elements to regulate the function of specific cells by modulating target proteins or target genes.

[0086] Based on the interaction between UVR8 and PYR1, various UV-B light-controlled switches can be designed to explore the specificity of UV-B in regulating other ABA receptors.

[0087] By integrating knowledge from multiple disciplines such as molecular biology, genetic engineering, genetics, and biochemistry, and combining experimental techniques such as genetic material construction, expression vector construction, phenotypic observation, interaction experiments, kinase activity assays, and gene expression assays, a UV-B light control switch based on the UVR8-PYR1 interaction can be constructed.

[0088] Based on the inventors' new findings, this invention provides a method for improving plants, the method comprising: regulating the UVR8-PYR1 photosignaling element within the plant, thereby regulating the plant's drought resistance. On one hand, a method for enhancing plant drought resistance is provided, comprising upregulating the expression or activity of UVR8 and / or PYR1, which can enhance their activity and interaction under UV-B activation. On another hand, a method for enhancing plant drought resistance is provided, comprising promoting the interaction between UVR8 and PYR1 under UV-B activation. The UVR8 and PYR1 form a complex, which promotes the interaction between PYR1 and ABI1 and inhibits the phosphatase activity of ABI1. Further, the UVR8-PYR1 photosignaling element activates SnRK2s kinase by inhibiting the phosphatase activity of ABI1 and relieving the inhibition of SnRK2s kinase by ABI1.

[0089] It should be understood that, once the function of the UVR8-PYR1 photosignaling element (optionally including its upstream and downstream genes) is known, various methods well known to those skilled in the art can be used to modulate the UVR8-PYR1 photosignaling element. For example, various methods well known to those skilled in the art can be used to enhance the expression of UVR8 and / or PYR1.

[0090] In this invention, the introduction of specific genes into plants (plant cells) includes introduction based on transgenic technology or introduction based on hybridization (breeding) technology. Transgenic technology, for example, involves transferring nucleic acid fragments into plants, recombinating them with their own genome, and then artificially selecting plants with inserted specific genes, enabling precise introduction of specific genes. Hybridization (breeding) technology, for example, involves mating individuals with different genotypes to obtain individuals with recombination of certain parental genes, from which specific genes are selected for introduction to exhibit superior traits (in this invention, this refers to improving the drought resistance of plants under arid conditions).

[0091] In this invention, the upregulators of the UVR8-PYR1 photoelectro-signaling element protein or its encoding gene include promoters, agonists, and activators. Any substance that can increase the activity of the photoelectro-signaling element protein, improve its stability, upregulate the expression of the photoelectro-signaling element gene, increase the effective duration of the photoelectro-signaling element protein, or increase the phosphorylation / activation level of various proteins can be used in this invention as substances useful for upregulating UVR8 or photoelectro-signaling elements. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0092] Having learned the functions of the upstream and downstream genes / proteins of the UVR8-PYR1 photosignal element, they can be used as molecular markers for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that regulate plant drought resistance by modulating this mechanism.

[0093] Therefore, the present invention provides a method for targeted selection or identification of plants, the method comprising: identifying the expression or sequence characteristics or interactions of UVR8-PYR1 photosignal element proteins in test plants.

[0094] This invention also provides a method for screening substances (potential substances) that enhance plant drought resistance, comprising: (1) adding candidate substances to a system containing UVR8 and / or PYR1; preferably, the system is under drought (stress) conditions; (2) detecting the system, observing the expression or activity of UVR8 and / or PYR1 in the system of (1), or analyzing the interaction between UVR8 and PYR1; if the candidate substance upregulates the expression or activity of UVR8 and / or PYR1, or the interaction between UVR8 and PYR1 is significantly enhanced, then the candidate substance is a substance that enhances plant drought resistance; preferably, the system also includes ABI1, and the method further includes: analyzing A The method further includes analyzing the phosphatase activity of BI1. If the phosphatase activity decreases, it indicates that the candidate substance is a substance that enhances plant drought resistance. Preferably, the system also includes SnRK2s kinase, and the method further includes analyzing the activity of SnRK2s kinase (including phosphorylation level). If the kinase activity increases (phosphorylation level increases), it indicates that the candidate substance is a substance that enhances plant drought resistance. Preferably, the system also includes drought response genes, and the method further includes analyzing the drought response genes. If the expression of the drought response genes is promoted, it indicates that the candidate substance is a substance that enhances plant drought resistance. Preferably, the drought response genes include DREB2A and ABF4.

[0095] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0096] Through large-scale screening, a class of potential substances that specifically act on the UVR8-PYR1 photosignal element or its upstream and downstream genes / proteins can be obtained, which have a regulatory effect on plant drought resistance traits.

[0097] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0098] Materials and methods

[0099] 1. Plant materials

[0100] In this invention, the model plants Arabidopsis thaliana and Nicotiana benthamiana were used as research materials:

[0101] (1) Wild type: Columbia-0 (Col-0).

[0102] (2) Mutants: uvr8-6 (SALK_033468) is a T-DNA insertion mutant ordered from ABRC (Arabidopsis Germplasm Resource Center, USA). The tetramutant pyr1 pyl124 (deletion of PYR1, PYL1, PYL2, and PYL4) was kindly provided by Professor Zhao Yang's research group; see Abscisic acidinhibits type 2C protein phosphatases via the PYR / PYL family of START proteins. Science. 2009; 324:1068-1071. The pentamutant uvr8 pyr1pyl124 (deletion of UVR8, PYR1, PYL1, PYL2, and PYL4) was obtained through genetic hybridization and genotyping was performed.

[0103] (3) Transgenic material 1: Nicotiana benthimiana, used for transient conversion experiments of various tobacco leaves.

[0104] (4) Genetically modified material 2:

[0105]

[0106] 2. Plant material cultivation

[0107] Planting on 1 / 2 MS plates: Sterilize all necessary experimental supplies, such as pipette tips, pipettes, and filter paper, under UV light for 20 minutes in a clean bench. Take an appropriate amount of Arabidopsis seeds and sterilize them with 75% ethanol for 5 minutes. Then, suspend them in an appropriate amount of anhydrous ethanol and transfer them from a centrifuge tube to sterilized filter paper. After drying, use a sterile toothpick to spot-plant the seeds onto a 1 / 2 MS plate, or add sterile water and use a pipette tip to spot-plant the seeds onto the 1 / 2 MS plate. The culture plates should be placed under specific conditions according to the experimental purpose and requirements.

[0108] Planting in soil: Place the required amount of seeds in a centrifuge tube, add water to soak the seeds, and treat at 4℃ for three days. Prepare the soil one day before planting: black soil:vermiculite = 1:1, with an appropriate amount of perlite. Soak the prepared soil in water containing a fungicide and insecticide until fully saturated. Sow the seeds in the soil according to the experimental purpose and requirements, and place it in an artificial climate chamber. The artificial climate chamber conditions are 22℃ long-day conditions, 16 hours of light / 8 hours of darkness. Short-day growth conditions are 22℃, 8 hours of light / 16 hours of darkness. Tobacco seed sowing is similar to Arabidopsis thaliana. Tobacco prefers heat and can be placed on the top shelf of a greenhouse for cultivation, during which thinning and watering should be carried out.

[0109] Unless otherwise stated, low-intensity narrowband UV-B with an intensity range of 1-10 μmol m² was used for UV-B treatment in the examples. –2 s –1 Specifically, it is described as a drought phenotype (1 μmol m –2 s –1 Immunoprecipitation Co-IP experiment (3 μmol / m³) –2 s –1 ), stomatal phenotype (5 μmol m –2 s –1 Transcriptome analysis and qPCR experiments (5 μmol / m³) –2 s –1 ).

[0110] 3. Observation of stomatal phenotype

[0111] First, prepare an open buffer containing 50 mM KCl and 10 mM MES-Tris at pH 6.15. Cut rosette leaves from seedlings grown under long-day conditions for three weeks from the petiole and soak them in the open buffer. Treat them separately under white light and white light plus UV-B for 3 hours each. Before observation, apply glue to one side of a coverslip, then gently wipe the water off the treated leaves with tissue paper. Place the leaves, epidermis side up, on the glued side of the coverslip, peel off the epidermis leaving only the epidermis, and then place them on a slide. After preparation, observe and photograph the slides using a Zeiss inverted microscope. Observe at least three leaves for each genotype, counting at least 50 stomata. Analyze the aspect ratio of the stomata using Digimizer software; a larger aspect ratio indicates a smaller stomatal diameter and a greater degree of stomatal closure.

[0112] 4. Observation of drought phenotypes

[0113] Before sowing, ensure that the soil in each pot is of equal weight. Then soak the soil in water containing a fungicide and insecticide. Sow the seeds after the soil is completely soaked. Maintain the same amount of watering during the growing season, and rotate the pots several times. For the first three days of drought treatment (stop watering), replenish water to ensure that the weight of each pot is consistent. Short-day seedlings grow for 21 days under conditions of two hours of UV-B treatment per day. After 21 days, stop watering, which marks the start of the drought treatment. The drought treatment lasts for 21 days. After the drought ends, rewater. Three days after rewatering, observe the phenotypic results, count the survival rate, and take photos.

[0114] 5. Extraction and genotyping of plant genomic DNA

[0115] Plant genomic DNA was extracted using the CTAB method. Genotyping was performed using polymerase chain reaction (PCR) with 2×PCR Mix from Yisheng Company. The 10 μL PCR reaction mixture consisted of: 5 μL 2×PCR Mix; 0.5 μL each of 10 μM forward and reverse primers; 100 ng DNA template; and water to a final volume of 10 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 20 sec, 57℃ annealing for 30 sec. The extension time was determined based on gene size and enzyme amplification efficiency; for 1 kb fragments, an extension at 72℃ for 30 sec was sufficient. After 35 cycles, a final extension at 72℃ for 10 min was performed. The reaction products were added to GelSafe and subjected to agarose gel electrophoresis, and images were taken using a gel imaging system.

[0116] 6. Extraction, reverse transcription, and real-time quantitative PCR of plant RNA

[0117] RNA was extracted, reverse transcribed, and then subjected to real-time quantitative PCR.

[0118] 7. Extraction and Western blot identification of plant proteins

[0119] Take one leaf from a two-week-old plant and place it in a centrifuge tube containing a steel ball. Place the sample in liquid nitrogen and shake vigorously up and down three times using a cryovial or a sample grinder to thoroughly break the sample into powder. Preheat a metal bath to 100°C. Add an appropriate amount of protein extraction buffer (4×Sample buffer) according to the sample volume. After all the sample has been added, shake the centrifuge tube to thoroughly mix the sample. Centrifuge briefly for a few seconds, then place the sample in the metal bath for 5 minutes and centrifuge at 12000 rpm for 5 minutes. Perform routine blotting analysis on the obtained protein.

[0120] 8. Prokaryotic cell-induced protein expression and purification

[0121] 8.1 Prokaryotic cell-induced protein expression

[0122] As previously described, the prokaryotic protein expression vector used was *Escherichia coli* BL21, and the heat shock transformation method was employed. The constructed plasmid was transformed into BL21 and cultured overnight at 37°C. Positive single clones were picked and placed in the corresponding antibiotic liquid LB medium, and cultured overnight at 37°C and 200 rpm. The next morning, the bacterial culture was amplified at a 1:100 ratio and cultured at 37°C and 200 rpm for approximately 3 hours. During this period, the OD600 was measured until it reached 0.4-0.6, not exceeding 0.6. At this point, 2 mL of the bacterial culture was taken and centrifuged as a control for subsequent identification. For the pCold series expression vectors (prokaryotic protein expression vectors with HIS tags), protein induction must be performed at 16°C. The bacterial culture was placed in a 16°C shaker for pre-cold induction for 30 min, and 0.5 mM IPTG inducer was added. Induction was performed at 160 rpm for at least 16 hours. For pGEX4T vectors (prokaryotic expression protein vectors with GST tags) and pMAL5c vectors (prokaryotic expression protein vectors with MBP tags), 16°C induction is not required. Once the bacterial OD reaches a suitable value, IPTG inducer can be added, and induction on a shaker at 28°C for 6 to 8 hours is sufficient. After induction, 2 mL of the induced bacterial culture is transferred to a centrifuge tube and compared with the bacterial culture collected before induction to determine if protein induction was successful. The remaining bacterial culture is collected by centrifugation in a 50 mL centrifuge tube and can be cryopreserved or used for protein purification. The obtained protein is then processed using standard methods and purified in vitro.

[0123] 9. Pull-down analysis of protein interactions

[0124] Two proteins with different tags were purified in vitro and incubated with antibody beads of a certain tag for a period of time. Then, Western blot analysis was performed to determine whether the proteins interacted. Taking HIS-UVR8 and MBP-PYR1 as examples, the concentrations of in vitro purified HIS-UVR8, MBP-tagged proteins, and MBP-PYR1 proteins were determined. The HIS-UVR8 and MBP-PYR1 combination was the experimental group, and MBP and HIS-UVR8 were the control group. The protein amount in each combination should not exceed 500 ng, as adding too much protein will produce false positives. Binding buffer was prepared, and HIS beads were used for pull-down. 30 μL of HIS beads were placed in a 1.5 mL centrifuge tube, and then 100 μL of binding buffer was added to each centrifuge tube to wash the beads. The tubes were centrifuged at 2000 rpm for 2 min at 4 °C. The amount of protein added to the binding buffer system was determined according to the concentration. The binding buffer system and beads were incubated at 4 °C for at least 1 hour. After incubation, wash buffer was prepared, and the tubes were centrifuged at 2000 rpm for 2 min at 4 °C, washed five times, and then the supernatant was removed. 30 μL of 4×Sample was added. The buffer was used as the IP sample. The amount of protein used before incubation of different combinations was taken into a new centrifuge tube, and 30 μL of 4×Sample buffer was added as the input sample. Then, Western blot was performed, and the samples were incubated with UVR8 antibody and MBP antibody, respectively. After development, the results were analyzed to determine whether the two proteins interacted.

[0125] 10. Yeast two-hybrid screening of cDNA libraries

[0126] There are five bait proteins used in yeast two-hybrid screening of cDNA libraries: pGBKT7-UVR8, pGBKT7-UVR8, and pGBKT7-UVR8. N396 pGBKT7-UVR8 C44 pGBKT7-UVR8 W285A pGBKT7-UVR8 R338A The cDNA library vector pACT2 (derived from Dr. Joe Ecker, Salk Institute, California) is used for yeast two-hybrid screening of cDNA libraries. The process involves two steps: cDNA library amplification and yeast two-hybrid screening and validation. Yeast two-hybrid validation of interactions:

[0127] Activate yeast strains: Activate yeast strains in advance before the yeast two-hybrid experiment. Yeast strains are AH109 or 190; prepare competent yeast cells; revive; perform heat shock transformation; and plate culture.

[0128] Interaction verification: After single colonies grow on a double-celled plate, pick a single colony and transfer it to a 96-well plate containing water. Set up four dilution gradients for yeast concentration, ensuring that the yeast colonies in each combination are as uniform in size as possible. Then, use a pipette to spot 5 μL onto a triple-celled or quadruple-celled plate and observe for plaque growth. If plaque growth occurs, it indicates that the two target proteins are interacting. The results should also be analyzed in conjunction with a negative control. To check whether UV-B affects the interaction, perform the same procedure on two plates, treating one plate under UV-B for 6 hours for two days, and then observe the growth.

[0129] 11. Protein co-precipitation assay (Co-IP)

[0130] (1) Sample preparation: Prepare tagged overexpression material seeds and sterilize them. Then spot them on a petri dish and incubate them in a long-day incubator for fourteen days, taking Col-0 and GFP-PYR1 as examples. If UV-B or ABA treatment is required, prepare an extra batch of material according to the experimental needs. Perform UV-B or ABA treatment on the material one day before Co-IP. The treatment time depends on the specific experimental requirements. Take 1g of each sample.

[0131] (2) Prototyping: After the sample processing is completed, take a sample in aluminum foil and place it in liquid nitrogen. Treat the prototyping container with liquid nitrogen, then transfer the sample in the aluminum foil to the corresponding prototyping container, and prototypify at 45Hz for 60 seconds. Repeat the prototyping once.

[0132] (3) Prepare the Co-IP extraction buffer.

[0133] (4) Incubate beads to enrich proteins.

[0134] (5) Wash the beans.

[0135] 12. Nucleocytoplasmic separation experiment

[0136] The nucleus-cytoplasm separation experiment is an experiment to separate the cell nucleus from the cytoplasm. 30 mL of EB1 buffer is needed for every 5-10 g of sample. After thoroughly mixing the sample with the EB1 buffer, filter the sample solution into a centrifuge tube using a two-layer microcloth filter. Either a large centrifuge tube or a small 2 mL centrifuge tube can be used. Centrifuge at 2100 g for 20 min at 4 °C, remove the supernatant, add 2 mL of EB2 buffer to each sample, collect the precipitate, mix gently to avoid bubble formation, let stand for 10 min, centrifuge at 12000 rpm for 10 min at 4 °C, repeat twice, remove the supernatant. At this point, the precipitate will appear white. Then prepare the Co-IP extraction buffer for subsequent Co-IP.

[0137] 13. Tobacco Instantaneous Conversion Experiment

[0138] Shake 2 mL of the corresponding Agrobacterium beforehand, then expand to 4-6 mL. Centrifuge at 3000 rpm for 15 min, then discard the supernatant. Resuspend the Agrobacterium in tobacco activation buffer, and then measure the OD using a spectrophotometer, ensuring the OD is between 0.8 and 1.0. Mix the experimental combinations in equal proportions, let stand in the dark for two hours, and then inject the Agrobacterium mixture into the back leaves of tobacco using a 1 mL needle-free syringe, ensuring even force. Cover the injected tobacco with a black plastic bag and treat in the dark overnight. Remove the black plastic bag the next day, and after two days of normal culture, the tobacco leaves can be harvested for experiments.

[0139] 14. Bimolecular Fluorescence Complementation Assay (BiFC)

[0140] Taking UVR8 and PYR1 as examples, their CDS sequences were constructed in vectors containing nYFP and cCFP, respectively. Then, the vector plasmids were transformed into Agrobacterium. After Agrobacterium identification, experimental and control Agrobacterium mixtures were set up, and then a tobacco transient transformation experiment was carried out. After two days of tobacco culture, the injected tobacco leaves were taken and observed under a fluorescence microscope.

[0141] 15. Bimolecular luciferase complementation assay (BiLC)

[0142] Taking UVR8 and PYR1 as examples, their CDS sequences were constructed in vectors containing nLUC and cLUC, respectively. Then, the vector plasmids were transformed into Agrobacterium. After Agrobacterium identification, experimental and control Agrobacterium mixtures were set up, and then a tobacco transient transformation experiment was carried out. After two days of tobacco culture, the injected tobacco leaves were taken, and the substrate of firefly luciferase (LUC) was injected into the leaves with a 1 mL needleless syringe. Then, the interaction signal was observed and the results were recorded by cold CCD.

[0143] 16. Colocation Analysis

[0144] Taking UVR8 and PYR1 as examples, their CDS sequences were amplified and then constructed into vectors containing fluorescent proteins, such as the vector pEARLY-104-GFP containing GFP, to obtain GFP-PYR1. This was then constructed into the mCherry vector to obtain mCherry-UVR8. The constructed vectors were transformed into Agrobacterium, identified, and then subjected to a transient tobacco transformation experiment. After two days of culture, the fluorescence signal was observed and photographed under a fluorescence microscope to analyze whether the two proteins co-localized.

[0145] 17. Detection of SnRK2s kinase phosphorylation level

[0146] The required reagents and protein extraction steps were performed according to the samples preparation section of the literature *Assessing Kinase Activity in Plants with In-Gel Kinase Assays. Methods MolBiol. 2016; 1363:189-197*. Primary antibody: The primary antibody used recognized the phosphorylation site at S175 of SnRK2s. Milk was added to the primary antibody solution (0.05 g milk per 10 mL of diluted primary antibody solution). The mixture was incubated overnight at 4°C on a shaker. Secondary antibody: The primary antibody could be reused. It was washed three times with TBST for 10 min each time. Secondary antibody dilution buffer was prepared with TBST, 0.5% milk, and rabbit antibody diluted 1:5000 (2 μL rabbit antibody per 10 mL of solution). The buffer was incubated at room temperature for 2 hours. After incubation, the buffer was washed three times with TBST for 10 min each time. Development: Developing was performed with developing solution. Exposure for 2 min was generally sufficient, but the specific settings should be adjusted according to the experimental conditions.

[0147] 18. CPRG Quantitative Experiment on Yeast Tri-Hybrid Interaction Signals

[0148] 1. Shaking culture: Select the yeast combination to be transformed as needed, pick out colonies and put them into 5 mL of the corresponding defective yeast liquid culture medium, and incubate overnight at 28°C and 200 rpm in a shaker.

[0149] 2. OD expansion measurement: The yeast culture broth that has been cultured overnight is expanded using YPDA liquid culture. During the expansion period, the OD600 value is measured to keep it between 0.5 and 0.8, and the OD values ​​of each combination should be kept as consistent as possible.

[0150] 3. Take samples and record the OD values.

[0151] 4. Reaction: Remove the sample from liquid nitrogen and place it in a 37℃ water bath for 0.5-1 min. Repeat the freeze-thaw cycle 2-3 times. After the freeze-thaw cycle, add 700 μL of buffer 2 and mix thoroughly. At this point, the solution will initially appear yellow; record the start time of the reaction. Once the solution turns red, add 3M ZnCl2 to terminate the reaction; record the termination time at this point. 20 mL buffer 2 formulation: 27.1 mg of CPRG is diluted to 20 mL with buffer 1 solution.

[0152] 5. Calculation: After terminating the reaction, centrifuge at 14000 rpm for 1 min, take 1 mL of supernatant, measure OD578 and record the value. Calculate using the formula: β-galunits=1000×OD578 / (t×V×OD600), where t represents the reaction time, and according to the experimental conditions, V=0.5.

[0153] Example 1: UV-B promotes drought resistance and stomatal closure in Arabidopsis thaliana, which depends on UVR8.

[0154] The Arabidopsis UV-B receptor UVR8 senses UV-B through its tryptophan group, and upon dissociation, the dimer depolymerizes to form the active monomer UVR8. The monomer UVR8 mediates UV-B photomorphogenesis in Arabidopsis, including hypocotyl elongation, lateral root elongation, stomatal closure, and UV-B tolerance, by regulating factors such as COP1-HY5, BES1 / BIM1, WRKY36, MYB73 / 77, and MYB13. Based on existing research, the inventors considered whether UV-B regulates plant adaptation to drought and whether new regulatory factors are involved in the UV-B signaling pathway to regulate plant responses to UV-B.

[0155] Drought phenotypic observation experiments revealed that wild-type Col-0 and UVR8-overexpressing transgenic plants treated with UV-B showed stronger drought tolerance and higher survival rates compared to plants without UV-B treatment. The uvr8 mutant lacked this response, indicating that UV-B promotes drought resistance in Arabidopsis thaliana through UVR8. Figure 1 A, B).

[0156] UV-B promotes the depolymerization of UVR8 dimers to form active monomers, which accumulate in the cell nucleus. Using GR-YFP-UVR8 / uvr8 transgenic plants (GR is a glucocorticoid receptor located in the cytoplasm; DEX treatment promotes the localization of the GR receptor to the nucleus) as material, stomatal phenotype observation experiments were conducted. It was found that the stomata of GR-YFP-UVR8 / uvr8 transgenic plants only showed a reversal of the UV-B response in the uvr8 mutant stomata after UV-B treatment and DEX treatment, exhibiting a stomatal closure phenotype. This indicates that UV-B-induced stomatal closure depends on UV-B-activated UVR8 in the cell nucleus. Figure 1 (C, D). The UVR8 protein responds to UV-B by converting from a dimer to a monomer and accumulates in the cell nucleus. Developing UV-B optogenetic tools using the UVR8 protein to regulate plant drought resistance and stomatal closure has promising applications in agricultural production.

[0157] It is known that ABA hormones play an important role in regulating stomatal closure and drought adaptation in plants. Through drought and stomatal experiments, the inventors also found that the ABA receptor tetramutator pyr1 pyl124 loses its UV-B response in stomatal and drought adaptation. This indicates that the ABA receptor PYR1 / PYLs, as positive regulators, participate in the physiological processes by which UV-B and UVR8 promote drought resistance and stomatal closure in Arabidopsis thaliana. Figure 1 (A, B, E, F).

[0158] Example 2: UVR8 directly interacts with the PYR1 receptor that regulates stomatal closure and drought adaptation, and UV-B promotes the accumulation of the UVR8-PYR1 complex in the cell nucleus.

[0159] To explore the regulatory factors involved in UV-B regulation of plant drought adaptation and stomatal closure, and to find novel proteins interacting with UVR8, we investigated pGBKT7-UVR8 and pGBKT7-UVR8... N396 (396 amino acids at the N-terminus of the UVR8 protein), pGBKT7-UVR8 C44 (44 amino acids at the C-terminus of the UVR8 protein), pGBKT7-UVR8 W285A and pGBKT7-UVR8 R338A Two persistently activated UVR8 mutants, with W / R to A mutations at amino acid positions 285 and 338 of the UVR8 sequence respectively, were used as bait proteins in a yeast two-hybrid screening cDNA library. The UVR8 interacting protein, the ABA receptor PYL8, was obtained through screening. Arabidopsis thaliana has 13 ABA receptors, PYR1 / PYL1-PYL12, which are functionally diverse and redundant. Yeast two-hybrid assays revealed that UVR8 can interact with multiple ABA receptors (…). Figure 2 A).

[0160] PYR1 is a key factor in the regulation of stomatal opening and closing in plants by light and ABA signals. MBP-PYR1 protein with an MBP tag and HIS-UVR8 protein with an HIS tag were purified in vitro and subjected to in vitro pull-down experiments. These experiments revealed that UVR8 and PYR1 directly interact in vitro. Figure 2 B).

[0161] Combine nYFP-PYR1 with UVR8-cCFP, UVR8 W285A -cCFP、UVR8 W285F GV3101 bacterial culture transformed with the -cCFP plasmid was transiently transformed into tobacco leaves for BiFC experiments. PYR1-nLUC, cLUC-UVR8, and cLUC-UVR8 were used in the experiments. W285A cLUC-UVR8 W285F BiLC experiments were conducted on tobacco leaves after transient transformation of GV3101 bacterial suspensions transformed with plasmids. It was found that regardless of the continuously activated form of monomeric UVR8... W285A Or the continuously inactivated form of the dimer UVR8 W285F Both interact with PYR1, and UV-B promotes enhanced UVR8-PYR1 interaction signaling in the cell nucleus, suggesting that UV-B promotes the formation of the UVR8-PYR1 complex. Figure 2 CE).

[0162] UVR8 protein accumulates in the cell nucleus in response to UV-B. Nuclei were extracted for intracellular protein infiltration (IP) experiments. GFP-PYR1 protein, obtained via IP from UVR8 beads, also accumulated in the cell nucleus, indicating that UV-B promotes the accumulation of the UVR8-PYR1 complex in the cell nucleus. Figure 2 F). The above results demonstrate that UVR8 and PYR1 interact directly, and that UV-B promotes the accumulation of the UVR8-PYR1 complex in the cell nucleus. The response of the UVR8-PYR1 complex elements to UV-B provides support for the development of UV-B optogenetic tools.

[0163] Example 3: UV-B and UVR8 promote drought resistance and stomatal closure in Arabidopsis thaliana in dependence on ABA receptors PYR1 / PYLs

[0164] To investigate the role of UV-B in promoting the formation of the UVR8-PYR1 complex in plant growth and development, the inventors constructed genetic materials such as the uvr8 pyr1 pyl124 five mutant, UVR8-FLAG / pyr1 pyl124, PYR1 overexpression material PYR1-FLAG, and GFP-PYR1, and observed stomatal phenotype and drought phenotype.

[0165] The results showed that the UVR8-overexpressing material UVR8-FLAG and the PYR1-overexpressing materials GFP-PYR1 and PYR1-FLAG exhibited stomatal closure and drought resistance phenotypes in response to UV-B. The uvr8 mutant and uvr8 pyr1 pyl124 lost their UV-B response. UVR8-FLAG / pyr1 pyl124 could not compensate for the stomatal and drought adaptation responses of the pyr1 pyl124 mutant to UV-B, indicating that UV-B and UVR8 promote drought resistance and stomatal closure in Arabidopsis thaliana dependent on the ABA receptor PYR1 / PYLs (…). Figure 3 AC).

[0166] This result suggests that the development of the UV-B optogenetic tool UV-B-UVR8-PYR1 has practical functional value in regulating stomatal opening and closing and plant drought adaptability, and can be used in agriculture.

[0167] Example 4: UV-B and UVR8 activate ABA response gene expression

[0168] The inventors discovered that UV-B promotes the interaction of the UVR8-PYR1 complex and that UV-B's promotion of plant drought resistance depends on ABA receptors PYR1 / PYLs. This embodiment explores the role of UV-B in promoting the formation of the UVR8-PYR1 complex at the gene regulatory level.

[0169] Using 10-day-old Col-0 and uvr8 (uvr8-6) seedlings as materials, samples were taken after UV-B treatment and ABA treatment for RNA-seq sequencing. The results showed that UV-B regulates the expression of 462 ABA-responsive genes in a manner dependent on UVR8. Figure 4 A). Analysis of heatmaps and gene regulation revealed that 243 genes (53% of 462 genes) were upregulated by both ABA and UV-B. GO analysis showed that most of these genes were related to responses to abiotic stimuli such as light and drought. Figure 4 BD). Further analysis using qRT-PCR to detect the expression trends of ABA-responsive genes such as DREB2A and ABF4 revealed that UV-B significantly induced the expression of DREB2A and ABF4 genes in wild-type Col-0, UVR8-FALG, and GFP-PYR1. This response trend disappeared in the uvr8 mutant, uvr8 pyr1pyl124 mutant, and GFP-PYR1 / uvr8, indicating that UV-B and UVR8 directly activate the expression of ABA-responsive genes. Figure 4 E, F).

[0170] The above results suggest that the UV-B optogenetic tool UV-B-UVR8-PYR1 can be used to regulate gene expression.

[0171] Example 5: UV-B promotes PYR1-ABI1 interaction through UVR8-PYR1 complex elements.

[0172] The ABA receptor PYR1 undergoes a conformational change upon binding to ABA, interacting with the negative regulator phosphatase ABI1 in an ABA-dependent manner. This relieves the inhibitory effect of ABI1 phosphatase on SnRK2s kinase, activates downstream SnRK2s kinase activity, promotes the expression of ABA-responsive genes, and enhances plant adaptability to drought stress. The inventors discovered that UV-B-activated UVR8 can directly interact with PYR1, and that UV-B and UVR8 can activate the expression of ABA-responsive genes. This study investigates whether UV-B directly activates PYR1 through the UVR8-PYR1 complex, thereby affecting the interaction between PYR1 and phosphatase ABI1.

[0173] The interaction was verified using a yeast three-hybrid assay, involving pBridge-ABI1 and pBridge-ABI1-UVR8. C27 (where UVR8) C27 The C-terminal 27 amino acids of the UVR8 protein, specifically amino acids 397-423aa in the sequence shown in SEQ ID NO:1, and pBridge-ABI1-UVR8 C44 (where UVR8) C44The C-terminal 44 amino acids of the UVR8 protein (amino acids 397-440aa) were used in a yeast three-hybrid experiment with pGADT7-PYR1 co-transfected yeast Y190. The β-galactosidase activity was used as an indicator of the strength of the interaction. The results showed that UVR8... C27 Or UVR8 C44 Its presence can significantly promote the interaction between ABI1 and PYR1.

[0174] The addition of MET methionine can inhibit the second protein, UVR8, in the pBridge vector of the yeast three-hybrid system. C27 Or UVR8 C44 The expression of UVR8 was found after applying MET treatment. C27 Or UVR8 C44 The promoting effect on ABI1-PYR1 interaction was significantly weakened. Figure 5 A, B) indicate that the yeast three-hybrid system is working and UVR8 C27 or UVR8 C44 (The C-terminal 397-423aa and 397-440aa of UVR8 (SEQ ID NO:1) can promote ABI1-PYR1 interaction.

[0175] Using MYC-ABI1 and MYC-ABI1 / uvr8 seedlings grown for 14 days as materials, in vivo Co-IP experiments were conducted with and without UV-B treatment. It was found that MYC-ABI1 treated with UV-B accumulated more PYR1 protein, a trend that disappeared in the MYC-ABI1 / uvr8 material. Figure 5 (C, D) This indicates that UV-B promotes PYR1-ABI1 interaction through UVR8. Whether in yeast cells or Arabidopsis leaf cells, the UV-B-UVR8-PYR1 receptor element can function in vivo to promote PYR1-ABI1 interaction, providing a pathway for the in vivo application of UV-B optogenetic tools.

[0176] Example 6: UV-B activates SnRK2s kinase activity via UVR8-PYR1 receptor element.

[0177] As mentioned earlier, UV-B promotes PYR1-ABI1 interaction through UVR8. The inventors further investigated whether UV-B and UVR8 regulate ABI1 phosphatase activity and SnRK2s kinase activity through the UVR8-PYR1 element.

[0178] First, the HIS-tagged ABI1 protein and UVR8 were purified in vitro. W285A Protein, UVR8, UVR8 C44The activity of ABI1 phosphatase was detected in vitro using MBP-tagged PYR1 protein, with absorbance at 630 nm as the quantitative indicator. PYR1 had no significant effect on ABI1 phosphatase activity in the absence of ABA, but the presence of UVR8 protein significantly inhibited ABI1 phosphatase activity. This indicates that the UVR8-PYR1 interaction promotes the PYR1-ABI1 interaction, thereby inhibiting ABI1 phosphatase activity. Figure 6 A).

[0179] In vivo kinase extraction experiments were conducted using 10-day-old OST1-FLAG seedlings. Phosphorylation levels of SnRK2s kinase were detected using phosphorylation antibodies that specifically recognize the S171 and S175 sites of SnRK2s kinase. The results showed that UV-B treatment significantly increased the phosphorylation level of SnRK2s kinase in OST1-FLAG, indicating that UV-B promotes SnRK2s kinase phosphorylation. Figure 6 B).

[0180] The inventors used OST1-FLAG transgenic plants as material and performed phosphorylation mass spectrometry analysis with and without UV-B treatment. They found that UV-B treatment could detect phosphorylation of serine at position 171 of the OST1 protein. Figure 6 (CE). The results showed that UV-B activated the OST1 protein and promoted its phosphorylation.

[0181] The above results indicate that the activity level of kinase SnRK2s can be regulated using the UV-B-UVR8-PYR1 optogenetic tool.

[0182] Example 7: UV-B Response of UVR8-PYR1 Receptor Elements to Promote Drought Resistance in Arabidopsis thaliana and Its Agricultural Application

[0183] PYR1 not only acts as an ABA receptor and can be activated by ABA, but the inventors also discovered that PYR1 can be activated by UV-B and UVR8. UV-B-activated UVR8 interacts with PYR1 to form the UVR8-PYR1 interaction complex, thereby promoting PYR1-ABI1 interaction, inhibiting ABI1 phosphatase activity, increasing SnRK2s kinase phosphorylation levels, promoting the expression of genes responsible for regulating drought response, and promoting stomatal closure. Ultimately, UV-B activates the ABA response, improving plant drought adaptability. Figure 7 A).

[0184] The results indicate that constructing a UV-B-UVR8-PYR1 optogenetic tool holds great promise for improving plant drought adaptability in agriculture. At the spatial level, the UVR8-PYR1 complex element can respond to UV-B by altering the spatial localization of the cytoplasm and nucleus. Figure 7B); At the molecular level, the application of the UVR8-PYR1 complex element in yeast cells can promote ABI1-PYR1 interaction and regulate reporter gene expression without the application of exogenous ABA. Figure 7 C) The basic application of the UVR8-PYR1 interaction in plant cells is that it can use UV-B to regulate the activity of important core proteins ABI1 phosphatase and SnRK2s kinase, and use the ABI1-SnRK2s regulatory hub to regulate the function of downstream proteins such as transcription factors or the expression of target genes.

[0185] In summary, UV-B activated UVR8-PYR1 elements activate the ABA response in a manner independent of ABA, enabling UV-B light signals and ABA hormone signals to synergistically regulate plant adaptation to drought environments through the dual receptor UVR8-PYR1. This provides a theoretical basis for constructing UV-B-UVR8-PYR1 optogenetic tools and utilizing UV-B light signals to improve crop drought resistance in production applications.

[0186] Sequence information:

[0187] UVR8 polypeptide sequence (SEQ ID NO:1):

[0188] MAEDMAADEVTAPPRKVLIISAGASHSVALLSGDIVCSWGRGEDGQLGHGDAEDRPSPTQLSALDGHQIVSVTCGADHTVAYSQSGMEVYSWGWGDFGRLGHGNSSDLFTPLPIKALHGIRIKQIACGDSHCLAVTMEGEVQSWGRNQNGQLGLGDTEDSLVPQKIQAFEGIRIKMVAAGAEHTAAVTEDGDLYGWGW GRYGNLGLGDRTDRLVPERVTSTGGEKMSMVACGWRHTISVSYSGALYTYGWSKYGQLGHGDLEDHLIPHKLEALSNSFISQISGGWRHTMALTSDGKLYGWGWNKFGQVGVGNNLDQCSPVQVRFPDDQKVVQVSCGWRHTLAVTERNNVFAWGRGTNGQLGIGESVDRNFPKIIEALSVDGASGQHIESSNIDPSS GKSWVSPAERYAVVPDETGLTDGSSKGNGGDISVPQTDVKRVRI

[0189] PYR1 polypeptide sequence (SEQ ID NO:2):

[0190] MPSELTPEERSELKNSIAEFHTYQLDPGSCSSLHAQRIHAPPELVWSIVRRFDKPQTYKHFIKSCSVEQNFEMRVGCTRDVIVISGLPANTSTERLDILDDERRVTGFSIIGGEHRLTNYKSVTTVHRFEKENRIWTVVLESYVVDMPEGNSEDDTRMFADTVVKLNLQKLATVAEAMARNSGDGSGSQVT

[0191] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for improving plant drought resistance, comprising enhancing the function of plant UVR8-PYR1 phototransistors, wherein the UVR8-PYR1 phototransistors respond to a UV-B signal and activate PYR1.

2. The method as described in claim 1, characterized in that, Under UV-B activation, in the UVR8-PYR1 optical signaling element, UVR8 and PYR1 interact to form a complex; preferably, the complex promotes the interaction between PYR1 and ABI1 and inhibits ABI1 phosphatase activity; more preferably, a region containing at least 27 aa of the C-terminus of the UVR8 protein promotes the interaction between PYR1 and ABI1; and / or Under UV-B activation, the UVR8-PYR1 photosignaling element activates SnRK2s kinase; preferably, the UVR8-PYR1 photosignaling element activates SnRK2s kinase by inhibiting the phosphatase activity of ABI1 and relieving the inhibition of SnRK2s kinase by ABI1; preferably, the activation of SnRK2s kinase promotes the expression of drought response genes, promotes stomatal closure, and improves plant drought resistance; Preferably, the drought response genes include: DREB2A and ABF4.

3. The method as described in claim 1 or 2, characterized in that, The UV-B is a narrow-band UV-B; preferably 1-10 μmol / m². –2 s –1 Low-intensity UV-B.

4. The method as described in claim 1, characterized in that, The enhancement of the plant's UVR8-PYR1 photoelectrochemical signaling element function includes upregulating the expression or activity of UVR8 and / or PYR1, or promoting the interaction between UVR8 and PYR1 to form a complex; preferably, the upregulation of UVR8 and / or PYR1 includes: introducing the coding gene of UVR8 and / or PYR1 or an expression construct or vector containing the coding gene into the plant; performing gain-of-function mutations on UVR8 and / or PYR1, preferably, obtaining functional UVR8 and / or PYR1 by gene modification or mutation in plants with lost or reduced UVR8 and / or PYR1 function; preferably, the introduction of the coding gene of UVR8 and / or PYR1 into the plant includes introduction based on transgenic technology, or introduction based on hybridization technology.

5. The use of a UVR8-PYR1 optical signal element or its upregulated molecule for: Improve plant drought resistance; among them, The UVR8-PYR1 optical signal element responds to the UV-B signal and activates PYR1; It promotes the interaction between PYR1 and ABI1 and inhibits the phosphatase activity of ABI1; wherein, under UV-B activation, UVR8 interacts with PYR1 to form a complex and exerts the aforementioned promoting effect. Activation of SnRK2s kinase; wherein, under UV-B activation, the UVR8-PYR1 photosignaling element activates SnRK2s kinase by inhibiting the phosphatase activity of ABI1 and relieving the inhibition of SnRK2s kinase by ABI1; preferably, the activation of SnRK2s kinase promotes the expression of drought response genes, promotes stomatal closure, and improves plant drought resistance; preferably, the drought response genes include: DREB2A and ABF4.

6. The use as described in claim 5, characterized in that, The upregulating molecules of UVR8-PYR1 include: exogenous UVR8 and / or PYR1 encoding genes or expression constructs or vectors containing such encoding genes; reagents for gain-of-function point mutations of UVR8 and / or PYR1; or reagents for obtaining functional UVR8 and / or PYR1 by gene modification or mutation in plants with lost or reduced UVR8 and / or PYR1 function.

7. The method as described in any one of claims 1 to 4 or the use as described in any one of claims 5 to 6, characterized in that, The plant is a plant containing UVR8-PYR1 photosignal elements, preferably also including ABI1 and SnRK2s kinases; preferably, the plant includes: dicotyledonous plants, monocotyledonous plants or gymnosperms; preferably, it includes: cruciferous plants, grasses, malvaceae plants, solanaceous plants; or, the plant includes: crops, flowering plants or forestry plants.

8. Provide the use of the UVR8-PYR1 optical signaling element as a biomarker for identifying plant drought resistance, or for targeted selection or identification of plants; Preferably, the method for targeted selection or identification of plants includes: Identify the expression or activity of UVR8 and / or PYR1 in the test plant, or analyze the interaction between UVR8 and PYR1 under UV-B activation: If the expression of UVR8 and / or PYR1 in the test plant is higher than the average expression value of UVR8 and / or PYR1 in this type of plant, or if the interaction between UVR8 and PYR1 under UV-B activation is higher than the average level of interaction in this type of plant, then it is a plant with improved drought resistance.

9. A method for screening substances that enhance plant drought resistance, comprising: (1) Add the candidate material to a system containing UVR8 and / or PYR1; Ideally, the system is in an arid environment; (2) Detect the system, observe the expression or activity of UVR8 and / or PYR1 in the system of (1), or analyze the interaction between UVR8 and PYR1; if the candidate substance upregulates the expression or activity of UVR8 and / or PYR1, or the interaction between UVR8 and PYR1 is significantly enhanced, it indicates that the candidate substance is a substance that can improve the drought resistance of plants. Preferably, the system further includes ABI1, and the method further includes: analyzing the phosphatase activity of ABI1; if its phosphatase activity decreases, it indicates that the candidate substance is a substance that can improve plant drought resistance. Preferably, the system further includes SnRK2s kinase, and the method further includes: analyzing the activity of SnRK2s kinase; if its kinase activity is increased, it indicates that the candidate substance is a substance that can improve plant drought resistance. Preferably, the system further includes drought-response genes, and the method further includes: analyzing the drought-response genes; if the expression of the drought-response genes is promoted, it indicates that the candidate substance is a substance that improves plant drought resistance; preferably, the drought-response genes include: DREB2A and ABF4.

10. A separated composite, said composite being a combination of UVR8 and PYR, constituting a UVR8-PYR1 optical signal element; said composite being formed under UV-B activation; preferably, said composite further includes: ABI1; the complex is used for: as a regulatory target to improve plant drought resistance, to prepare regulatory molecules for plant drought resistance, as a biomarker to identify plant drought resistance, and as a screening target or tool to improve plant drought resistance.

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